Introduction

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Octamethylcyclotetrasiloxane (D₄)³ is a clear, odorless, silicone fluid of molecular weight 296 with alternating silicon-oxygen bonds connected in a ring (cyclic) arrangement with two methyl groups covalently bonded to each silicon atom (-(CH₃)₂SiO-) (Fig. 1). D₄ is an intermediate in the industrial manufacture of polydimethylsiloxane, a silicone polymer that is used widely in industrial and consumer applications (Stark et al., 1982). D₄ is also an ingredient in selected personal care products, including antiperspirants and skin care products. In addition, workplace exposures occur in the production of D₄ and other silicone materials via the respiratory route. The chemical and physical properties of D₄ are well known (Varaprath et al., 1996), and the distribution and persistence of mixtures of low molecular weight silicones after s.c. injections in mice were reported by Kala et al. (1998). However, there is little published data on the biological fate and toxicological effects of D₄ after relevant routes for human exposure.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Chemical structure of D4.

McKim et al. (1998) reported that repeated inhalation exposure to high concentrations of D₄ produces a reversible and dose-related liver enlargement with significant induction of cytochrome P-450 CYP2B1/2. In addition, the enzyme induction profile produced during exposure to D₄ was comparable to that observed with phenobarbital (PB). The results of these studies provided compelling evidence that D₄ was a PB-like inducer of hepatic microsomal enzymes in the Fischer 344 rat. Preliminary results presented in abstract form demonstrated that pretreatment of female rats with PB before administration of D₄ increased the excretion rate and metabolism of D₄ (Salyers et al., 1996). Thus enzyme induction, either by D₄ itself or by other exogenous chemicals, may influence disposition of D₄ in the rat.

Because of the widespread use of D₄ and the potential for human exposure, a comprehensive program has been initiated to assess the kinetics, metabolism, induction, and toxicity of D₄ in rats after relevant routes of exposure. In addition, studies have been initiated to examine the pharmacokinetics of D₄ in humans (Utell et al., 1998).

Specific objectives of these studies were to assess the effects of dose and gender on the distribution, persistence, and pathways for elimination of D₄ and its radioactive metabolites after either a single inhalation exposure to [¹⁴C]D₄ or multiple inhalation exposures to unlabeled D₄ followed by a single inhalation exposure to [¹⁴C]D₄. These studies provide information that may be helpful in characterizing the risk of human populations exposed to D₄.

	Experimental Procedures

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Test Animals. Young adult male and female Fischer 344 rats weighing approximately 125 to 210 g were obtained from Charles River Canada Inc. (St. Constant, Quebec, Canada). Animals were housed individually in suspended stainless steel, wire mesh bottom cages in a room designed to be maintained at 65-78°F, and 30 to 70% relative humidity. A commercial diet (PMI Certified Rodent Chow, 5002; PMI Feeds Inc., St. Louis, MO) and municipal drinking water were available ad libitum except during exposure periods. The photoperiod alternated 12 h of light with 12 h of darkness. During inhalation exposures, animals were housed in polycarbonate restrainers to achieve nose-only exposure. After exposure, animals allocated to the elimination components of the experiments were individually housed in Roth-type glass metabolism cages designed for the separate collection of urine, feces, and expired air. These animals were acclimated to the metabolism caging for 48 h before being placed on study. Airflow through the metabolism caging was kept at a minimum of 500 ml/min.

Test Chemicals. D₄ (CAS 000556672) was obtained from Dow Corning Corporation (Midland, MI). The purity was determined to be 99.8% by gas chromatography-mass spectrometry (GC/MS). [¹⁴C]Labeled D₄ was obtained from Wizard Laboratories (West Sacramento, CA). Radiochemical purity was confirmed by HPLC equipped with a radiometric flow detector. All dosing solutions had a radiochemical purity of 98.5% or greater.

[¹⁴C]Dose Solution Preparation and Analysis. [¹⁴C]Dose solutions were prepared by diluting [¹⁴C]D₄ with unlabeled D₄ to a target specific activity that allowed delivery of approximately 40 µCi of radioactivity to each animal over the 6-h exposure period. Specific activity was confirmed by liquid scintillation counting.

Administration of Test Material. Animals were exposed to D₄ vapor in a cylindrical flow-past nose-only inhalation chamber. All animals were conditioned to the exposure apparatus for 4 days before the start of the experiments. Targeted conditioning periods were 1, 2, 4, and 6 h on days 1 to 4, respectively. In single exposure studies, animals received one exposure to [¹⁴C]D₄ immediately after the conditioning period. In multiple exposure studies, after receiving the same conditioning, animals were subjected to fourteen 6-h exposures to unlabeled D₄ followed on the 15th day by a 6-h exposure to [¹⁴C]D₄.

Experimental Design. Rats were weighed and assigned to subsets within each single or multiple exposure dose group by a computer-based stratified randomization procedure. Subset groups were established for body burden, distribution, and elimination components of the study, with four or five male or female animals per subset group, per dose. For both single exposure and multiple exposure studies, one male and one female animal per dose group were selected randomly to assess background radioactivity.

Sample Collection and Storage.

Body burden subset For the single exposure studies, rats were removed from the exposure chamber, removed from the exposure restraint tube, injected i.p. with sodium pentobarbital, and euthanized by cervical dislocation approximately 3 to 5 min after removal from the inhalation chamber (when the animals appeared to have stopped breathing). The carcass, along with urine and feces collected from the exposure tube, were then immediately solubilized together. In the multiple exposure studies, to minimize any loss of radioactivity in the period between removal from the exposure chamber and the cessation of breathing, body burden animals were anesthetized by i.v. (tail vein) or i.p. injection of sodium pentobarbital and allowed to stop breathing before they were removed from the inhalation chamber. Thereafter the animals were processed as described above.

Distribution subset. Rats were injected with sodium pentobarbital and euthanized by exsanguination from the abdominal aorta for collection of blood and tissues at 0, 1, 2, 3, 12, 24, 48, 72, 96, 120, and 168 h postexposure. Blood samples were transferred to heparinized tubes, mixed, and cooled on ice for approximately 10 min. Aliquots were transferred to scintillation vials and the remaining blood was centrifuged to prepare plasma. Blood and plasma were processed immediately for radioactivity measurement. In addition, various tissues (liver, lungs, perirenal fat, ovaries, vagina, and testes)⁴ were excised, weighed, and processed for radioactivity measurements as described below.

Elimination subset. Immediately after exposure, rats were placed in Roth-type glass metabolism cages for collection of excreta (urine and feces) and expired air (¹⁴CO₂ and other volatiles). Excreta deposited in the exposure tube of each animal were collected at the end of exposure and counted separately. Urine and feces (collected over dry ice) and expired CO₂ (trapped in a single 4 N KOH trap) were collected at 6, 12, and 24 h, and subsequently at 24-h intervals up to 168 h postexposure. Other expired volatiles were collected in two 2-ethoxyethanol (2-EE) traps (the first maintained on ice, the second on dry ice) at 1, 2, 4, 6, 9, 12, and 24 h, and at 24-h intervals up to 168 h postexposure. Cage rinses (70% ethanol/water followed by distilled water) were performed at 72, 120, and 168 h postexposure. After 168 h of excreta collection, the animals were anesthetized by pentobarbital injection and subsequently euthanized by exsanguination. Whole blood, plasma, and tissues including the remaining carcass were collected as in the distribution group. To facilitate comparisons of the different routes of elimination in different dose/gender groups, data from the metabolism cages were normalized. In each case the percentages of radioactivity eliminated by a particular route were calculated relative to the total radioactivity recovered in that particular elimination group subset.

Control subset. Control rats were housed in Nalgene metabolism cages for collection of urine and feces for approximately 24 h. The animals were euthanized in the same manner as the elimination subset and blood, plasma, tissues, and carcasses were collected and processed to establish background radioactivity.

Sample Processing and Analysis. Weighed aliquots of plasma, urine, cage washes, trapping media for expired volatiles, and 4 N KOH solutions were mixed directly with liquid scintillation fluid without additional processing. Except for liver, lung, and feces, the tissues were entirely digested in 35% tetraethylammonium hydroxide or Soluene 350 (whole blood only). Liver, lung, and feces were homogenized in isotonic saline, and aliquots of the homogenates were then solubilized and processed for radioactivity. To minimize color quench, whole blood and feces samples were decolorized using hydrogen peroxide.

Pharmacokinetic Analysis. C_max and t_max were obtained by data inspection (mean values at each sampling time). Area under the concentration curve versus time (AUC) values were calculated by the trapezoidal rule using an in house computer program in SAS Version 6.07. This calculation relied on mean concentration values and target sampling time over the following intervals: initiation of exposure to 168 h postexposure for plasma, and end of exposure to 168 h postexposure for tissue samples. The apparent terminal elimination half-lives (t_l/2) were calculated by regression analysis (SAS Version 6.07 computer software) on at least three data points in the terminal phase of the plasma concentration versus time curve.

Statistical Analysis. Where appropriate, means ± S.D. values were calculated. To aid in elucidating any gender or exposure level differences in total radioactivity recovered in the excreta, expired volatiles, and expired CO₂, these data were analyzed using the SAS Version 6.07 computer program. The Bartlett test, used to verify the homogeneity of variances, indicated that the variances were homogeneous for all the parameters (except expired ¹⁴CO₂) at a P value of  .001. For expired ¹⁴CO₂, the gender effect was evaluated separately. Trends across exposure levels were assessed using linear and quadratic contrasts.

Qualitative Analysis of Urine for D₄ and Metabolites. The urine samples collected at 12, 24, and 48 h postexposure or containing at least 10,000 dpm/ml were centrifuged to obtain a clear supernatant. [¹⁴C]D₄ and its metabolites in these supernatants were separated by HPLC with radiometric detection (Radiomatic Instruments, Meriden, CT) at a flow rate of 4 ml/min (3:1 ratio of ULTIMA-FLOM M cocktail/mobile phase). The urinary metabolite profile was obtained on a 5 µm Alltima C₁₈ HPLC column (4.6 × 250 mm; Alltech Associates, Inc., Deerfield, IL) eluted with 100% H₂O from 0 to 20 min, a linear gradient between 100% H₂O and 100% acetonitrile from 20 to 40 min, 100% acetonitrile from 40 to 50 min, followed by a gradient between 100% acetonitrile and 100% H₂O from 50 to 60 min.

Direct Chemical Analysis of D₄. Quantitation of D₄ in fat and expired air samples was performed using a GC/MS system calibrated with solvent standards containing D₄ and tetrakis(trimethylsiloxy) silane as an internal standard. Extraction of D₄ from fat aliquots was done with tetrahydrofuran according to the method of Varaprath et al. (1998). The amount of D₄ in the solvent extracts was determined according to a validated GC/MS method with a calibration range of 1 to 16,000 ng D₄/ml of tetrahydrofuran. Quantitation of D₄ collected in cold traps of 2-ethoxyethanol (2-EE) was done according to a validated GC/MS method with a calibration range of 50 to 12,000 ng D₄/g 2-EE. Linear regression analysis was performed for both methods from solvent standards to obtain calibration curves. Quantitation was done by comparing the peak area response ratios from selected ion monitoring of m/z 281 for both D₄ (M-15) and tetrakis(trimethylsiloxy) silane (M-103) to the calibration curves. The limits of quantitation determined for these methods were approximately 30 ng D₄/g fat and 20 ng D₄/ml 2-EE.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Exposure Concentrations. Targeted and actual concentrations of D₄ vapor in the single exposure studies (groups 1, 2, and 3) and multiple exposure studies (groups 1A and 2A) were within ±7% of the target value (Table 1). Mean chamber concentrations of D₄ as measured by gas chromatography during the 6-h exposure for the single exposures were: 716 ppm (group 1), 70.4 ppm (group 2), and 7.52 ppm (group 3). In multiple exposure studies, mean chamber concentrations were 705 ppm during days 1 to 14 and 703 ppm during day 15 for group 1A, and 7.52 ppm during days 1 to 14 and 6.81 ppm during day 15 for group 2A.

Body Burden. Specific radioactivity of the [¹⁴C]D₄ vapor administered to the rats was adjusted so that each animal inhaled 30 to 40 µCi/animal during the 6-h exposure (see Experimental Procedures for details of calculations). The rats retained 1.6 to 2.2 µCi/animal (5.0-6.1% of the inhaled radioactivity) at the end of the exposure. Prior exposure of the rats to unlabeled D₄ (14 consecutive exposures to D₄ vapor for 6 h/day) did not significantly affect the retention of [¹⁴C]D₄ on the 15th day. The percentage of inhaled D₄ retained by the animals was independent of concentration and gender over the range of 7 to 700 ppm (Table 1).

Distribution of Radioactivity. Groups of rats were euthanized at various times postexposure to study the distribution of radioactivity derived from [¹⁴C]D₄ (comprising both parent and metabolites). Radioactivity was widely distributed through rat tissues at the end of the exposure. Highest concentrations (microgram equivalents of D₄ per gram of tissue) were found in lung, liver, fat, and ovary (0 h; Tables 2 and 3). Radioactivity observed in uterus, vagina, and testes of rats immediately after D₄ exposure was lower than in lung, liver, fat, and ovary similar to that present in plasma or whole blood.⁵

Proportionality studies. The amounts of radioactivity retained during single exposures to [¹⁴C]D₄ (body burden) were directly proportional to exposure concentration over the range of 7 to 700 ppm (Table 1). The concentrations of radioactivity in the fat of male and female rats immediately after single exposures to 716 ppm [¹⁴C]D₄ were 188- and 137-fold higher than in animals exposed to 7.52 ppm, respectively (a 95-fold difference in exposure concentrations). In contrast, the end exposure plasma concentrations in these animals only increased 46- and 36-fold, respectively, during the 95-fold increase in exposure concentrations (Table 2), whereas the end exposure concentrations of radioactivity in the liver were almost exactly proportional to exposure concentration (Tables 2 and 3).

Gender effects. Concentrations of radioactivity in the tissues of female rats immediately after exposure (0 h) were generally similar to those observed in males. Female rats had slightly lower plasma concentrations (60-89%) and higher fat concentrations (171-249%) than males after single exposures (Table 2). These small gender effects were less apparent after multiple exposures where the concentrations of radioactivity in female rats were 83 to 100% and 115 to 190% of those in male rat plasma and fat, respectively (Table 3).

Kinetic Analyses of Radioactivity Data. For plasma and most of the solid tissues, the highest concentration of radioactivity (C_max) was typically measured 0 to 1 h postexposure (t_max, Tables 4 and 5). However, the C_max for fat occurred at a later time in many cases (3-24 h postexposure).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Semilogarithmic plot of the mean plasma radioactivity concentration after a single exposure to [14C]D4 at various exposure levels (a) or after a single and repeat exposure to [14C]D4 (b). Each value represents the mean of data from three or four animals ± S.D. (168-h value is the mean from five animals).

Tissue kinetics. The time course of radioactivity in solid tissues was similar to that seen in plasma for the period 0 to 168 h postexposure⁶, exhibiting multiphasic behavior with a rapid decline during the first 24 h postexposure and a slower decline thereafter. Although the elimination half-lives varied between groups (Tables 4 and 5), there were no apparent gender or dose effects on the rate of radioactivity elimination for tissues other than fat.

Fat (perirenal) kinetics. C_max for fat occurred up to 24 h postexposure (Tables 2 and 3). Furthermore, elimination of radioactivity from fat tissue did not show the initial rapid decline seen in plasma and other solid tissues in the first 24 h postexposure. Instead, the decline in fat concentration was monoexponential for both male and female rats over the period 0 to 168 h, which comprised slightly more than one half-life (Fig. 3).

View larger version (39K): [in this window] [in a new window]   Fig. 3.   Time course of radioactivity in the fat compartment of male () and female () after 6-h exposures to 7, 70, or 700 ppm of D4, followed for 168 h postexposure.

Elimination Routes for Radioactivity.

Single exposure studies Most of the recovered radioactivity was found in the urine and expired volatiles (Fig. 4, a and b). In females (but not in males), a small but statistically significant dose dependence in the relative importance of the two major elimination routes was noted. As exposure increased from 7 to 700 ppm, relatively higher amounts of radioactivity were recovered in expired air from female rats whereas relatively lower amounts were recovered in urine from these animals.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Disposition of radioactivity after single exposure to 7, 70, and 700 ppm of [14C]D4. Data expressed as a mean from five animals ± S.D. L*, significant linear trend among means (P < .05). *, significant difference (P < .05) from other exposure levels.

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Cumulative excretion of radioactivity in the urine (a) or expired volatiles (b) after a single exposure to [14C]D4. Data expressed as a mean from five animals ± S.E.

Multiple exposures. Disposition of radioactivity in the multiple exposure experiments was similar to that seen in the single exposure studies. Most of the recovered radioactivity was found in the urine and expired volatiles (Fig. 6). Statistical analysis, evaluating gender and group effects in the radioactivity recoveries in the excreta, indicated that the percentage of radioactivity recovered in the urine was independent of gender and dose.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   Disposition of radioactivity after repeat exposure to 14 consecutive days of unlabeled D4 and one exposure (day 15) to [14C]D4. Data expressed as a mean from five animals ± S.D. *Statistically significantly different (P < .05) from 7-ppm exposure group.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Cumulative excretion of radioactivity in the urine (a) or expired volatiles (b) after repeat exposure to [14C]D4. Data expressed as a mean from five animals ± S.E.

Chemical Analyses for Parent D₄. Selected samples of fat and expired volatiles were directly analyzed for D₄ using techniques outlined in Experimental Procedures. These analyses indicated that almost all of the radioactivity present in expired air (analyzed up to 9 h postexposure) was parent D₄ (Table 8). Similarly, all of the radioactivity present in fat tissues was D₄, even in samples collected 168 h postexposure.

Qualitative Analysis of Urine for D₄ and Metabolites. Representative urine samples (collected in the period 0-48 h postexposure) were examined for [¹⁴C]D₄ and its metabolites by HPLC with on-line radioactivity detection. Multiple urine samples from male and female animals exposed to 7, 70, or 700 ppm in single or multiple exposure studies were characterized (Fig. 8). In contrast to expired volatiles (which were almost all parent D₄), no unmetabolized D₄ (retention time 50 min; Fig. 8) was detected in any of the urine samples examined. HPLC analysis revealed that two major metabolites and at least five minor metabolites were present. Identification of the metabolites has been reported separately (Varaprath et al., 1999). The same spectrum of metabolites was observed in every sample of urine analyzed, therefore, only a representative chromatogram showing the typical profile is presented in Fig. 8. Although small differences in the relative rates of elimination of radioactivity by different routes have been noted between different gender/exposure concentration groups, there were no significant differences with gender or dose effects on either the types of urinary metabolites observed or their relative abundance in the urine samples.

View larger version (22K): [in this window] [in a new window]   Fig. 8.   Typical HPLC radiochromatogram of urine from a rat exposed to [14C]D4.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Pharmacokinetic parameters and elimination pathways obtained in these studies of radioactivity are composite parameters for D₄ and various metabolites. Kinetic analyses of these data provide a basis for estimating the amount of time required to achieve a specified level of clearance of D₄ and/or its metabolites from rats. Data developed using [¹⁴C]D₄ provide general information about possible pathways for metabolism and may establish parameters for designing more specific studies. Ultimately a quantitative description of the mechanisms involved in the entry, distribution, metabolism, and elimination of parent chemical and/or metabolites should be helpful in assessing the safety of D₄.

Single and multiple inhalation exposure studies were conducted with male and female Fischer 344 rats using D₄ vapor at concentrations of 7 to 700 ppm. Retention of [¹⁴C]D₄ (5-6% of inhaled radioactivity) was independent of dose and gender after single exposure and was unaffected by 14 consecutive days of exposure to unlabeled D₄. Radioactivity was widely distributed throughout the body. Some of the solid tissues (fat, liver, lung, ovary) exhibited higher levels of radioactivity than the plasma (3- to 10-fold), but a few tissues (testes, uterus, vagina) contained levels of radioactivity that were similar to or only slightly higher than plasma.

Levels of radioactivity in plasma and tissues other than fat rose throughout the exposure and, in many cases, for a short period (1 h) postexposure. Elimination of radioactivity from the plasma and tissues other than fat was multiphasic. Most of the radioactivity was eliminated within 24 h postexposure, with a slow log-linear terminal elimination phase beginning from about 48 to 72 h postexposure (Fig. 2, a and b).

In contrast to plasma and the solid tissues investigated in this study, maximum concentrations of radioactivity in fat occurred as late as 24 h postexposure. In addition, the time course for elimination of radioactivity from fat was monophasic rather than multiphasic (Fig. 3). The slope of the terminal (and only) phase in the fat curves was similar to the terminal slopes for the other tissues, suggesting that fat served as a reservoir of D₄, releasing it into circulation as the concentrations in other tissues were reduced by metabolism and exhalation.

The plasma and tissue kinetic behavior is similar to that observed with other volatile lipophilic chemicals such as styrene, methylchloroform, and chloroform after inhalation exposures (Ramsey and Andersen, 1984; Reitz et al., 1988; Corley et al., 1990). Fat has a higher affinity for these chemicals than plasma or other tissues. However, because fat is poorly perfused by blood, concentrations of volatile lipophilic compounds in fat rise slowly during inhalation exposures and take several hours or days to reach saturation. However, once the inhalation exposure is terminated, concentrations of lipophilic chemicals in plasma and tissues fall rapidly due to metabolism and loss in expired air. When plasma levels drop low enough, the equilibrium changes and small amounts of chemical are slowly released into plasma from fat stores until these are depleted. In these studies, terminal half-lives for radioactivity observed in the fat compartment ranged from 81 to 226 h. Given that 8 to 10% of the retained radioactivity is still present in the rat at the end of 168 h and using a typical half-life of 150 h, it can be calculated that the body burden will decrease approximately 10× (to 1% of the initial body burden) after an additional 500 h have passed.

The primary routes for elimination of radioactivity from rats exposed to [¹⁴C]D₄ vapor were excretion in the urine and exhalation. Major elimination routes were the same in single and multiply exposed animals, regardless of gender or exposure concentration. In each case, 75 to 80% of the radioactivity retained by the animals was eliminated by 168 h postexposure. Most of the radioactivity eliminated in urine or exhaled air was recovered in the first 48 h.

Analyses of expired air revealed that all the radioactivity was present as the parent compound (D₄). In contrast, radioactivity eliminated in the urine consisted entirely of polar metabolites of D₄. Two major metabolites comprising 75 to 85% of the urinary radioactivity were identified as dimethylsilanediol [Me2Si(OH)2] and methylsilanetriol [MeSi(OH)₃] (metabolites A and D, Fig. 8; Varaprath et al., 1999). Formation of MeSi(OH)₃ clearly established oxidative demethylation at the silicon-methyl bonds of D₄. Five additional minor metabolites were also identified. These metabolites are believed to arise by additional hydrolysis and/or oxidation after the original (rate-limiting) oxidative metabolism of D₄ by P-450 enzymes. This metabolic pathway appears to be present in all dose/gender/exposure groups, because the chromatographic profiles of urinary radioactivity from these groups were virtually identical.

Small dose and gender effects on the distribution of retained radioactivity to the tissues were observed. As the exposure concentration increased from 7 to 700 ppm, higher percentages of the radioactivity were found in the fat compartment rather than plasma and other tissues. This dose dependence existed in both sexes but was more pronounced in females than males after single exposures to [¹⁴C]D₄ vapor. In addition, small dose-dependent shifts in elimination pathways were seen in singly exposed female rats with higher percentages eliminated in expired air versus urine at higher exposure concentrations of D₄. These shifts in elimination pathways were not seen in singly exposed male rats or multiply exposed male or female rats. It is likely that these small differences were related to small changes in the relative rates of metabolism by the different dose/gender/exposure groups.

Repeated inhalation exposure to D₄ produced a reversible and dose-related liver enlargement with significant induction of cytochrome P-450 CYP2B1/2 in male and female Fischer rats in a manner similar to that observed for PB (McKim et al., 1998). In terms of CYP2B1/2 activity, as determined by pentoxyresorufin O-depentylation assays, male rats exposed to D₄ had 1.5- to 2-fold higher activity than females. A similar dependence of metabolic rates on gender and induction in Fischer 344 rats treated with PB was reported by Nims et al. (1993). After repeated exposures to D₄, the male/female difference diminishes because female rats are more sensitive to induction than male rats (i.e., the ratio of induced/control activities is higher for females than males). Consequently, it would be predicted that multiple exposures to high concentrations of D₄ would diminish both the dose dependence of the routes of elimination and the gender difference in susceptibility to this dose dependence, as observed.

Although the gender difference and dose dependencies noted above were statistically significant, they are relatively small in magnitude. In general, the rates and routes of elimination were similar in males and females, at high and low concentrations of D₄, and administration of multiple doses of D₄ before [¹⁴C]D₄ exposure produces quantitative rather than qualitative changes in elimination pathways and rates.