Page 9: Guidelines for Canadian Drinking Water Quality: Guideline Technical Document – Toluene, Ethylbenzene and the Xylenes

8.0 Kinetics and metabolism

8.1 Absorption

8.1.1 Toluene

Toluene is well absorbed by the lungs and the gastrointestinal tract and to a lesser extent through the skin.

Baelum et al. (1993) reported that gastrointestinal absorption of toluene was complete in human volunteers, as indicated by the presence of toluene in exhaled air and toluene metabolites (hippuric acid and o-cresol) in urine following oral administration of toluene at 2 mg/minute via a 3-hour gastric tube. Animal studies, however, have shown that toluene is absorbed less rapidly than in humans by the oral route (Pyykkö et al., 1977; Ameno et al., 1992).

Toluene is rapidly absorbed by the inhalation route in both humans and experimental animals (Benignus et al., 1984; Hobara et al., 1984; Wigaeus Hjelm et al., 1988; Löf et al., 1993). In humans, Carlsson (1982) reported that exposure of exercising male individuals to 79.5 ppm (300 mg/m3) toluene for 2 hours resulted in an average uptake (percentage of inspired air) of 55%, which dropped to 50% following 2 hours at rest. Similar results were reported by Löf et al. (1990), who exposed 10 male individuals to 79.5 ppm (300 mg/m3) toluene for 4 hours at rest, resulting in absorption of approximately 50% of the dose. Benoit et al. (1985) reported that the exposure of four individuals (sex not specified) to 50 ppm (188 mg/m3) toluene for 90 minutes at rest resulted in an average retention of 83%. In humans, toluene appears in blood 10–15 minutes after the onset of exposure, and its concentration in blood is strongly correlated with alveolar concentration. In rats, peak blood and cerebral levels appear in less than 1 hour after the onset of exposure (Tardif et al., 1991; INRS, 2008).

The rate of absorption of toluene through human skin is slow (Dutkiewicz and Tyras, 1968); it has been reported to range from 14 to 23 mg/cm2 per hour (forearm skin). Brown et al. (1984) calculated that bathing in water containing a toluene concentration of 5–500 µg/L (15 minutes/day) would result in an absorbed dermal dose ranging from 0.2 to 20 µg/kg body weight (bw) per day for a 70 kg adult and from 0.4 to 40 µg/kg bw per day for a 10.5 kg infant. Soaking the skin in a solvent containing 65% toluene for 5 minutes produced a maximum concentration of toluene in blood of 5.4 µmol/L (Aitio et al., 1984). This latter experiment, conducted with two volunteers, revealed individual differences in absorption, which is consistent with the high variability reported by Sato and Nakajima (1978). Monster et al. (1993) measured toluene levels in alveolar air samples of six rotogravure printing workers who washed their hands with toluene for 5 minutes; the next morning, toluene levels in alveolar air ranged between 0.5 and 10 mg/m3. Morgan et al. (1991) reported that dermal absorption of toluene in aqueous solution (saturated, two-thirds saturated and one-third saturated solutions) and neat toluene in rats was significant, even though only 1% of the body surface was exposed; for example, 24 hours of exposure to neat toluene resulted in a peak blood concentration of 9.5 µg/mL.

8.1.2 Ethylbenzene

Ethylbenzene can be absorbed from the respiratory tract, from the gastrointestinal tract and through the skin.

Data pertaining to the oral absorption of ethylbenzene are from animal studies only. For instance, the recovery of ethylbenzene metabolites in the 24-hour urine of rabbits following exposure to a single oral dose of 593 mg/kg bw was between 72% and 92% of the administered dose, suggesting rapid and effective absorption by this route (El Masry et al., 1956). Consistent with those results, 84% of the radioactivity from a single oral dose of 14C-labelled ethylbenzene at 30 mg/kg bw administered to female rats was recovered within 48 hours (Climie et al., 1983). More recently, Faber et al. (2006) reported that ethylbenzene was detected at 0.49, 3.51 and 18.28 mg/L in maternal blood of rats 1 hour after the last of four daily administrations of ethylbenzene by gavage at equal doses of 0, 8.67, 30 and 114 mg/kg bw, respectively (total doses: 0, 26, 90 and 342 mg/kg bw per day); ethylbenzene was not detected in blood of weanlings from the same dams.

Inhalation studies in humans have shown that ethylbenzene is rapidly absorbed via this route of exposure (Bardodej and Bardodejova, 1970; Gromiec and Piotrowski, 1984; Tardif et al., 1997; Knecht et al., 2000). Volunteers exposed to 23–85 ppm (approximately 100-370 mg/m3) ethylbenzene for 8 hours were shown to retain 64% of the inspired dose, with only trace amounts of ethylbenzene being detected in expired air at the end of the exposure period (Bardodej and Bardodejova, 1970). A mean retention of 49% was reported by Gromiec and Piotrowski (1984) when humans were exposed by inhalation to ethylbenzene at 18–200 mg/m3 (approximately 4–46 ppm). The differences may be attributable to human variability in absorption rates as well as methodological differences. Tardif et al. (1997) reported a steady-state blood-to-alveolar air concentration ratio of approximately 30 within 1 hour of beginning exposure.

Animal studies also show a rapid absorption of ethylbenzene via the inhalation route of exposure. In a study by Chin et al. (1980), exposure of Harlan-Wistar rats for 6 hours to radiolabelled ethylbenzene resulted in a rapid absorption of the dose, with a reported retention of 44%. Radioactivity was detected in intestine, kidney, liver and adipose tissue for up to 42 hours post-exposure. Freundt et al. (1989) reported that blood concentrations of ethylbenzene in rats following 2 hours of inhalation exposure were proportional to its concentration in the atmosphere.

Overall, studies on dermal exposure to ethylbenzene showed rapid absorption of the liquid form and poor absorption of the vapour form (Dutkiewicz and Tyras, 1967; Gromiec and Piotrowski, 1984) and suggested that skin absorption could be a major route of uptake for liquid ethylbenzene or ethylbenzene in water; for instance, the average amounts of ethylbenzene absorbed after volunteers immersed one hand for up to 2 hours in an aqueous ethylbenzene solution at 112 or 156 mg/L were 39.2 and 70.7 mg, respectively. Morgan et al. (1991) reported that in Fischer 344 rats, the peak blood level of ethylbenzene (5.6 µg/mL) was reached within 2 hours of topical application of neat ethylbenzene to approximately 1% of the body surface and slowly declined during the 24-hour observation period. In contrast, the total amount absorbed was reduced when ethylbenzene was administered in aqueous solutions (saturated, two-thirds saturated and one-third saturated solutions).

8.1.3 Xylenes

In humans, xylene isomers are absorbed by the respiratory tract (60–65%), gastrointestinal tract (up to 90%) and skin (2%). However, limited information is available on the absorption of xylenes in humans and experimental animals following ingestion. In addition, xylenes can cross the placental barrier (Ghantous and Danielsson, 1986).

Ogata et al. (1979) reported that the absorption of xylene in human volunteers (oral ingestion of 40 mg/kg bw) was at least 34% for o-xylene and 53% for m-xylene, based on the recovery of urinary metabolites.

In male and female rats dosed with 0.15 mL of radiolabelled m-xylene (0.27 mg/kg bw) by oral gavage, peak blood levels of radioactivity were observed within 20 minutes, indicating rapid absorption (Turkall et al., 1992). Kaneko et al. (1995) reported that blood concentrations of m -xylene (peak concentration approximately 2.5 µM) rapidly increased within 5 hours following oral administration of 8.64 mg/kg bw in corn oil to rats.

Dermal absorption of xylene from water is not well known. However, a dermal absorption rate coefficient (Kp) of 0.08 cm/hour has been estimated (U.S. EPA, 1992). Results of experimental studies with humans indicate that the degree of penetration and absorption of m-xylene following dermal exposure is not as efficient as that following exposure by the respiratory tract (Engström et al., 1977; Riihimäki and Pfäffli, 1978; Riihimäki, 1979b). Morgan et al. (1991) reported that in Fischer 344 rats, a peak blood level of m-xylene (8.8 µg/mL) was reached within 2 hours of topical application of neat m-xylene and slowly declined during the 24-hour observation period. The total amount absorbed was reduced, however, when m-xylene was administered in aqueous solutions (saturated, two-thirds saturated and one-third saturated solutions).

Simulations of the Kaneko et al. (1991a) PBPK model estimated that increased physical activity increased the blood concentration of m-xylene. The concentration of m-xylene in the blood during exposure was lower in women than in men, but was higher in women 10 hours after exposure.

8.2 Distribution

8.2.1 Toluene

The highest concentration of toluene measured in a human (51 years old) who died 30 minutes following accidental ingestion of toluene at 625 mg/kg bw was found in the liver (433.5 µg/g), followed by the pancreas (88.2 µg/g), brain (85.3 µg/g), heart (62.6 µg/g), blood (27.6 µg/g), body fat (12.2 µg/g) and cerebrospinal fluid (11.1 µg/g) (Ameno et al., 1989). Following a presumed inhalation overdose, toluene has been measured in brain (highest concentration), liver, lung and blood in a human (Paterson and Sarvesvaran, 1983). Takeichi et al. (1986) reported similar findings in a 20-year-old male painter who died while working with a toluene-based paint; the highest concentrations were found in the brain, followed by the liver and blood.

In an experiment using rabbit tissue homogenates, tissue distribution of toluene has been reported as follows (from highest to lowest levels): adipose (fat), bone marrow, brain, liver, heart, lung, kidney and muscle (Sato et al., 1974). In dogs, following inhalation exposure, toluene concentrations in the liver and brain were higher than those found in the kidney (Endoh et al., 1989). In rats, however, oral and inhalation exposure to toluene resulted in higher toluene concentrations in the liver compared with the brain (Pyykkö et al., 1977).

Once in the blood, toluene is distributed between red blood cells and serum (1:1 in human; 1:2 in rat), and several studies have shown relationships between blood and tissue levels of toluene, particularly for the brain (Benignus et al., 1984; Harabuchi et al., 1993). In rats orally exposed to toluene at 400 mg/kg bw, the highest blood level occurred 1.5 hours after exposure (Ameno et al., 1992), and toluene distribution in the brain was similar after both inhalation and oral exposure (Ameno et al., 1992). In humans, data suggest that the accumulation of toluene in the brain is more important than that in the liver following inhalation exposure, whereas following oral exposure, toluene appears to have a greater affinity for the liver (ATSDR, 2000).

Animal studies involving the inhalation exposure of pregnant mice report that the distribution of toluene occurs via rapid uptake by lipid tissues (brain and fat); in addition, toluene easily crosses the placental barrier, and fetal concentrations reportedly correspond to approximately 75% of maternal blood levels (Ghantous and Danielsson, 1986). Toluene has also been detected in human breast milk (Pellizzari et al., 1982).

Fisher et al. (1997) used a generic lactation model developed for various VOCs to estimate the amount of toluene an infant would ingest through breast milk if the mother was occupationally exposed to toluene at the American Conference of Governmental Industrial Hygienists (ACGIH) Threshold Limit Value at the time of the study (50 ppm) throughout a workday. The model, which was not validated and did not include metabolic parameters for toluene, predicted that the intake for a 10 kg infant would be 0.46 mg/day.

8.2.2 Ethylbenzene

There is limited information regarding the distribution of ethylbenzene in humans following oral or dermal exposure. Engström and Bjurström (1978) reported that the distribution of ethylbenzene is rapid in humans after inhalation and that ethylbenzene is distributed throughout the body, including the fat, intestine, kidneys and liver. Inhalation studies in animals support these observations (Elovaara et al., 1982; Engström et al., 1985). Chin et al. (1980) reported that ethylbenzene is efficiently distributed throughout the body in rats following inhalation exposure to radiolabelled ethylbenzene; the highest amounts of radioactivity in tissues 42 hours following exposure to 230 ppm ethylbenzene for 6 hours were found in the carcass, liver and gastrointestinal tract, with lower amounts detected in the adipose tissue. In addition, ethylbenzene has been observed to cross the placental barrier in humans (WHO, 2003a; INRS, 2008).

The percentages of absorbed doses in hairless mice following dermal application of 14C-labelled ethylbenzene were as follows: 15.5%, carcass; 4.5%, application site; 14.3%, expired breath; and 65.5%, excreta (Susten et al., 1990).

8.2.3 Xylenes

Limited information is available on the distribution of xylenes in humans and experimental animals following ingestion. Xylene isomers are relatively soluble in blood.

Turkall et al. (1992) reported that adipose tissue contained the highest concentration of radioactivity after oral gavage: approximately 0.3% of the initial administered dose/mg tissue was reported for female rats and 0.1% was reported for male rats (the authors did not report the total weight of adipose tissue analysed).

Kumarathasan et al. (1998) determined tissue/blood partition coefficients in several tissues and organs (brain, muscle, kidney, liver and fat) and blood from Sprague-Dawley rats for each xylene isomer in a mixture. For m-xylene, the blood/air partition coefficient averaged 62. The highest tissue/blood partition coefficient was for fat (50), followed by kidney (2.1), liver and brain (2) and muscle (1.8). There were no differences between isomers.

Riihimaki and Savolainen (1980) found that 10–20% of a xylene dose was distributed to the adipose tissue. Adipose has the highest concentration of neutral fat and the highest affinity for xylene of all tissues. Astrand et al. (1978) reported that following rapid uptake of xylene vapours, the amount detected in the blood generally amounted to 2–3% of the total xylene dose absorbed; this may be explained by the high lipid solubility of xylenes, resulting in their distribution and storage in various tissues.

The highest concentration of xylene (combined concentration of xylene and its metabolites) following a 4-hour exposure to 14C-labelled p-xylene (48 ppm) was found in the kidneys of male rats, followed by the subcutaneous fat (Carlsson, 1981). Bergman (1983) investigated the distribution of 14C-labelled m-xylene in mice and found high levels of radioactivity in the body fat, bone marrow, white matter of the brain, spinal cord, spinal nerves, liver and kidney immediately following inhalation exposure. No radioactivity was detected in the body by 48 hours post-exposure.

Fisher et al. (1997) used a generic lactation model developed for various VOCs to estimate the amount of o-, p- and m-xylene an infant would ingest through breast milk if the mother were occupationally exposed to xylenes at the ACGIH Threshold Limit Value at the time of the study (100 ppm) throughout a workday. The model, which was not validated and did not include metabolic parameters for xylenes, predicted that the intake for a 10 kg infant would be 6.59 mg/day.

8.3 Metabolism

A number of animal and human PBPK models have shown that exposure to binary mixtures of aromatic solvents could result in metabolic interactions that modify their rates of metabolism and elimination (Tardif et al., 1991, 1992, 1993, 1997). These metabolic interactions probably result from the mutual competition between solvents for metabolizing enzymes (e.g., cytochrome P450 [CYP] 2E1). As a result, the half-lives of both solvents increase. Such interactions resulting from exposure to a ternary mixture (i.e., toluene, xylene, ethylbenzene) were also shown to occur in animals and humans (Tardif et al., 1997).

8.3.1 Toluene

Approximately 80% of absorbed toluene from inhalation exposure is recovered in urine as hippuric acid, the principal metabolite of toluene (Ogata, 1984; Löf et al., 1993; Tardif et al., 1998). Toluene metabolism, which takes place mainly in the liver, consists of sequential hydroxylation and oxidation by CYP enzymes (CYP2E1, CYP2B6, CYP2C8, CYP1A2 and CYP1A1) to benzoic acid (Phase I reactions), followed by conjugation of benzoic acid (Phase II) with glycine to form hippuric acid. Several studies of urinary metabolites in toluene-exposed humans (Angerer, 1979; Andersen et al., 1983; Dossing et al., 1983; Inoue et al., 1986; Baelum et al., 1987, 1993; Jonai and Sato, 1988; Löf et al., 1990, 1993; Ng et al., 1990; Kawai et al., 1992; Maestri et al., 1997; Angerer et al., 1998) and rats (Bray et al., 1949; Van Doorn et al., 1980; Wang and Nakajima, 1992) have identified hippuric acid as the major urinary metabolite of toluene. A minor CYP-related pathway involves a previous epoxidation of the aromatic ring to form either o- or p -cresol, which undergoes conjugation reactions to form mainly sulphate and glucuronide derivatives. Glutathione conjugation also occurs, resulting inS-benzylglutathione and S-benzylmercapturic acid (conjugation to benzyl alcohol) or S-p-toluyl glutathione and S-p-toluylmercaptic acid (conjugation to the epoxidated ring). Urinary excretion of these minor metabolites accounts for less than 5% of absorbed toluene (Nakajima et al., 1991, 1992a, 1992b, 1993, 1997; Nakajima and Wang, 1994; Tassaneeyakul et al., 1996). CYP2E1, one of the major CYP isozymes involved in the major toluene pathway (Tassaneeyakul et al., 1996; Nakajima et al., 1997), is reported to be expressed several hours after birth in humans and continues to increase during the first year of life (Vieira et al., 1996). Excretion of non-metabolized toluene in exhaled air can represent from 7% to 20% of absorbed toluene (Carlsson, 1982; Leung and Paustenbach, 1988; Löf et al., 1993).

8.3.2 Ethylbenzene

Ethylbenzene is metabolized mainly through oxidation (hydroxylation) by microsomal enzymes (Phase I), which is followed by conjugation reactions (Phase II) to form a number of metabolites that are excreted in urine. The first step involves the hydroxylation of ethylbenzene to 1-phenylethanol, which is catalyzed by specific isoforms of CYP (CYP2E1/CYP2B6) (Sams et al., 2004). No significant qualitative differences in metabolism between the oral and inhalation routes of exposure were reported in humans or experimental animals.

In humans exposed via inhalation, the major metabolites of ethylbenzene are mandelic acid (approximately 64–71%) and phenylglyoxylic acid (approximately 19–25%) (Bardodej and Bardodejova, 1970; Engström et al., 1984; Tardif et al., 1997; Knecht et al., 2000; Jang et al., 2001). Dutkiewicz and Tyras (1967) reported that following dermal exposure of human volunteers, maximum excretion of mandelic acid via the urine occurred at 3 hours after skin exposure and represented only 4.6% of the absorbed dose; the rate of absorption of an aqueous solution of ethylbenzene was greater than that of liquid (pure) ethylbenzene.

Differences in the biotransformation of ethylbenzene between species have been reported (El Masry et al., 1956; Bakke and Scheline, 1970; Climie et al., 1983; Engström et al., 1984, 1985). The most important difference concerns the major metabolites that differ in nature and percentages between species. In rats exposed by inhalation or orally to ethylbenzene, the major metabolites were identified as benzoic acid, hippuric acid, 1-phenylethanol, mandelic acid and phenylglyoxylic acid (Climie et al., 1983; Engström et al., 1984, 1985). Nakajima and Sato (1979) showed that the in vitro metabolic activity of rat liver microsomal enzymes on ethylbenzene is increased by fasting, despite a marked loss of liver weight. The authors also reported that the metabolic rate was significantly higher in fasted males than in fasted females.

8.3.3 Xylenes

The metabolism of the three xylene isomers occurs primarily in the liver and to a lesser extent in the lung and kidneys. The main metabolic pathway that accounts for almost the entire absorbed dose of xylenes (90%) in humans involves hydroxylation of a methyl group, which is catalyzed mainly by an isoform of CYP (CYP2E1), forming methylbenzyl alcohols. This methyl hydroxylation is both a saturable and an inducible metabolic process (Tassaneeyakul et al., 1996). In a subsequent step, the alcohol moieties are oxidized to form methylbenzoic acids, which conjugate with glycine to form methylhippuric acids, the main metabolites that are excreted into the urine (Ogata et al., 1970, 1979; Sedivec and Flek, 1976; Astrand et al., 1978; Senczuk and Orlowski, 1978; Riihimäki et al., 1979; Norström et al., 1989). Further metabolic pathways produced minor urinary metabolites that account for < 10% of the absorbed dose: methylbenzyl alcohols, o-toluylglucuronides, xylene mercapturic acids such as S-(o-methylbenzyl)-N-acetylcysteine (Norström et al., 1988) and xylenols.

In animals, the metabolism of xylene is qualitatively similar to that in humans. Quantitative differences, especially in the metabolism of methylbenzoic acids (toluic acids), have been reported (Bakke and Scheline, 1970; Sugihara and Ogata, 1978; Ogata et al., 1979; van Doorn et al., 1980). According to some authors, those differences may be explained in part by differences in the size of the doses given to humans and animals in experimental studies (David et al., 1979; Ogata et al., 1979; van Doorn et al., 1980).

Results from rat studies demonstrate that orally administered xylenes are subject to a first-pass metabolic effect that limits the amount of absorbed parent material reaching the general circulation (Kaneko et al., 1995).

Studies in animals showed that the metabolism of xylene may be influenced by prior exposures (inhalation or oral) to xylene (Elovaara et al., 1989). For instance, pretreatment of rats with m-xylene increased the percentage of m-methylhippuric acid and thioethers (glutathione conjugates) in the urine. Thioether excretion in 24-hour urine was enhanced about 10-fold after inhalation exposure to xylene and 20-fold after oral administration.

8.4 Excretion

8.4.1 Toluene

Toluene for the most part is eliminated in the urine (Löf et al., 1990, 1993; Turkall et al., 1991; Tardif et al., 1992, 1998), mainly as hippuric acid, in experimental animals and humans. Elimination from the blood is rapid (Sato and Nakajima, 1978; Carlsson, 1982; Löf et al., 1990, 1993), displaying three-phase elimination half-lives of 3, 40 and 738 minutes following a single inhalation exposure in humans (Löf et al., 1993). Elimination of unchanged (non-metabolized) toluene is also detected in the expired air (Carlsson, 1982; Leung and Paustenbach, 1988; Pellizzari et al., 1992; Laparé et al., 1993; Löf et al., 1993; Monster et al., 1993), especially immediately after exposure, and can represent from 7% to 20% of the absorbed dose; elimination via expired air subsequently decreases rapidly thereafter (Benoit et al., 1985).

Unchanged toluene was detected in alveolar air samples of eight human volunteers for up to 4 hours after cessation of exposure (2 mg/minute gastric infusion for 3 hours), and rates of urinary excretion of hippuric acid and o-cresol were elevated compared with those for unexposed volunteers (Baelum et al., 1993). Interestingly, co-administration of ethanol (0.32 g/kg bw, corresponding to two alcoholic drinks) with the 2 mg/minute dosage decreased hippuric acid urinary excretion and increased the area under the time versus concentration curve for alveolar toluene, which is consistent with previous studies showing that ethanol inhibits the major toluene metabolic pathway of side-chain oxidation (Dossing et al., 1984; Wallen et al., 1984). Urinary hippuric acid has generally been used as a biomarker of exposure to toluene; however, it best correlates with acute exposure given its short half-life (Lowry, 1987). Urinary o-cresol, which is more specific, is currently used as a biomarker of occupational exposure to toluene (Truchon et al., 1999). Both hippuric acid and o-cresol in urine have been found to serve as good markers for exposure by inhalation to toluene concentrations greater than 50 ppm (188.5 mg/m3) (Ikeda et al., 2008). Blood concentrations of toluene have been reported as the most reliable measure of toluene exposure (Kawai et al., 1993; Brugnone et al., 1995). Toluene blood levels appear to correlate well with air toluene levels below 1–3 ppm (3.8–11.3 mg/m3) (Kawai et al., 1994; Ikeda et al., 2008), for which hippuric acid as a marker of exposure is no longer useful. Finally, a small fraction of toluene is also excreted in urine and has been proposed as a biomarker for monitoring occupational exposure to toluene (Kawai et al., 2008).

In rats, approximately 22% of a single oral dose of 14C-labelled toluene (185 kBq) was reported to be eliminated in the expired air (Turkall et al., 1991); combined with urinary excretion, Turkall et al. (1991) reported that almost 100% of the single dose of radiolabelled toluene was eliminated within 48 hours after exposure.

8.4.2 Ethylbenzene

Elimination of ethylbenzene and its metabolites in animals after oral exposure has been shown to be similar to that following inhalation exposure (ATSDR, 2010). Female rats that received a single oral dose of 30 mg 14C-labelled ethylbenzene per kilogram body weight excreted 82% of radioactivity in the urine and 1.5% in the feces. The major metabolites were mandelic acid (23%), hippuric acid (34%) and 1-phenylethyl glucuronide (4%). Consistent with the differences observed in the biotransformation pathways, quantitative and qualitative metabolic differences between species and exposure routes were shown to exist in the percentages of metabolites excreted in the urine (Climie et al., 1983).

Rabbits orally exposed to ethylbenzene excreted large amounts of glucuronide conjugates (32%) in the urine (Smith et al., 1954; El Masry et al., 1956) instead of mandelic acid (2%), hippuric acid and phenylglyoxylic acid, which are the major metabolites in rats.

Susten et al. (1990) reported that the absorbed dose of 14C-labelled ethylbenzene collected in expired breath during the first 15 minutes of ethylbenzene application was 9.3% in hairless mice exposed by the percutaneous route.

8.4.3 Xylenes

In humans, approximately 95% of absorbed xylenes following inhalation are biotransformed and excreted as methylhippuric acids, while the remaining 5% are eliminated unchanged in the exhaled breath (Sedivec and Flek, 1976; Astrand et al., 1978; Senczuk and Orlowski, 1978; Ogata et al., 1979; Riihimäki et al., 1979; Pellizzari et al., 1992). A small fraction (< 0.005%) is eliminated unchanged in the urine, and < 2% is eliminated as xylenols (Sedivec and Flek, 1976).

Turkall et al. (1992) reported that excretion of radioactivity by rats following an oral dose of 14C-labelled m-xylene showed that urinary excretion occurred mostly during the first 12 hours after administration and approximated 50–59% of the dose. Whereas 96.2% was eliminated in the urine in males and 73.7% in the urine in females over 48 hours, approximately 8% and 22% of the total dose were excreted unchanged in exhaled air by males and females, respectively, during the first 12 hours. Overall, m-methylhippuric acid comprised 67–75% of the urinary radioactivity, with xylenol representing 2–18% and unchanged m-xylene comprising approximately 1%.

Simulations of the Kaneko et al. (1991a) PBPK model estimated that increased physical activity increased the rate of urinary excretion of the m- xylene metabolite m-methylhippuric acid. The rate of urinary excretion of the metabolite was lower in women than in men both during and after exposure.

8.5 Physiologically based pharmacokinetic models

In human health risk assessment for toluene, ethylbenzene and xylenes, PBPK modelling is useful, since no appropriate toxicity data are available for humans ingesting these chemicals in drinking water, many of the animal studies rely on inhalation exposure and metabolite generation over low to high parent compound exposures is non-linear. Several PBPK models have been developed for each of the compounds. The structural basis of these models is the initial Ramsey and Andersen (1984) styrene model, which compartmentalizes organs into liver, fat, richly perfused tissues and slowly perfused tissues.

In addition to the PBPK models for each of the compounds that are described in the following sections, several models have been designed to evaluate exposure to combinations of the compounds or to gasoline-like mixtures. The first model developed for this purpose was used to investigate the interactions between toluene and m-xylene; it considered the metabolism of both compounds by CYP2E1 and contained four organ compartments (liver, fat, slowly perfused tissues and richly perfused tissues), as well as equations for pulmonary exposure and excretion (Tardif et al., 1993, 1995). A subsequent model added ethylbenzene to the mixture (Tardif et al., 1997). PBPK models were also developed to consider mixtures of gasoline, which considered the three aforementioned compounds as well as other VOCs (Haddad et al., 1999, 2000, 2001) or toluene, ethylbenzene and o-xylene in combination with other VOCs (Dennison et al., 2003).

8.5.1 Toluene

PBPK models describing the kinetics of inhaled toluene have been developed and validated for rats (Tardif et al., 1993, 1995, 1997; DeJongh and Blaauboer, 1996; Haddad et al., 1999, 2000, 2001; Dennison et al., 2003) and scaled to—and validated for—humans (Pierce et al., 1996a; Tardif et al., 1997; Nong et al., 2006).Most models are based on the same four compartments (liver, fat, and richly and slowly perfused tissues), with saturable oxidative metabolism restricted to the liver. Modifications to this approach included the addition of a brain compartment to allow for estimates of toluene levels in the target organ (DeJongh and Blaauboer, 1996) or consideration of extrahepatic metabolism, specifically in the lungs (Pierce et al., 1996a). One model was also adapted to simulate pharmacokinetics in different stages of childhood using child-specific physiological and metabolic parameters (Nong et al., 2006); the same model also incorporated population distributions of various parameters for adults using Monte Carlo simulation.

Two additional generic human PBPK models for VOCs included toluene. Fisher et al. (1997) developed a lactation model, which added a milk compartment to estimate the transfer of toluene and other VOCs from women exposed by inhalation to their nursing infants; however, metabolic parameters for toluene do not seem to be incorporated, and the model was not validated. In a generic model for several non-polar organic non-electrolytes diluted in aqueous solution, dermal absorption of toluene (with three compartments of stratum corneum, viable epidermis and blood) was described (Shatkin and Brown, 1991); however, the model could not be validated for toluene because of large discrepancies between model estimates and empirical data.

8.5.2 Ethylbenzene

Several models have been developed and validated to simulate the kinetics of inhaled ethylbenzene in rats (Tardif et al., 1997; Haddad et al., 1999, 2000, 2001; Dennison et al., 2003), mice (Nong et al., 2007) and humans (Tardif et al., 1997). In general, all of the models have similar structures—with compartments for liver, fat, and slowly and richly perfused tissues, as well as the modelling of lungs for inhalation and excretion—with saturable oxidative metabolism occurring in the liver. Extrahepatic metabolism was also incorporated into the mouse model, with calculations for metabolic activity also being developed for the lungs and richly and poorly perfused tissues (Nong et al., 2007). Dermal exposure to ethylbenzene has also been simulated and validated in humans using a generic model developed for several non-polar organic non-electrolytes diluted in aqueous solution, which describes only the absorption through stratum corneum and viable epidermis into the blood and not the subsequent distribution to other organs (Shatkin and Brown, 1991).

8.5.3 Xylenes

PBPK models have been designed to describe the inhaled kinetics of m-xylene (Tardif et al., 1993, 1995; Haddad et al., 1999, 2000, 2001; Kaneko et al., 2000), o-xylene (Dennison et al., 2003) or all three xylene isomers (Adams et al., 2005). These models have been developed and validated for rats (Tardif et al., 1993, 1995; Haddad et al., 1999, 2000, 2001; Kaneko et al., 2000; Dennison et al., 2003) and humans (Kaneko et al., 2000; Adams et al., 2005). Only one model considered compartments other than the liver, fat, richly and slowly perfused tissues, and lung, in which equations for the distribution of m-xylene to muscle were included (Kaneko et al., 2000). Most of the models restricted saturable oxidative metabolism to the liver, with additional consideration of lung metabolism in one study (Adams et al., 2005). Although equations were designed to estimate only the rate of metabolism for most models, one model allowed for the estimation of concentrations of the metabolite m-methylhippuric acid excreted in urine (Kaneko et al., 2000).

Thrall and Woodstock (2003) also developed a model to simulate exposures to o-xylene via the dermal route. The model was based on the Tardif et al. (1993) model, with an extra skin compartment, and was validated in humans and rats.

In a generic lactation model, Fisher et al. (1997) added a milk compartment to estimate the transfer of o-, m- and p-xylene and other VOCs to nursing infants based on maternal inhalation exposures. The relevance of this model is limited, because metabolic parameters for xylenes do not seem to be incorporated, and the model was not validated.

8.5.4 Health Canada model

Health Canada developed a PBPK model based on the Tardif et al. (1997) model to facilitate inhalation to oral, experimental animal to human and high dose to low dose extrapolations for toluene, ethylbenzene and xylenes (Nong, 2014). The same basic compartments as in the Tardif et al. (1997) model (fat, liver, slowly perfused and richly perfused tissues, with exposure and excretion via the lungs) were used in the Health Canada model, but the model was updated to include equations for oral exposure as well as a dermal compartment that could be used in estimates of the contribution of showering and bathing to exposure to toluene, ethylbenzene and xylenes. Dermal uptake is based on a previous PBPK model for VOCs (Thrall et al., 2000, 2002, 2003), with the compounds being absorbed directly to the bloodstream with no first-pass effect. The multiroute model represented metabolism as a saturable oxidative process that occurs in the liver, with no extrahepatic metabolism included. Each compound was modelled separately, with no metabolic interaction between the compounds being considered. Since past work conducted by Nong et al. (2007) indicated that model predictions without extrahepatic metabolism refinements were still within a 2-fold variation from low to high dose exposure, the simulation for each compound separately is still valid. Validation of inhalation exposures in the model was performed by comparing model simulations for rats and humans with those presented in the previously validated Tardif et al. (1997) model; validation for the mouse was performed by comparing simulations with those presented in Nong et al. (2007). Human dermal exposure was validated against data from Thrall and Woodstock (2002) for toluene, Mattie et al. (1994) for ethylbenzene (determined from benzene in accordance with very similar partition coefficients for skin exposure) and Thrall and Woodstock (2003) for xylenes. The human oral component of the multiroute model was compared with equivalent dose estimates from inhalation exposures as a means to internally validate extrapolation between routes, even though there was a lack of human oral exposure data.

Data from Seeber et al. (2004, 2005), NTP (1999) and Korsak et al. (1994) were used for PBPK modelling of toluene, ethylbenzene and xylenes, respectively. The dose metric that was used for this modelling was average concentrations of the parent compounds, which are considered to be the toxic moieties, in the blood. Blood concentration is used as a proxy for brain concentration, which is considered to be appropriate, since the absorption and elimination curves for blood and brain concentrations of toluene have been previously demonstrated to be almost identical (DeJongh and Blaauboer, 1996; van Asperen et al., 2003; Bushnell et al., 2007).

For toluene, an external dose of 26 ppm (Seeber et al., 2003; 2004), which was identified as a no-observed-adverse-effect level (NOAEL) from a study of humans exposed occupationally, was inputted into a human PBPK model to estimate the lifetime average internal blood concentrations of toluene (in mg/L). The external human oral dose generated by the model corresponds to the level of exposure identified above, assuming daily consumption of 1.5 L of drinking water.

Lifetime internal blood concentrations of ethylbenzene were determined using PBPK models for mouse (using hyperplasia of the pituitary gland and liver cellular alterations as endpoints). For these effects, a NOAEL of 75 ppm from the NTP (1999) study was identified in mice and used to determine the lifetime average internal blood and liver concentrations of ethylbenzene (in mg/L) in the mouse PBPK model. External human oral doses were generated by the model to correspond to a blood and liver concentration of ethylbenzene equivalent to the external mouse NOAEL of 75 ppm, assuming daily consumption of 1.5 L of drinking water.

Ethylbenzene exposure was also shown to cause various tumours in rodents (NTP, 1999). However, only alveolar/bronchiolar tumours found in male mice were determined to be of relevance to humans. External doses were inputted into the mouse PBPK model in order to estimate the lifetime average internal blood concentrations of ethylbenzene (in mg/L). Using the log logistic model as the best fit model from the U.S. EPA's benchmark dose (BMD) software (U.S. EPA, 2010), these internal doses were then analysed to determine the most appropriate point of departure for inputting into the human model. This internal dose was then inputted into the human PBPK model in order to determine the human external dose required to result in blood concentrations similar to those in the mouse, assuming 1.5 L consumption of drinking water per day and 70 years of exposure.

For xylenes, an external dose of 50 ppm m-xylene was identified as a NOAEL in exposed rats based on performance on the rotarod test (Korsak et al. 1994). This dose was inputted into a rat PBPK model in order to estimate the lifetime average internal blood concentrations of m-xylene (in mg/L). The external human oral dose generated by the model corresponds to the level of exposure identified above, assuming daily consumption of 1.5 L of drinking water.

The PBPK model as described above was also used to estimate litre-equivalent contributions from dermal and inhalation exposures when showering and bathing (see Section 5.6). These litre-equivalent contributions were estimated by running the human PBPK model for a 30-minute bathing scenario. By comparing the internal doses generated from the dermal and inhalation routes of exposure with the internal dose from ingestion, the litre-equivalent contributions for dermal and inhalation exposures were estimated to be 0.2 L-eq and 0.43 L-eq for toluene, 0.21 L-eq and 0.44 L-eq for ethylbenzene, and 0.21 L-eq and 0.43 L-eq for xylenes, respectively.

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