Nonlinear kinetics of inhaled propylene glycol monomethyl ether in Fischer 344 rats following single and repeated exposures

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

89,19-28 ( 1987)

Nonlinear Kinetics of Inhaled Propylene Glycol Monomethyl Ether in Fischer 344 Rats following Single and Repeated Exposures’ DAVID Mammalian

A. MORGOTT

AND RICHARD

J. NOLAN’

and Environmental Toxicology Research Laboratories, Health and Environmental Sciences USA, The Dow Chemical Company, Midland, Michigan 486 74

Received August 27, 1986; accepted February 3, 1987 Nonlinear Kinetics of Inhaled Propylene Glycol Monomethyl Ether in Fischer 344 Rats following Single and Repeated Exposures. MORGOTT, D. A., AND NOLAN, R. J. (1987). Toxicol. Appl. Pharmacol. 89, 19-28. The kinetics of propylene glycol monomethyl ether (PGME) and its demethylated metabolite, propylene glycol (PGLY), were investigated with the aim of describing concentration- and treatment-related changes in absorption and clearance. Groups of Fischer 344 rats received either 1 or 10 daily 6-hr inhalation exposures to PGME. Single exposures were performed using both nose-only (300,750, 1500, and 3000 ppm) and wholebody (300 and 3000 ppm) inhalation techniques, whereas multiple exposures (300 and 3000 ppm) were confined to the whole-body procedure. PGME blood levels failed to plateau during a 6-hr inhalation exposure, indicating that absorption was limited by respiration. The clearance of PGME from the blood could be described as a pseudo-zero-order process following each exposure concentration and treatment regimen examined. PGLY blood levels indicated that the demethylation of PGME to PGLY was saturated at exposure concentrations exceeding 1500 ppm. PGME blood levels were higher in male than in female rats receiving a single 3000 ppm exposure. Unlike the results from a single exposure, PGME elimination was essentially complete 24 hr after the last of 10 consecutive 3000 ppm exposures. The changes in PGME elimination following multiple 3000 ppm exposures were associated with higher in vitro levels of cytochrome P-450 and mixed-function oxidase activity. Multiple exposures to 300 ppm did not affect PGME elimination or in vitro microsomal metabolism. 0 1987 Academic Press. Inc.

1-Methoxy-2-propanol, known commonly as propylene glycol monomethyl ether (PGME), is a moderately volatile (vapor pressure of 12.5 mm Hg at 25°C) colorless liquid marketed for general solvent use under the tradenames of Dowano13 PM and Arcosolve4 PM. Though it has physical and chemical properties similar to those of ethylene glycol monomethyl ether, PGME is metabolized

differently and is therefore significantly less toxic than the ethylene derivative (Hanley et al., 1984; Doe et al., 1983; Miller et al., 198 1). Consequently, PGME is increasingly being employed as a substitute for ethylene glycol monomethyl ether in many paint, ink, and dye formulations. Recent short-term and subchronic inhalation studies with PGME have established 3000 ppm as a minimal effect level for Fischer 344 rats. The most notable consequence of repeated exposures at this concentration was an increase in relative liver weight. Miller et al. (1981) found a 24% increase in the mean relative liver weights of male rats and a 6% increase in the relative liver weights of

’ Portions of this work were presented at the 25th Annual Meeting of the Society of Toxicology, March 3-7, 1986, New Orleans, LA. This work was cosponsored by Dow Chemical USA and the ARC0 Chemical Co. * To whom reprint requests should be addressed. ’ Trademark of The Dow Chemical Co. 4 Trademark of the ARC0 Chemical Co. 19

0041-008X/87

$3.00

Copyright Q 1987 by Academic Press, Inc. All rights of reproduction in sny form reserved.

20

MORGOTT

females receiving nine 6 hr/day exposures to 3000 ppm. Similarly, Landry et al. (1983) observed a 6-7% increase in the mean relative liver weights of male and female rats after a 13-week exposure (5 days/week) to 3000 ppm. Central nervous system (CNS) depression (lethargy, sedation, and ataxia) was also noted for rats receiving 3000 ppm exposures; however, the effect was temporary and accomodation occurred after only a few exposures. Exposure concentrations of 300 and 1000 ppm failed to cause treatment-related effects in either of these studies. The current study focuses on the absorption and clearance of inhaled PGME over the concentration range (300-3000 ppm) used in previous toxicity studies with Fischer 344 rats. The study was designed to determine (i) whether the inhalation of PGME vapor caused dose-dependent changes in PGME and propylene glycol (PGLY) kinetics following single and repeated exposures and (ii) the affect of repeated exposures on in vitro liver microsomal metabolism. MATERIALS

AND

METHODS

Animals. Fischer 344 rats of both sexeswere purchased from the Charles River Breeding Laboratories (Kingston, NY) and were acclimated to the laboratory environment for at least I week prior to use. Rats were housed in stainless steel cages (two per cage) with municipal tap water and a pelleted rodent chow available ad libitum. Animals were housed in rooms designed to maintain a temperature of 22 +- 2”C, a relative humidity of 40-60% and a photoperiod of 12 hr. At the time of use, the weight ranges for the male and female rats were 185-230 and 160- 190 g, respectively. Reagents and chemicals. PGME and ethylene glycol monoethyl ether were obtained through The Dow Chemical Co. (Midland, MI). Analysis by gas liquid chromatography (GLC) with name ionization detection (FID) showed the PGME to contain 98.6% of the alpha isomer ( I-methoxy-2-propanol) with the remaining 1.4% constituting the beta form (2-methoxy-2-propanol). Propylene glycol ( 1,2-propanediol) and ethylene glycol ( 1,2ethanediol) were purchased from Fisher Scientific Co. and J. T. Baker Chemical Co., respectively. The following enzyme substrates and products were purchased from the Fluka Chemical Corp. (Hauppauge, NY) and used at their stated purity: aniline hydrochlo-

AND NOLAN ride (99%); pnitroanisole (97%); paminophenol hydrochloride (99%); pnitrophenol(99%); and 4-nitrocatechol (98%). DL-Isocitric acid (97%) isocitrate dehydrogenase (porcine heart type VI, 25 units/mg), and NADP (monosodium salt, 98%) were obtained from Sigma Chemical Co. (St. Louis, MO). All other solvents, chemicals, and gaseswere of reagent-grade purity or greater. Exposure methodologies. Inhalation exposures were conducted under dynamic airflow conditions for approximately 6 hr/day using either a l- or lo-day treatment schedule. Single-day exposures were performed using both nose-only (300,750, 1500, and 3000 ppm PGME) and whole-body exposure chambers (300 and 3000 ppm PGME), whereas the repeated exposures (300 and 3000 ppm) were confined to the whole-body technique. Nose-only exposures were conducted at a flow rate of 5 liters/min using a custom-made l-liter acrylic chamber capable ofholding up to four rats (McKenna et a/., 1982). PGME vapor concentrations of 300 and 750 ppm were achieved by dilution of the vapor metered from loo-liter Saran bags containing stock concentrations of 3000 and 4500 ppm, respectively. The 1500 and 3000 ppm levels were attained with an integrated flameless heat torch/Jtube assembly, connected in series with the chamber airflow (Miller et al.. 1980). The concentration of PGME entering the nose-only chamber was monitored at lomin intervals using a Varian 1400 gas chromatograph (Varian Instrument Group, Palo Alto, CA) equipped with a FID and two 1.O-ml gas sampling loops on an 8port switching valve. Analyses were performed at 110°C using a stainless steel column (2 ft X l/l6 in.) packed with 0.8% THEED (tetrahydroxyethylenediamine) on a 80/100 mesh Carbopack C support (Supelco, Inc., Bellefonte, PA). Whole-animal exposures were performed in 112-liter stainless steel and glass Rochester-type chambers operated at an airflow of 30 liters/min. Exposure concentrations of 300 and 3000 ppm were attained with the previously described heat torch/J-tube assembly. PGME chamber concentrations were determined at 25- to 50min intervals using a Miran I infrared gas analyzer (Foxboro-Wilks, Norwalk, CT) operated at a wavelength of either 3.45 or 8.80 pm. Untreated control animals were placed in an air-only control chamber. Analytical instruments were calibrated with three PGME vapor standards which bracketed the exposure concentration being examined. The vapor standards were prepared in preconditioned Saran bags using measured volumes of PGME and clean dry air from a calibrated Singer dry gas meter (American Meter Co., Philadelphia, PA). Treatment regimens. Single nose-only inhalation exposures were performed on groups of four rats for 6 hr. After the chamber was equilibrated to the desired vapor concentration, the animals were secured in individual wire mesh restrainers and positioned at the chamber ports. After the 6-hr exposure, the animals were removed

NONLINEAR

KINETICS

from the chamber and placed in a holding cage containing an ad libitum supply of food and water. Blood samples were collected from a jugular vein cannula both during and after the exposure. Whole-body exposures were used for both the kinetic studies and the in vitro experiments with the liver microsomal fraction. The kinetic studies were conducted on groups of 12 rats receiving either a single or the last of 10 consecutive whole-body exposures. The animals were placed in the chamber just prior to starting the exposure. At 2,5, and 6 hr into the final exposure session, the chamber was opened and 4 animals were quickly removed and bled. The rats removed at 2 and 5 hr were bled once, while the animals removed after 6 hr of exposure were bled serially. Since the chamber used for the whole-body exposures required about 20 min to equilibrate upon startup, the actual exposure period was somewhat less than 6 hr. Liver microsomes were isolated from groups of 5 rats approximately 18 hr afier the last of ten 6 hr/ day exposures. Blood collection and analysis. Blood samples of lOO200 ~1 were collected either from an indwelling jugular vein cannula for the nose-only exposure studies or from the retroorbital sinus (Riley, 1960) for the whole-body exposure segments. The cannulae were surgically implanted under methoxyflurane anesthesia approximately 48 hr prior to treatment (Harms and Ojeda, 1974). Blood samples were divided into two weighed ahquots for separate analysis of PGME and PGLY. The PGME samples were diluted with 0.9% saline containing ethylene glycol monoethyl ether as an internal standard, whereas the PGLY samples were diluted with acetone containing ethylene glycol as an internal standard. The diluted blood samples were spun for l-2 min in a high-speed microcentrifuge (Brinkman Instruments, Inc., Westbury, NY) prior to analysis. Analyses were performed isothermally at 110°C on a dual-channel Varian 3700 gas chromatograph equipped with a flame ionization detector, strip chart recorder, and Varian CDS 111 integrator on each channel. A glass analytical column (3 ft X 2 mm) packed with 0.8% THEED on an 80/100 Carbopack C support was used for the PGME analysis, while a fused silica capillary column (15 m x 0.33 mm) coated with a 0.5-pm film of DB-wax (J & W Scientific, Inc., Ranch0 Cordova, CA) was used for PGLY. Nitrogen at a flow rate of 30 ml/min was used as the carrier gas for both columns. The within-day precision for both methods was approximately 2-3%, and the limit of quantitation was about 3 pg/g of blood. Liver microsome analysis. Liver microsomes were isolated from groups of treated and control rats approximately 18 hr after the final exposure. Animals were anesthetized with carbon dioxide and killed by exsanguination before the liver was excised and weighed. Microsomes were isolated by differential centrifugation of a 25% liver homogenate prepared in 100 mM Trisacetate buffer (pH 7.4) containing 100 mM potassium

OF A GLYCOL

ETHER

21

chloride and 1 mM EDTA (Guengerich, 1982). The washed microsomal pellet was suspended in 100 mM T&acetate buffer containing 20% glycerol and 1 mM EDTA and then frozen at -70°C until assayed. Cytochrome P-450 was measured according to the method of Omura and Sato ( 1964) as reported by Guengerich (1982), using the dithionite-reduced carbon monoxide difference spectrum ofdetergent solubilized microsomes. Aniline hydroxylase activity was measured by the method of Imai et al. ( 1966) as described by Schenkman et al. (1967), and pnitroanisole O-demethylase activity was determined by the method of Netter and Seidal ( 1964) as modified by Reinke et al. ( 1979). Microsomal protein concentrations were determined by the method of Lowry et al. (195 1). Assays were performed on a dualbeam Beckman Acta CIII recording spectrophotometer (Beckman Instrument Co., Chicago, IL). Data analysis. The blood concentration of PGME during the absorption and clearance phases was described separately by the line of best fit using a leastsquares linear regression analysis. Based on the results of an F test (Draper and Smith, 1966) there was no significant lack of fit for the blood concentration-time data to a straight line. The area under the PGME blood concentration-time curves was calculated by the trapezoidal rule after extrapolating to a blood concentration of zero. The average daily exposure concentrations of PGME in the inhalation chamber were calculated from measurements taken at equal time intervals over the course of the exposure period. Body and organ weight data, P-450 measurements, and MFO activities were statistically analyzed using Student’s t test (p < 0.05).

RESULTS Nose-only exposures. The average concentrations of PGME in the inhalation chamber during the 300,750,1500, and 3000 ppm exposures were (mean + SD) 304 + 9,734 k 67, 1554 + 43, and 305 1 f 63 ppm, respectively. PGME blood levels continued to rise throughout each 6-hr exposure and did not reach an apparent steady state at any of the four exposure concentrations (Fig. 1). The disappearance of PGME from the blood was well described as a zero-order process at each exposure concentration, as determined by linear least-squares regression of the postexposure results. The average end-exposure blood levels of PGME increased disproportionately as the exposure concentration increased relative to the rise expected for first-

22

MORGOTT

loooo /I =y-%z-%zF

wm 1500 wm

9 750 kwm

;^*,

A

0

5

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15

20

25

30

TIME (hrs)

FIG. 1. PGME blood levels resulting from a single 6hr nose-only exposure to 300,750, 1500, and 3000 ppm PGME. The points represent the means (*SD) for four rats exposed to 300,750, and 1500 ppm and the means for two rats exposed to 3000 ppm of PGME. The lines were drawn based on the least-squares linear regression of the average PGME blood levels. A 17-hr value of 7 rg/ g for the 750 ppm exposure has not been plotted. A 42hr blood sample from rats receiving 3000 ppm contained insufficient PGME to quantify (i.e., ~3 &g). The inset depicts the zero-order kinetics for the 300 ppm exposure using linear coordinates.

AND NOLAN

3000 ppm exposure were indistinguishable from the PGLY maximum at 1500 ppm. Only a few of the blood specimens collected following the 300 ppm exposure contained quantifiable amounts of PGLY (i.e., >3 pg/g). Whole-body exposures. The average concentrations of PGME in the inhalation chamber during the single 6-hr exposures to 300 and 3000 ppm were 296 +- 10 and 2885 f 268 ppm, while the average chamber concentrations during the 10 daily 300 and 3000 ppm exposures were 303 -+ 7 and 3005 f 43 ppm. As was observed for the nose-only exposures, PGME blood levels failed to plateau during a 6-hr whole-body exposure, and the elimination of PGME from the blood could be described as a zero-order process for each of the treatment regimens (Fig. 4). There were no discernible differences in PGME kinetics for rats exposed once or for 10 consecutive days

2500 1 2000

E S y

1500

8

order kinetics (Fig. 2). Table 1 shows that the area under the blood concentration-time curves also rose disproportionately compared to the changes in exposure concentration. Furthermore, the values for the zero-order elimination rate of PGME, the calculated clearance, and the half-time (time required for a 50% reduction in the average end-exposure blood level) did not remain constant as expected for a first-order process. The maximum blood PGLY levels resulting from the 750,1500, and 3000 ppm exposures were 10.5, 20.7, and 16.4 rg/g, respectively. The 750 and 1500 ppm exposures produced a proportional increase in the maximum blood concentration of PGLY (Fig. 3) while the highest blood PGLY levels for the

g

loo0

I I? 500

0

1000

EXPOSURE

2000

CONC

3000

(ppm)

FIG. 2. Relationship between exposure concentration and the end-exposure blood concentrations of PGME in rats receiving a single 6-hr nose-only exposure. The dashed line indicates the theoretical linear increase expected for first-order elimination. The points are the mean (*SD) end-exposure blood concentrations of PGME for four rats exposed to 300,750, and 1500 ppm and the means for two rats exposed to 3000 ppm of PGME.

NONLINEAR

KINETICS

OF A GLYCOL

23

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TABLE I

SUMMAR~OFPROPYLENEGLYCOLMONOMETHYLETHERBLOODK~NETICSFOLLOWINGNOSE-ONLY ANDWHOLE-BODYINHALATIONEXPOSURES

Exposure conditions Nose-only Single (male)

Whole-body Single (male) Single (female) Whole-body Multiple (male)

Exposure concentration (mm)

End-exposure blood concn btig)

Zero-order elimination rate k&W

Blood clearance” (ml/min-kg)

Halftime b IW

Area under blood concn time curve W-w/g)

300 750 1500 3000

109 344 818 2113

22.3 30.5 43.0 67.2

3.1 1.3 0.8 0.5

2.4 5.6 9.5 15.7

0.6 2.9 10.0 32.7

300 3000 3000

14 1816 1322

15.1 58.7 55.2

3.1 0.5 0.6

2.4 15.5 12.0

0.4 30.1 20.7

300 3000

67 1002

15.5 51.4

3.5 0.8

2.2 9.7

0.4 14.2

ciCalculated as follows using a volume of distribution of 900 ml/kg (Stott and McKenna, 1984): Elimination rate (&g-hr) X volume distribution (ml/kg) end-exposure blood concentration (&g) b Time taken for the end-exposure blood concentration to decline by 50%.

at the 300 ppm level. In contrast, rats repeatedly exposed to 3000 ppm showed lower PGME blood levels than those receiving a single exposure. The average end-exposure blood levels of PGME were 45% lower after multiple exposures at 3000 ppm; and unlike the singly exposed rats, PGME was not detected in blood samples collected 24 hr after the 10th exposure. Blood propylene glycol levels resulting from a single or repeated exposure to 3000 ppm PGME are presented in Fig. 5. Following a single 3000 ppm exposure, PGLY blood levels rose slowly from a mean of 5.5 pg/g at 2 hr to an average maximum of 22 &/g at 30 hr. The PGLY blood levels resulting from repeated exposures at 3000 ppm peaked at 12 hr (26 pg/g) and were nearly the same at 2 and 24 hr (14.5 vs 18.4 pg/g). The PGLY results obtained at 300 ppm were at or below the quantitation limit for the analytical method.

Liver microsome analysis. The average chamber concentrations of PGME in the inhalation chamber over the lo-day treatment period were 302 +- 7 and 3022 t- 49 ppm. Rats repeatedly exposed to 300 ppm had a slight but statistically significant decrease in average body weight, absolute and relative liver weight, and hepatic cytochrome P-450 content relative to control animals (Fig. 6). Multiple exposures at 3000 ppm resulted in a significant increase in absolute and relative liver weight. These liver weight increases were accompanied by an induction of microsomal cytochrome P-450 and a two- to threefold rise in aniline hydroxylase and pnitroanisole Odemethylase activities. Sex differences. The average end-exposure PGME blood concentration for male rats was about 40% higher than that for females, and after 24 hr blood PGME levels were nearly 8 times greater in males than in females (Fig.

24

MORGOTT

AND NOLAN

1000 ppm and ventilation rates ranging from 50 to 100 ml/min, these authors found that the absorption of PGME in anesthetized Fischer 344 rats was directly proportional to the airflow through the upper respiratory tract. The clearance of PGME from the blood of Fischer 344 rats following the inhalation exposure was well described as a pseudo-zeroorder process at each exposure concentration and treatment regimen examined. PGME end-exposure blood levels, clearance times, and areas under the blood concentrationtime curves all showed the changes characteristic of nonlinear kinetics (Levy, 1968). Although the elimination of PGME was consistent with the saturation of a capacity-limited 0

5

10

15

20

25

30

TIME (hrs)

FIG. 3. Propylene glycol blood levels resulting from a single 6-hr nose-only exposure to 750, 1500, and 3000 ppm PGME. The points represent the means (*SD) for four rats exposed to 750 and 3000 ppm and the means for two rats exposed to 3000 ppm of PGME. The lines were drawn by inspection. A 42-hr blood sample from rats receiving 3000 ppm contained insufficient PGLY to quantify (i.e., <3 j&g).

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7). PGLY blood levels were approximately twofold higher for males over females throughout the 24-hr postexposure monitoring period (Fig. 8).

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DISCUSSION loI.;';';';.;';

PGME blood levels failed to plateau during a 6-hr exposure at concentrations ranging from 300 to 3000 ppm. The high polarity and complete miscibility of PGME in aqueous solutions give rise to a very high blood/air partition coefficient (403; Stott and McKenna, 1984) that favors PGME uptake from the respiratory tract. These observations indicate that the absorption of PGME was limited by pulmonary ventilation in the rat. Stott and McKenna ( 1984) have also reported data that indicate PGME absorption was limited by respiration, At an exposure concentration of

0

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30

TIME (hrs) FIG. 4. PGME blood levels resulting from either a single or 10 consecutive 6 hr/day whole-body exposures to 300 and 3000 ppm PGME. Each point represents the mean (*SD) from three to four male rats. The lines were drawn based on the least-squares linear regression of the average PGME blood levels. A IO-hr value of 6 erg/g for rats receiving multiple 300 ppm exposures has not been plotted. The following blood samples contained insufficient PGME to quantify (i.e., <3 pg/g): the 42-hr sample for a single 3000 ppm exposure and the 30-hr sample for multiple 3000 ppm exposures. The inset depicts the zeroorder kinetics for the 3000 ppm exposures using linear coordinates.

NONLINEAR

1.0,. 0

;. 5

;. 10

; 15

; 20

.

KINETICS

; 25

; 30

TIME (hrs)

FIG. 5. Propylene glycol blood levels resulting from either single or 10 consecutive 6 hr/day whole-body exposures to 3000 ppm PGME. Each point represents the mean (*SD) from three to four male rats. The lines were drawn by inspection. The following blood samples contained insufficient PGLY to quantify (i.e., <3 &g): the 42-hr sample for a single 3000 ppm exposure and the 30hr sample for multiple 3000 ppm exposures.

OF A GLYCOL

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mately equivalent to the amount of PGME that would be absorbed during a 6-hr exposure at 1200 ppm. The concentrations of PGLY observed in the blood following the single 6-hr nose-only exposures suggest that the demethylation of PGME to form PGLY was saturated in rats exposed to over 1500 ppm of PGME. The maximum concentration of PGLY in the blood of rats exposed to 750 and 1500 ppm was proportional to the exposure concentration, while the maximum blood PGLY concentration in rats exposed to 3000 ppm was lower than that in rats exposed to 1500 ppm. Recognizing that peak blood levels would be lower in rats following a 6hr inhalation exposure than if an equivalent amount of PGME was administered as a bolus oral dose, the oral (Miller et al., 1983) and inhalation data are in reasonable agreement as to the amount of PGME that will saturate the O-demethylation of PGME. However, since blood PGLY levels were proportional to the exposure concentrations in rats exposed to 750 and 1500 ppm, saturation of the O-demethylation of PGME does not explain the nonlinear kinetics observed in rats ex300

process, the rate-limiting step(s) could not be determined from these data. Miller et al. (1983) found that 55-65% of the [l-r4C]PGME administered orally was excreted as 14C02, presumably via the oxidation of PGLY to lactate and pyruvate (Ruddick, 1972). Another lo-20% of the oral dose was eliminated in the urine as PGLY and as the glucuronide and sulfate conjugates of PGME. Dose-dependent decreases in 14C02 elimination, and observed changes in the urinary metabolite profile, led Miller et al. to conclude that the demethylation of PGME to PGLY was saturated at a dose of 8.7 mmol/kg. When the near complete absorption of PGME vapor from the lung (87% as shown by Stott and McKenna, 1984) and the ventilation rate of 7.3 liter/hr for 225-g rats (Fiserova-Bergerova and Hughes, 1983) are considered, the 8.7 mmol/kg oral dose is approxi-

250

W 300 200 d E fzj 0 ap

q

3000

ppm ppm

150

100

50

w-r.

e.ooY RATIO

~450

ACTIVITY ANISOLE ACTIVITY

FIG. 6. Changes in body and liver weights, liver cytochrome P-450 content, and microsomal aniline hydroxylase andpnitroanisole O-demethylase activities resulting from 10 consecutive 6 hr/day exposures to either 300 or 3000 ppm of PGME. Each column depicts the average for five male rats plotted as a percentage of a similar number of control animals. t, Statistically significant differences from control values using Student’s t test (p > 0.05).

MORGOTT

26

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100

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exposure ____-____10-p 0

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; 10

; 15

; 20

; 25

30

TIME (hrs)

FIG. 7. PGME blood levels in male and female rats receiving a single 6-hr whole-body exposure to 3000 ppm PGME. Each point represents the mean @SD) from three rats. The lines were drawn based on the leastsquares linear regression of the average PGME blood levels. The 42-hr blood samples from both the male and female rats contained insufficient PGME to quantify (i.e., ~3 pg/g). The inset depicts the zero-order kinetics using linear coordinates.

posed to between 300 and 1500 ppm of PGME. The kinetics of PGME and PGLY were affected by both the number of inhalation exposures and the exposure concentration. The zero-order rate of elimination of PGME increased with the vapor concentration after a single nose-only exposure (Table I), suggestive of an adaptive metabolic response. Increases in liver cytochrome P-450 content and MFO-mediated oxidations of aniline and p-nitroanisole suggest that autoinductive increases in microsomal-mediated metabolism may be responsible for the lower blood levels of PGME observed after repeated exposures and that this induction prevented the day-today accumulation of PGME that would otherwise have been predicted based on the single-exposure data. Though several studies have shown that high oral dosages of PGLY

AND NOLAN

induced liver microsomal metabolism in the rat, PGLY does not appear to be responsible for the P-450 induction observed in the present study. Male Wistar rats given 8- 10 ml/kg/day of PGLY for 3-7 days responded with increases in microsomal p-nitroanisole O-demethylase and aniline hydroxylase activities relative to untreated controls (Yamamoto and Adachi, 1978; Dean and Stock, 1974). However, these changes in MFO activity were not accompanied by increases in either liver weight or cytochrome P-450 content. In addition, the PGLY blood levels attained in rats after repeated exposures to 3000 ppm PGME (Fig. 5) were far below the blood concentrations (< 1 mg/ml) occurring in species given large oral dosages of PGLY (Lehman and Newman, 1937; Yu et al., 1985). The induction of liver microsomal P450 by 10 whole-body exposures to 3000 ppm was accompanied by increases in rela-

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female

limit of quantitation ,_-____________________________________

t SXpOS”rS ..-.---_-_-

1.0 0

;

;

;.

;

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5

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25

; 30

TIME (hrs)

FIG. 8. Propylene glycol blood levels in male and female rats receiving a single 6-hr whole-body exposure to 3000 ppm PGME. Each point represents the mean (*SD) from three rats. The lines were drawn by inspection. The 42-hr blood samples from both the male and female rats contained insufficient PGLY to quantify (i.e., <3 Hdd.

NONLINEAR

KINETICS

tive liver weight that were equivalent to those found by Miller et al. (198 1) in male rats receiving 9 exposures at 3000 ppm. The increased liver weights observed in previous toxicity studies were undoubtedly accompanied by autoinductive increases in microsomal metabolism; otherwise, given the nonlinear kinetics observed in the present study, potentially lethal amounts of PGME would have accumulated in these rats. It is interesting that higher blood PGME levels were observed in male than in female rats exposed to 3000 ppm and that Miller et al. (198 1) found that the increase in relative liver weights was greater in male (24%) than in female (6%) rats following nine daily 6-hr exposures to 3000 ppm of PGME. The larger increase in relative liver weights observed in male rats could be a response to the higher levels of PGME in their blood. However, sexrelated differences in the activity and inducibility of microsomal enzymes are frequent observations in the rat and, in many cases, are under hormonal control (Kato, 1974). Because the absorption of inhaled PGME was limited by pulmonary ventilation, factors affecting respiration would be expected to influence the rate and amount of PGME absorbed during an inhalation exposure. For example, the concentration-dependent increases in PGME end-exposure blood levels were, at maximum, less than twice the expected concentration assuming linear kinetics. These disproportionate changes are relatively small considering the markedly nonlinear clearance of PGME from the blood. However, these measurements are being compared under non-steady-state conditions, where the overall accumulation of PGME is restricted by the ventilation rate. Likewise, the PGME blood levels resulting from a single nose-only exposure were approximately 50% higher than those obtained from a similar whole-body exposure. A portion of this increase may be attributed to the slightly longer exposure period associated with the nose-only procedure; however, the magnitude of the difference suggests that hy-

OF A GLYCOL

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27

perventilation in response to the stress of being restrained may have contributed significantly to the increased absorption of PGME during the nose-only exposures. The kinetics of inhaled PGME in the rat are relatively unique and distinctly unlike those observed with most aromatic solvents or halogenated hydrocarbons with limited water solubility. At exposure concentrations exceeding 300 ppm, the absorption of PGME from the lung was limited by pulmonary ventilation, and the blood levels failed to plateau during the course of a 6-hr exposure. In addition, PGME blood levels declined in an apparent zero-order fashion and showed no evidence of a transition to first-order at blood concentrations ranging as low as 20 pg/g. The data also suggest that PGME induced its own metabolism in the rat, as a function of both the exposure concentration and the number of exposure sessions. This induction of P450-mediated MFO activity provides the most likely explanation for the increased liver weight resulting from repeated exposures of the rat to PGME.

REFERENCES M. E., AND STOCK, B. H. ( 1974). Propylene glycol as a solvent in the study ofhepatic microsomal enzyme metabolism in the rat. Toxicol. Appl. Pharmacol. 28, 44-52. DOE, J. E., SAMMUELS, D. J., TINSTON, D. J., AND WICKRAMARATNE, G. A. (1983). Comparative aspects of the reproductive toxicology by inhalation in rats of ethylene glycol monomethyl ether and propylene glyco1 monomethyl ether. Toxicol. Appl. Pharmacol. 69, 43-47. DRAPER, N. R., AND SMITH, H. (1966). Applied Regression Analysis, pp. 26-3 1. Wiley, New York. FISEROVA-BERGEROVA, V., AND HUGHES, H. C. (1983). Species differences on bioavailability of inhaled vapors and gases. In Modeling oflnhalation Exposure to Vapors: Uptake, Distribution, and Elimination (V. Fiserova-Bergerova, Ed.), Vol. 2, pp. 97-106. CRC Press, Boca Raton, FL. GUENGERICH, P. F. (1982). Microsomal enzymes involved in toxicology-analysis and separation. In Principles and Methods of Toxicology (A. W. Hayes, Ed.), pp. 609-634. Raven Press, New York. DEAN,

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