(a) Humans

2-Butoxyethanol is well absorbed by ingestion, inhalation and through the skin.

Several cases of ingestion of 2-butoxyethanol from suicide attempts have been reported (see Table 16). Rambourg-Schepens et al. (1988) described a case report of a suicide attempt by ingestion of 250–500 mL of a window cleaner that contained 12% 2-butoxyethanol. The subject survived but had moderate haemoglobinuria on the 3rd day and peak urinary levels of butoxyacetic acid of approximately 40 g/g creatinine [estimated from a graph]. In another report that involved the ingestion of 8 oz (8 × 28.4 cm³ = 227.2 mL) of a cleaner that contained 10–30% 2-butoxyethanol and 10–40% isopropanol, the peak blood concentration of butoxyacetic acid was 900 µg/L (6.8 µM) and declined with a half-life of 12.7 h in samples collected 100 min after ingestion and at different times over 3 days (McKinney et al., 2000). [This is longer than other reported half-lives and may be due to simultaneous treatment of the patient with ethanol to inhibit metabolism.] The subject survived with no reported haemolysis. A third case also involved ingestion of 360–480 mL of a window cleaner that contained 22% 2-butoxyethanol. The highest measured blood concentration of butoxyacetic acid was 4.86 mM in samples collected approximately 16 h after ingestion and at different times over 3 days (Gualtieri et al., 2003).

Table 16

Toxic effects of 2-butoxyethanol after its ingestion by humans.

The blood-to-air partition coefficient of 2-butoxyethanol was reported to be 7965 (Johanson & Dynésius, 1988) and, as a consequence, it is well absorbed by inhalation. Kumagai et al. (1999) measured the concentration of 2-butoxyethanol in inspired and expired air of four volunteers exposed to 25 ppm [120.5 mg/m³] for 10 min at rest and found that 80% of the 2-butoxyethanol was absorbed. Johanson et al. (1986a) exposed seven male volunteers by inhalation to 20 ppm [96.4 mg/m³; 0.85 mmol/m³] 2-butoxyethanol for 2 h during physical exercise. Respiratory uptake averaged 10.1 µmol/min or 57% of the amount inspired. The concentration of 2-butoxyethanol in blood reached a plateau of 7.4 µmol/L after 1–2 h and the apparent elimination half-life was 40 min. Jones and Cocker (2003) reported a similar apparent elimination half-life of 2-butoxyethanol from blood of 56 min (range, 41–84 min) after exposure of four volunteers to 50 ppm [241 mg/m³] 2-butoxyethanol for 2 h at rest. Peak levels of the metabolite butoxyacetic acid were seen in blood 20 min after the 2-h exposure with a mean concentration of 35 µM (range, 28–43 µM) that declined with a half-life of 13 min (range, 2–42 min). Johanson et al. (1986a) reported that total blood clearance of 2-butoxyethanol was 1.2 L/min and that the steady-state volume of distribution was 54 L.

In-vivo studies have investigated the dermal absorption of 2-butoxyethanol liquid (Johanson et al., 1988; Jakasa et al., 2004) and vapours (Johanson & Bowman, 1991; Corely et al., 1997; Jones et al., 2003). Johanson et al. (1988) conducted experiments of dermal exposure among five male volunteers; two or four of their fingers were exposed to neat 2-butoxyethanol for 2 h. Capillary blood samples were collected from the other arm and were analysed for 2-butoxyethanol. Urine was collected for 24 h and analysed for butoxyacetic acid. The percutaneous uptake rates of 2-butoxyethanol ranged from 7 to 96 nmol/min/cm². The authors estimated that, on average, 17% of the absorbed dose was excreted as butoxyacetic acid within 24 h. However, their method for determining butoxyacetic acid did not include hydrolysis of the glutamine conjugate and may therefore underestimate the total amount of butoxyacetic acid excreted. The percutaneous uptake rate ranged from 1 to 16 µmol/min when four fingers were exposed while the respiratory rate ranged from 8 to 14 (average, 10) µmol/min when the same volunteers were exposed to 20 ppm [96.4 mg/m³; 0.85 mmol/m³] butoxyethanol vapour during light work (Johanson et al., 1986a).

In a more recent study, six male volunteers were exposed by dermal application twice to 50%, once to 90% or once to undiluted (neat) 2-butoxyethanol for 4 h on a 40-cm² area of skin. Inhalation exposure with a known input rate and duration was used as a reference dose. Dermal absorption parameters were calculated from 24-h excretion of total (free plus conjugated) butoxyacetic acid in urine and 2-butoxyethanol in blood, measured after both inhalation and dermal exposures. These exposures correlated to pulmonary uptake and dermal uptake. The dermal absorption of 2-butoxyethanol was higher from the aqueous solutions than from neat 2-butoxyethanol. The dermal fluxes obtained from 24-h cumula-tive excretion of butoxyacetic acid were 1.34 ±0.49, 0.92 ±0.6 and 0.26 ±0.17 mg/cm²/h for 50%, 90% and neat 2-butoxyethanol, respectively. The permeation rates into the blood reached a plateau between 60 and 120 min after the start of exposure, which indicated the achievement of steady-state permeation. The apparent permeability coefficient was 1.75 ±0.53 ×10⁻³ and 0.88 ±0.42 ×10⁻³ cm/h for 50% and 90% 2-butoxyethanol, respectively. These results show that percutaneous absorption of 2-butoxyethanol increases markedly in aqueous solutions and that a water content as low as 10% can increase per-meation rates fourfold. The uptake after dermal exposure to aqueous solutions substantially exceeds pulmonary uptake (Jakasa et al., 2004).

In-vitro studies with human epidermal membranes in glass diffusion cells with water as a receptor fluid show steady-state absorption rates of 0.198 (± 0.7) mg/cm²/h for neat 2-butoxyethanol applied as an infinite dose. The permeability constant was 2.14 ×10⁴ cm/h and the lag time was less than 1 h (Dugard et al., 1984). Lower rates of absorption were seen with full thickness human skin and a tissue culture medium that contained 2% bovine serum albumin (used as receptor fluid), in which the steady-state flux was 544 ± 64 nmol/cm²/h (0.046 mg/cm²/h) (Wilkinson & Williams, 2002).

2-Butoxyethanol vapour is also well absorbed through the skin and can contribute significantly to the systemic dose. Corley et al. (1997) exposed one arm of each of six volunteers to 50 ppm [24.1 mg/m³] 2-butoxyethanol vapour for 2 h. Blood was collected from both the exposed and unexposed arms and was analysed for 2-butoxyethanol and butoxyacetic acid. Urine was collected and analysed for butoxyacetic acid and its conjugates, ethylene glycol and glycolic acid. The concentration of 2-butoxyethanol was 1500 times greater in finger-prick blood from the exposed arm than that in venous blood from the unexposed arm. This confirmed that the previous estimates of 75% dermal absorption of 2-butoxyethanol calculated by Johanson and Bowman (1991) were probably overestimates due to contamination of the blood sample with 2-butoxyethanol during collection. Estimates of dermal absorption based on butoxyacetic acid and a physiologically based pharmacokinetic model showed that dermal absorption of 2-butoxyethanol probably contributed 15–27% of the total systemic dose. After dermal exposure of one arm to 50 ppm for 2 h, the peak 2-butoxyethanol concentration was 0.07 µM [8.26 µg/L] and occurred at the end of exposure. The half-life for 2-butoxyethanol in blood was 0.66 h. The peak blood concentration of butoxyacetic acid was 0.59 µM [70 µg/L] and occurred 3.7 h after the end of exposure. The half-life of butoxyacetic acid in blood was 3.3 h.

The significance of dermal absorption of 2-butoxyethanol vapour was confirmed by Jones et al. (2003) who exposed four volunteers on nine occasions by either ‘whole body’ (inhalation and dermal absorption) or ‘skin only’ (breathing clean air) exposure to 50 ppm 2-butoxyethanol for 2 h at different temperatures and humidities. At 25 °C and 40% rela-tive humidity, dermal absorption of vapour accounted for an average of 11% of the total absorbed dose. Dermal absorption increased slightly with increased temperature or humidity; when both were combined (30 °C and 60% relative humidity), dermal absorption contributed 39% of the ‘total’ absorbed dose.

In humans, the elimination of 2-butoxyethanol is mostly by excretion of butoxyacetic acid in the urine. Johanson et al. (1986a) showed that less than 0.03% of the dose was excreted as unchanged 2-butoxyethanol in urine and that butoxyacetic acid (without hydrolysis) accounted for 15–55% of the dose. This value probably underestimates the percentage of 2-butoxyethanol that is excreted as butoxyacetic acid since this metabolite is also excreted as a glutamine conjugate. Sakai et al. (1994) showed that the percentage of conjugation of butoxyacetic acid in the urine of workers exposed to 2-butoxyethanol varied from 44 to 92% with a mean value of 71%, a value supported by Corely et al. (1997) who estimated butoxyacetic acid–glutamine conjugation to be around 67% and found no ethylene glycol or glycolic acid in the urine. Jones and Cocker (2003) showed that conjugation of butoxyacetic acid was variable both between and within workers and, based on urine samples from 48 workers, that the average level of conjugation was 57% (95% CI, 44–70%).

Studies of volunteers showed that peak urinary excretion of butoxyacetic acid occurs 3–6 h after the end of a 2-h exposure to 2-butoxyethanol by inhalation and then declines with a half-life of approximately 6 h (Johanson et al., 1986a; Jones & Cocker, 2003).

The low renal clearance of butoxyacetic acid (23–39 mL/min) indicates extensive binding to protein and absence or low efficiency of tubular secretion of butoxyacetic acid. The low apparent volume of distribution (15 L) is an additional indication of binding of butoxyacetic acid to blood proteins (Johanson & Johnsson, 1991).

(b) Animals

(a) Oxidation of 2-butoxyethanol to 2-butoxyacetaldehyde and 2-butoxyacetic acid

Although it was considered for a long time to be an intermediate step in the conversion of 2-butoxyethanol to butoxyacetic acid, the actual formation of the aldehyde metabolite has only very recently been demonstrated in experimental animals. Deisinger and Boatman (2004) administered a single oral dose of 600 mg/kg bw 2-butoxyethanol to male and female B6C3F₁ mice and detected low but measurable levels of 2-butoxyacetal-dehyde in the blood, liver and forestomach at all time-points (5, 15 and 45 min) up to 90 min after exposure. Concentrations were highest in all tissues 5 min after administration and declined thereafter. Initial levels were approximately 10-fold higher in the forestomach than in the blood and liver. Levels of the aldehyde intermediate were signi-ficantly higher in tissues of female mice than in those of male mice, and were one to three orders of magnitude less than those of the acid metabolite.

An extensive database of studies in exposed humans and experimental animals shows that the formation of 2-butoxyacetic acid is a principal metabolic end-point for 2-butoxyethanol. Although it mainly occurs in the liver, there is potential for the formation of 2-butoxyacetic acid in tissues at or near the site of contact that contain ALDH and ADH, e.g. the skin, forestomach or glandular stomach (Sabourin et al., 1992a). Aasmoe et al. (1998) demonstrated that ADH3 was the only ADH isoenzyme that effectively oxidized 2-butoxyethanol in the liver of rats. No information was found on the isoenzyme(s) of ADH that is involved in the metabolism of 2-butoxyethanol to 2-butoxyacetic acid in humans. There are several classes of ADH, for some of which interindividual variation occurs in humans (i.e. polymorphism); thus, there may be substantial variability in the extent to which 2-butoxyethanol is metabolized to 2-butoxyacetic acid in the human popu-lation, but this has not been investigated extensively.

Green et al. (2002) compared the distribution of ADHs in the forestomach and glandular stomach of rodents with that in the stomach of humans. The authors noted that the enzymes are largely concentrated in the stratified squamous epithelium of the forestomach of rats and mice but are more diffuse in the glandular stomach, and have greater activity in these tissues in mice than in rats. This greater activity corresponds to the greater sensitivity of mice to 2-butoxyethanol-induced effects in the forestomach. In the human stomach, ADH and ALDH are evenly distributed throughout the gastric epithelial cells of the mucosa.

2-Butoxyacetic acid appeared in the urine or was measured in the blood of humans following incidental ingestion or occupational or controlled exposure via inhalation or dermal contact (Johanson et al., 1986a, 1988; Rambourg-Schepens et al., 1988; Angerer et al., 1990; Rettenmeier et al., 1993; Sakai et al., 1993; Söhnlein et al., 1993; Sakai et al., 1994; Corley et al., 1997; Haufroid et al., 1997; Laitinen, 1998; McKinney et al., 2000; Jakasa et al., 2004). In these studies, 2-butoxyacetic acid was the major metabolite identified and was found to correspond to up to 70% of the amount of 2-butoxyethanol absorbed (Jakasa et al., 2004). Some of the 2-butoxyacetic acid is eliminated as the free acid, while a portion is conjugated before elimination (Jakasa et al., 2004). Concentrations of free 2-butoxyacetic acid in the blood of five men exposed to 20 ppm [97 mg/m³] 2-butoxyethanol by inhalation for 2 h ranged from 18.5 to 56.5 µM [2 to 7 mg/L] (Johanson & Johnsson, 1991). Levels of 2-butoxyacetic acid at the end of a shift in the urine of workers who used glass cleaners that contained 2-butoxyethanol increased linearly with ambient concentration of 2-butoxyethanol (exposure range, < 0.1–7.33 ppm [< 0.5– 35 mg/m³]) (Vincent et al., 1993), which suggests that saturation of this metabolic pathway does not occur at these exposure concentrations. However, in an experimental study, Johanson et al. (1986a, 1988) observed substantial variation in the urinary levels of 2-butoxyacetic acid between volunteers exposed to 2-butoxyethanol by dermal application (2.5–39%; five subjects) or by inhalation (15–55%; six subjects). Similarly, Jakasa et al. (2004) observed interindividual variations of 42–70% in urinary levels of total 2-butoxyacetic acid (i.e. free and conjugated) of six volunteers exposed by inhalation to approximately 93 mg/m³ 2-butoxyethanol.

2-Butoxyacetic acid is also the major metabolite of 2-butoxyethanol in experimental animals. In rats administered up to 2590 ppm [2.6 mg/L] 2-butoxyethanol in the drinking-water (1.2 mmol/kg bw), 2-butoxyacetic acid eliminated in the urine accounted for up to 60% of the administered dose (Medinsky et al., 1990). 2-Butoxyacetic acid also comprised more than 75% of the radioactivity in the urine of rats administered a single dose of up to 500 mg/kg bw [¹⁴C]2-butoxyethanol by gavage (Ghanayem et al., 1987a). Inhibition of ADH or ALDH by pyrazole or cyanamide, respectively, significantly reduced the extent of conversion of 2-butoxyethanol to 2-butoxyacetic acid in rats administered a single oral dose of 500 mg/kg bw (i.e. from 75–90% of total radioactivity in the urine to 5–13%); the reduc-tion in levels of 2-butoxyacetic acid corresponded with a reduction in toxicity (Ghanayem et al., 1987b) (see also Section 4.1.1). Similarly, competitive inhibition of ADH by ethanol, n-propanol or n-butanol reduced the production of 2-butoxyacetic acid from 2-butoxyethanol by 43, 33 and 31%, respectively (consistent with the greater affinity of the enzyme for alcohols than for glycol ether), which was accompanied by a corresponding reduction in the toxicity of 2-butoxyethanol (Morel et al., 1996). Exposure of rats to airborne concentrations of up to 483 ppm [2333 mg/m³] 2-butoxyethanol did not appear to exceed the saturation level of its conversion to 2-butoxyacetic acid, based on the increase in concentrations of this metabolite measured in the urine (Sabourin et al., 1992a; Johanson, 1994).

In an investigation of the metabolism of 2-butoxyethanol to 2-butoxyacetic acid during long-term inhalation exposure of rats and mice (Dill et al., 1998; National Toxicology Program, 2000), the rate of elimination of 2-butoxyethanol from blood followed linear kinetics, which suggests that the production of 2-butoxyacetic acid in both species was also linear. However, the rate of disappearance of 2-butoxyethanol from blood was greater in mice than in rats. Similarly, mice were more efficient at eliminating 2-butoxyacetic acid from the blood, although elimination followed non-linear kinetics in both species (see also Section 4.1.1(b)). In addition, the rate of elimination of both 2-butoxyethanol and 2-butoxyacetic acid from blood decreased with increasing duration of exposure, particularly in rats. Thus, although mice may metabolize 2-butoxyethanol to 2-butoxyacetic acid at a greater rate than rats, the metabolite is subsequently cleared much more rapidly in mice, which is consistent with the apparently greater sensitivity to 2-butoxyethanol-induced haematological effects of rats than mice in both short- and long-term studies. Similarly, the greater sensitivity of female rats than male rats is probably related to slower clearance of the active acid metabolite than to sex-related differences in its production, although the activity of the hepatic ADH isoenzyme involved (ADH3) was reported to be greater in female rats than in male rats (Aasmoe et al., 1998; Aasmoe & Aarbakke, 1999). The activities of gastric ADH are greater in male than in female rats [however, the sex difference was only significant when octanol was the substrate; 2-butoxyethanol was not tested in this study] (Aasmoe & Aarbakke, 1999). Consistent with the less pronounced sex-specific sensitivities to 2-butoxyethanol-induced toxicity in mice, little sex difference in the formation or elimination of 2-butoxyacetic acid was observed in this species.

Ghanayem et al. (1987c) observed that older rats metabolized 2-butoxyethanol to 2-butoxyacetic acid to a greater extent than younger rats following oral administration of 500 mg/kg bw, based on a comparison of the levels of 2-butoxyacetic acid in the urine. Similar results were obtained in rats that were administered a single intravenous dose of up to 125 mg/kg bw 2-butoxyethanol, based on greater values of the AUC and C_max of 2-butoxyethanol and in C_max and half-life of 2-butoxyacetic acid in older rats compared with younger rats (Ghanayem et al., 1990). In mice, although no significant difference in the rate of clearance of 2-butoxyethanol (and presumably the formation of 2-butoxyacetic acid) was observed between older and younger animals exposed to up to 125 ppm [603 mg/m³] by inhalation for 1 day, younger mice cleared 2-butoxyacetic acid at a 10-fold greater rate than older mice after 1 day. However, this difference was less obvious after 3 weeks of exposure (Dill et al., 1998) (see also Section 4.1.1(b)).

In an in-vitro investigation of the comparative metabolism of 2-butoxyethanol in hepatocytes from humans and rats, Green et al. (1996) observed that cells from rats were more efficient at converting 2-butoxyethanol to 2-butoxyacetic acid than those from humans (four men and three women). Saturation of this pathway appeared to occur at much lower doses in hepatocytes from humans than in those from rats, since the percentage of total radioactivity identified as 2-butoxyacetic acid was only 1.5-fold greater in rat hepatocytes than in human hepatocytes at 0.02 mM, while the difference was 6.1-fold at 10 mM. The percentage that was converted to 2-butoxyacetic acid increased with up to 10 mM glycol ether in rat hepatocytes but decreased in human hepatocytes between 0.02 and 0.2 mM.

(b) Conjugation of 2-butoxyacetic acid with glutamine or glycine

Conjugation of 2-butoxyacetic acid with glutamine via acyltransferase has been demonstrated in humans. Rettenmeier et al. (1993) reported that N-butoxyacetylglutamine accounted for a mean of 48% (range, 16–64%) of the 2-butoxyacetic acid that was detected in urine collected at the end of the work week from six lacquerers who were exposed to 2-butoxyethanol (the remainder was free 2-butoxyacetic acid). Hence, conjugation with glutamine may represent an important route of removal of 2-butoxyacetic acid. Sakai et al. (1994) reported that a larger proportion (mean, 71%; range, 44–92%) of total 2-butoxyacetic acid was present as conjugates [not further identified] in the urine of six workers who were exposed to 2-butoxyethanol. They also determined that the fraction of 2-butoxyacetic acid eliminated as conjugates decreased throughout the work week, which may reflect a decline in the capacity for conjugation with continued exposure. These data are consistent with the lower proportions of conjugated 2-butoxyacetic acid observed by Rettenmeier et al. (1993) in the urine of workers after several days of exposure. More recently, Jones and Cocker (2003) determined that the mean extent of conjugation of 2-butoxyacetic acid [not further identified] was 57% (95% CI, 44–70%) in a group of 48 exposed workers, in six of whom there was no evidence of conjugation. These authors also observed substantial intra-individual variability in volunteers who were exposed repeatedly to 50 ppm [242 mg/m³] 2-butoxyethanol for 2 h (i.e. nearly 0–100%) and hypothesized that this large variability was not related to polymorphisms but to other factors such as levels of glutamine. Corley et al. (1997) observed that about two-thirds (67%) of the 2-butoxyacetic acid excreted in the urine of male volunteers exposed der-mally (one arm) to airborne concentrations of 50 ppm [242 mg/m³] 2-butoxyethanol was present as N-butoxyacetylglutamine.

Little information was available on the conjugation of 2-butoxyacetic acid with glycine. No evidence of conjugation with this amino acid was found in rats that were administered up to 2590 ppm [2590 µg/mL] 2-butoxyethanol in the drinking-water for 1 day and observed for 3 days after exposure (Medinsky et al., 1990) or in rats exposed dermally to up to 2530 µmol/kg bw [298 mg/kg bw] (Sabourin et al., 1992b). However, glycine conjugation with the alkoxyacetic acid metabolite has been observed for other glycol ethers (2-methoxyethanol and 2-ethoxyethanol) (Jönsson et al., 1982; Cheever et al., 1984; Moss et al., 1985). Corley et al. (1994) did not observe conjugation of 2-butoxyacetic acid with amino acids in samples of blood or urine from rats that were administered single doses of up to 126 mg/kg bw by gavage.

On the basis of these data, it appears that clearance of 2-butoxyacetic acid via conju-gation occurs to a greater extent in humans than in rats, although data on this metabolic step in rodents are sparse.

(c) Dealkylation of 2-butoxyethanol to ethylene glycol and butyraldehyde

Only limited data on the occurrence of O-dealkylation of 2-butoxyethanol to ethylene glycol and (presumably) butyraldehyde in humans are available. Ethylene glycol, or its metabolite (oxalate), have been detected in the plasma or urine from two individuals who had ingested cleaning products that contained 2-butoxyethanol (250–500 mL of a solution that contained 12% 2-butoxyethanol) (Rambourg-Schepens et al., 1988) and 150 mL of a solution that contained 9.1% 2-butoxyethanol (Nisse et al., 1998). Metabolic acidosis, which is commonly associated with ethylene glycol poisoning, was observed in both cases (Rambourg-Schepens et al., 1988; Nisse et al., 1998). Conversely, neither ethylene glycol nor its metabolites were detected in the serum or urine from two people who had ingested 2-butoxyethanol (200–250 mL of a product that contained 12.7% 2-butoxyethanol (∼25–30 g) or 360–480 mL of a product that contained 22% 2-butoxyethanol (∼80–105 g)) (Gijsenbergh et al., 1989; Gualtieri et al., 1995), although metabolic acidosis occurred in both cases. Similarly, Corley et al. (1997) did not detect ethylene glycol or its metabolic products in the urine of a group of volunteers who were exposed dermally to 50 ppm [242 mg/m³] 2-butoxyethanol for 2 h.

There is stronger evidence for the occurrence of this metabolic pathway in rats. Medinsky et al. (1990) reported that ethylene glycol comprised 14–22% of total urinary radioactive metabolites in rats that were administered up to 2590 ppm [2590 µg/mL] [¹⁴C]2-butoxyethanol in the drinking-water for 24 h. It also accounted for 2–11% of total ¹⁴C in the urine of rats that were administered doses of 8.6 or 126 mg/kg bw by gavage (Corley et al., 1994), but was not detected in the urine of rats that were administered up to 500 mg/kg bw 2-butoxyethanol by gavage (Ghanayem et al., 1987a). [This discrepancy may have been due to the ¹⁴C radiolabel being on the butoxy moiety instead of the ethanol moiety (National Toxicology Program, 2000).] Similarly, Sabourin et al. (1992b) iden-tified 4.1–6.5% of urinary metabolites as ethylene glycol in rats that were exposed dermally to up to 2530 µmol/kg bw [298 mg/kg bw] 2-butoxyethanol. Contrary to the increase in the proportion of urinary ethylene glycol observed in rats exposed orally or dermally to increasing levels of 2-butoxyethanol (Medinsky et al., 1990; Sabourin et al., 1992b), the fraction of urinary metabolites identified as ethylene glycol decreased with increasing exposure level in rats exposed to airborne concentrations of 4.3, 49 and 438 ppm [21, 237 and 2119 mg/m³] 2-butoxyethanol for 6 h (i.e. 16, 13.4 and 8%, respectively). The majority of ethylene glycol was excreted after cessation of exposure (Sabourin et al., 1992a).

In an in-vitro study, hepatocytes from both rats and humans (four men and three women) metabolized 2-butoxyethanol to ethylene glycol. However, ethylene glycol represented a greater proportion of total radioactivity in rat cells than in human cells, which indicates a possible greater propensity for this metabolic step in rats than in humans. In both rat and human hepatocytes, the percentage of 2-butoxyethanol that was metabolized to ethylene glycol decreased with increasing dose, which the authors suggested was indicative of saturation of this pathway at relatively low concentrations of 2-butoxyethanol (Green et al., 1996).

Haufroid et al. (1997) hypothesized that polymorphism for CYP2E1 may influence the extent to which 2-butoxyethanol may be dealkylated to ethylene glycol and butyralde-hyde. This was based on the observation that the level of free 2-butoxyacetic acid in the urine of a worker who expressed the heterozygous c2 allele (c1/c2) did not change subs-tantially throughout the workday, unlike the 30 other workers who expressed the homozygous c1 allele (c1/c1) in whom substantial increases in levels of urinary free 2-butoxyacetic acid occurred. The authors suggested that the lack of increase in urinary 2-butoxyacetic acid may be due to greater activity of the c2 allele compared with that of the c1 allele in O-dealkylation of 2-butoxyethanol as observed by Hayashi et al. (1991). It was noted that urine samples were not analysed for amino acid conjugates of 2-butoxyacetic acid, which could also have influenced the excretion of free 2-butoxyacetic acid. The genetic polymorphism that affects transcriptional regulation of human CYP2E1 could also contribute to interindividual differences in the metabolism of 2-butoxyethanol via the dealkylation pathway.

Although no information was available regarding the formation of butyraldehyde or the subsequent metabolic product, butyric acid, in humans or experimental animals, it is predicted that butyraldehyde is the other metabolite that would result from O-dealkylation of 2-butoxyethanol to ethylene glycol.

(d) Glucuronide and sulfate conjugation of 2-butoxyethanol

Information on the in-vivo formation of glucuronide or sulfate conjugates with 2-butoxyethanol was only available for rats, although the glucuronide conjugate has been tentatively identified in hepatocytes from both rats and humans exposed to 2-butoxyethanol in vitro (Green et al., 1996). In rats administered single oral doses of 125 or 500 mg/kg bw radiolabelled 2-butoxyethanol, the glucuronide conjugate represented up to 24% of total urinary metabolites. This conjugate was no longer detectable in the urine 48 h after administration of 125 mg/kg bw, while levels were similar at 8 and 48 h following ingestion of 500 mg/kg bw. The sulfate conjugate was detected in samples of urine taken 8 h after administration of 125 mg/kg bw but was not found in the urine from rats exposed to 500 mg/kg bw. The glucuronide conjugate was the major metabolite in bile and constituted up to 89% of biliary radioactivity 30 min after exposure; however, this proportion declined to 54% after 8 h. The sulfate conjugate was not detected in the bile (Ghanayem et al., 1987a).

The glucuronide conjugate has also been identified as a minor metabolite in the urine of rats exposed to 290–2590 ppm [290–2590 µg/mL] [¹⁴C]2-butoxyethanol in the drinking-water for 24 h (8–11% of total radioactive metabolites measured in urine) (Medinsky et al., 1990) and of rats administered 8.6 or 126 mg/kg bw [¹⁴C]2-butoxyethanol by gavage (10–15% of total radioactive metabolites) (Corley et al., 1994). The proportion of urinary metabolites identified as 2-butoxyethanol~glucuronide increased with the level of exposure in rats exposed to airborne concentrations of 4.3–438 ppm [21–2119 mg/m³] 2-butoxyethanol for 6 h (i.e. from 10.6 to 62.3%). The glucuronide conjugate is apparently cleared rapidly, since it constituted only 3.6–6.5% of urinary metabolites during the 7–16 h after cessation of exposure (Sabourin et al., 1992a). However, the proportion of urinary metabolites identified as the glucuronide conjugate did not increase in rats administered dermal doses of 520–2530 µmol/kg bw, but was similar at all doses (i.e. 13–15%) (Sabourin et al., 1992b).

Inhibition of the principal pathway of metabolism of 2-butoxyethanol (i.e. oxidation to 2-butoxyacetic acid) by injection of pyrazole or cyanamide (inhibitors of ADH and ALDH, respectively) into rats administered a single oral dose of 500 mg/kg bw [¹⁴C]2-butoxyethanol (labelled at C1) resulted in a shift to increased conjugation with glu-curonide and sulfate. The proportions of these conjugates increased from 9–24% (glucu-ronide) and undetectable (sulfate) to 75–85% and 7.5–20%, respectively, of the total radioactivity in the urine. The shift towards the conjugation pathway corresponded with a reduction in 2-butoxyethanol-induced haematotoxicity (Ghanayem et al., 1987b). Following intravenous administration of lower single doses of 2-butoxyethanol to rats (62.5 or 125 mg/kg bw), the glucuronide conjugate was only detected in the blood of animals that had also been administered pyrazole as an inhibitor of ADH (Ghanayem et al., 1990).

Ghanayem et al. (1987c) demonstrated that young rats produce a greater proportion of glucuronide conjugates and (as discussed above) proportionally less 2-butoxyacetic acid than older rats, which is consistent with the apparent lower sensitivity of young rats to the haematotoxic effects of 2-butoxyethanol. However, the sulfate conjugate was not detected in the urine of young rats.

(a) Oral exposure

Several individual cases that involved consumption of a few hundred millilitres of glass cleaners that contained 6.5–35% 2-butoxyethanol have been reported (Gijsenbergh et al., 1989; Rambourg-Schepens et al., 1988; Litovitz et al., 1991; Bauer et al., 1992; Burkhart & Donovan, 1998; Nisse et al., 1998; McKinney et al., 2000; Osterhoudt, 2002; Gualtieri et al., 2003). The main symptoms were hypotension, poor ventilation, coma, metabolic acidosis (probably due to the formation of butoxyacetic acid), renal impairment, haematuria and effects on the blood (erythropenia, reduced haematocrit and haemoglobin levels, haemoglobinuria and/or hypochromic anaemia) (Table 16). [Treatment by haemodialysis can lead to haemodilution that might account for a lower blood cell count but not for haemoglobinuria.] In a report of 24 cases of ingestion of up to 300 mL of glass-cleaning products that contained 0.5–10% 2-butoxyethanol that were included in a survey of childhood poisonings, all of the children were reported to be asymptomatic. Even at the highest estimated dose of 1850 mg/kg bw (300 mL of a glass cleaner that contained 8% 2-butoxyethanol (∼24 g)) that was consumed by a 2-year-old child, no signs of metabolic acidosis or of haematological, renal or neurological adverse effects were manifest (Dean & Krenzelok, 1992).

(b) Dermal exposure

Percutaneous absorption of 2-butoxyethanol was investigated experimentally in five men, who kept two or four fingers immersed in 2-butoxyethanol for 2 h. None of the subjects exhibited an adverse reaction (Johanson et al., 1988). No evidence of strong adverse dermal effects or skin sensitization was observed in 201 individuals exposed dermally to 0.2 mL 10% 2-butoxyethanol in patches, although repeated applications did produce increasing erythema in several subjects (Greenspan et al., 1995).

(c) Inhalation exposure

The effects reported by subjects who were exposed to 113 ppm [546 mg/m³] 2-butoxyethanol for 4 h (two men), 195 ppm [942 mg/m³] for two 4-h periods separated by a 30-min interval (the same two men and one woman) or 98 ppm [474 mg/m³] for 8 h (two men and two women) included irritation to the eyes (probably due to direct contact with the vapours), nose and throat, a disturbance of taste, a slight increase in nasal mucous discharge and headache. In this study, the women appeared to be more sensitive to the induction of these effects than the men. In none of these trials was there any evidence of changes from pre-exposure values in erythrocyte fragility, blood pressure, pulse rate or urinary levels of glucose or albumin (Carpenter et al., 1956).

In two studies in which groups of four or seven male volunteers were exposed by inhalation to 20 or 50 ppm [97–242 mg/m³] 2-butoxyethanol for 2 h, no overt signs of toxicity were observed (Johanson et al., 1986a; Johanson & Boman, 1991). In the earlier investigation, in which the intake of 2-butoxyethanol was estimated to be 2 mg/kg bw for the 2-h exposure at 20 ppm (during which time the men performed light physical exercise), no consistent effects on the lungs (ventilation or breathing rate) or the heart (electro-cardiogram readings or heart rate) were observed (Johanson et al., 1986a). In the sub-sequent study, the investigators distinguished between vapour inhalation (through the mouth alone) and dermal exposure to the vapour (with a mask to prevent inhalation); no toxic effects were evident following either exposure protocol (Johanson & Boman, 1991). In a more recent study, four volunteers exposed to 25 ppm [121 mg/m³] 2-butoxyethanol for 10 min also did not show overt signs of toxicity shortly after treatment (Kumagai et al., 1999).

Signs of respiratory or ocular irritation, headache, sore throat and a sore nose that would become raw and bleed were reported in small groups of workers exposed to 5 ppm [24 mg/m³] or less 2-butoxyethanol vapours (Apol, 1986).

The effect of occupational exposure to 2-butoxyethanol on various haematological, renal and hepatic parameters was investigated in 31 male workers at a beverage production plant who were exposed to mean concentrations of 3.64 mg/m³ 2-butoxyethanol (range, 1.77–6.14 mg/m³; 20 samples) or 2.20 mg/m³ (range, 0.75–3.35 mg/m³; 11 samples) for 1–6 years, based on limited exposure data. The workers were also exposed to unspecified concentrations of methyl ethyl ketone. These parameters were also measured in a comparison population of 21 unexposed workers, matched for sex, age and smoking habits. Blood samples were collected at the end of the shift, while urine samples were taken before and after the workshift. No differences were observed in erythrocyte count, reticulocyte count, haemoglobin concentration, mean corpusclar volume, mean corpuscular haemoglobin or erythrocyte osmotic resistance between exposed and unexposed workers. However, haematocrit was significantly lower (p = 0.03) in the exposed group, while their mean cell haemoglobin concentration was significantly higher (p = 0.02) than that of controls, although the absolute differences in mean values were small (i.e. 43.9 versus 45.5% and 336 versus 329 g/L, respectively) and the ranges of values overlapped. No correlation was found between any of the haematological parameters investigated and levels of butoxyacetic acid in the urine at the end of the workshift or the difference in levels of this acid before and after the workshift (used as parameter of internal exposure), and no differences were observed in the renal parameters investigated (serum creatinine or urinary retinol binding protein). With regard to hepatic parameters, no statistically significant differences in levels of aspartate aminotransferase (20.5 ± 4.9 versus 21.7 ± 6.3 IU/L) or alanine aminotransferase (20.5 ± 13.1 versus 28.6 ± 16.3 UI/L) in the plasma were found between exposed and unexposed workers. However, plasma concentration of alanine aminotransferase and levels of butoxyacetic acid in the urine were statistically significantly correlated (r = 0.60; p = 0.0004) (Haufroid et al., 1997).

Several hours after the floor in an unventilated office had been stripped using a solvent that contained 2-butoxyethanol and other substances, seven workers were exposed to airborne levels estimated to be 100–300 ppm (483–1449 mg/m³) (based on symptoms) for 0.5–4 h. Subjects had immediate severe irritation of the eye and upper respiratory tract, nausea and presyncope which subsided within 3 days. [It was not specified in the brief account of these cases whether haematological parameters were examined immediately after exposure.] All but one of these workers reported recurrent upper respiratory irritation and the appearance of cherry angiomas 3 months later. Eight months after exposure, six of the seven workers had an increased blood cell sedimentation rate and mild hypertension, while skin lesions continued to appear and persisted. Follow-up for an additional 5 years in four of these workers revealed gradual disappearance of most of these effects, with the exception of the cherry angiomas that continued to persist and develop (Raymond et al., 1998).

Collinot et al. (1996) investigated the potential of 2-butoxyethanol to induce hepatic enzymes in a group of 17 male foundry workers who were exposed to a maximum concentration of 7.5 ppm (36 mg/m³) 2-butoxyethanol. Levels of D-glucaric acid (which is the end product of the glucuronic acid pathway and is an indicator of enzyme activity in the liver) in the urine of exposed workers were compared with those in 18 unexposed controls. Uri-nary excretion of D-glucaric acid was significantly greater in exposed subjects than in controls, although significant seasonal variability was also found among exposed workers (increase in D-glucaric acid of 165% in winter and 85% in summer when the doors were open and the 2-butoxyethanol concentration was lower). [The Working Group noted that many of these occupational studies involve mixed exposures, the effects of which cannot be ascribed specifically to 2-butoxyethanol.]

(a) In-vivo studies with single doses (acute effect)

Based on LD₅₀s and LC₅₀s that range from 530 to 6700 mg/kg bw and from 450 to 700 ppm [2173–3381 mg/m³], respectively, 2-butoxyethanol produces low to moderate toxicity in experimental animals following acute exposure (Smyth et al., 1941; Werner et al., 1943a; Carpenter et al., 1956; Smyth et al., 1962; Weil & Wright, 1967; Gingell et al., 1998). Haematological effects, as well as effects on the liver, kidney, lung and spleen, some of which may be secondary to haematotoxicity, have been observed in animals exposed acutely to lower doses or concentrations. For example, alterations in haematological parameters that are characteristic of haemolytic anaemia have been observed in rats administered single oral doses as low as 125 mg/kg bw, while haemoglobinuria was noted in older rats following administration of 32 mg/kg bw by gavage (Ghanayem et al., 1987c). Female rats were more sensitive to 2-butoxyethanol-induced haemolysis than male rats following administration of a single dose of 250 mg/kg bw by gavage, as the magnitude of changes in haematological parameters was greater in females than in males; in addition, the onset of haemolysis was faster in females than in males (Ghanayem et al., 2000). Exposure to airborne concentrations of 62 ppm [299 mg/m³] for 4 h resulted in increased osmotic fragility of erythrocytes (Carpenter et al., 1956), whereas dermal exposure to 260 mg/kg bw for 6 h induced haemolysis in rats (Bartnik et al., 1987). Ghanayem et al. (1992) and Sivarao and Mehendale (1995) demonstrated that younger blood cells were more resilient to 2-butoxyethanol-induced haemolysis than older cells, as rats that had been bled several days before exposure to a single oral dose were less severely affected than rats that had not been bled.

2-Butoxyethanol is considered to be a mild-to-severe irritant to the skin and eyes of rabbits (range doses, 0.1–0.5 mL neat compound), and severity increased with duration of exposure (range, 1–72 h) (Carpenter & Smyth, 1946; Smyth et al., 1962; Kennah et al., 1989; Jacobs, 1992; Zissu, 1995). There was no evidence of skin sensitization in one investigation in guinea-pigs (Zissu, 1995).

(b) In-vivo studies with multiple exposures

(c) In-vitro investigations of haemolytic effects

Differences in species sensitivity to haemolysis induced by 2-butoxyethanol and its metabolites have been investigated in several studies in vitro. Consistent with the results of in-vivo studies, 2-butoxyacetic acid was more potent than either the parent compound or the acetaldehyde metabolite (Bartnik et al., 1987; Ghanayem, 1989; Sivarao & Mehendale, 1995). Although slight species differences were observed in erythrocytes exposed to 2-butoxyethanol (humans were less sensitive than rats, mice, dogs and guinea-pigs) (Carpenter et al., 1956; Bartnik et al., 1987), variability between species was much more pronounced when cells were exposed to 2-butoxyacetic acid (erythrocytes from humans were less sensitive than those of rats). In a comparison across multiple mammalian species (three to five animals per species), Ghanayem and Sullivan (1993) observed that red blood cells from rats, mice, hamsters, rabbits and baboons were more sensitive to the effects of 2-butoxyacetic acid than those of pigs, dogs, cats, guinea-pigs and humans.

Bartnik et al. (1987) reported that 1.25 mM [165 µg/mL] 2-butoxyacetic acid (the lowest concentration administered) resulted in 25% haemolysis in male Wistar rat ery-throcytes after 180 min, while, in contrast, 15 mM [1980 µg/mL] 2-butoxyacetic acid did not produce measurable haemolysis in erythrocytes from healthy humans over the same time. [This study was conducted on washed erythrocytes rather than whole blood, which indicates that the species difference in sensitivity in vitro was probably due to an inherent difference in the erythrocytes, rather than in the extent of plasma protein binding of 2-butoxyacetic acid.] Ghanayem (1989) exposed pooled whole blood from male Fischer 344 rats and healthy human volunteers (three men and women) to 2-butoxyacetic acid for 0.25–4 h and measured haematocrit and free plasma haemoglobin levels as indicators of swelling of the erythrocytes and haemolysis, respectively. Based on observations after 4 h of incubation, 8.0 mM [1056 µg/mL] 2-butoxyacetic acid had less effect in humans than 0.5 mM [66 µg/mL] 2-butoxyacetic acid had in rats. [However, it is not clear from the data presented whether the slight changes in human erythrocytes were produced in relation to the initial control value or were compared with data for a 4-h incubation in the absence of 2-butoxyacetic acid.] Similarly, Udden and Patton (1994) observed no effects in human erythrocytes exposed to 2 mM [264 µg/mL] 2-butoxyacetic acid (the maximum concentration tested) for 4 h, although this concentration induced rapid haemolysis in rat erythrocytes. Exposure of rat erythrocytes to 0.2 mM [26.4 µg/mL] 2-butoxyacetic acid did not result in haemolysis, although reduced cell deformability and increased mean cell volume were noted. In a subsequent investigation, similar sub-haemolytic changes were observed, including loss of deformability and an increase in mean cell volume and cell sodium levels, in human erythrocytes (from up to 14 individuals) that were incubated with 7.5 or 10.0 mM [990 or 1320 µg/mL] 2-butoxyacetic acid and in rat erythrocytes exposed to lower concentrations of 2-butoxyacetic acid (i.e. 0.05, 0.075 or 1.0 mM [6.6, 9.9 or 132 µg/mL]). A slight, but statistically significant increase in haemolysis was observed in red blood cells obtained from 40 hospitalized adults that were exposed in vitro to 10 mM 2-butoxyacetic acid; significant increases in haemolysis were not noted in exposed cells from 11 healthy adults or 46 hospitalized children (Udden, 2002).

Following incubation for 4 h with 2 mM [264 µg/mL] 2-butoxyacetic acid, Udden (1994) reported a lack of haemolysis in human red blood cells from groups of different human subjects, including nine healthy young adults (31–56 years old), nine older subjects (64–79 years old), seven patients who had sickle-cell disease and three patients who had spherocytosis. However, these groups differed with regard to the extent of spontaneous haemolysis that occurred after incubation of their red blood cells for 4 h in the absence of 2-butoxyacetic acid.

Unlike the sex-related differences in sensitivity to 2-butoxyethanol-induced haematological effects that have been observed in rats exposed in vivo, in-vitro exposure of rat ery-throcytes to the metabolite 2-butoxyacetic acid revealed no differences in sensitivity between males and females, as measured by packed cell volume (Ghanayem et al., 2000), which provides further support to the supposition that differences in vivo are related to differences in metabolism and toxicokinetics of 2-butoxyethanol rather than to differences in toxicodynamics.

(d) Mode of action that induces haematological effects

Extensive data from comparative toxicity studies with the parent compound and its metabolites, as well as from studies in which oxidation of 2-butoxyethanol to 2-butoxy-acetic acid is inhibited, indicate that 2-butoxyacetic acid is the metabolite that is principally responsible for the haematological effects observed in experimental animals exposed to 2-butoxyethanol (Ghanayam et al., 1987b; Morel et al., 1996). The changes in haematological parameters induced by 2-butoxyacetic acid are characteristic of haemolytic anaemia, although the mode(s) of action by which 2-butoxyacetic acid induces these effects has (have) not been established conclusively. Before the occurrence of haemolysis in red blood cells from rats, erythrocyte swelling, morphological changes (including a change from typical discocytic to spherocytic appearance and stomatocytosis) and decreased deformability have been observed (Udden, 1996). Udden (2000) observed similar morphological changes in rat erythrocytes following in-vivo exposure to 125 mg/kg bw 2-butoxyethanol and in-vitro exposure to 1.0 mM [132 µg/mL] 2-butoxy-acetic acid which are suggestive of a progression from stomatocytosis, or cupping, to spherocytosis and, finally, to lysis. These morphological changes were not observed in human erythrocytes incubated with up to 2.0 mM [264 µg/mL] 2-butoxyacetic acid. The severity of morphological changes in erythrocytes, including stomatocytosis, macrocytosis, moderate rouleaux formation, spherocytosis, schistocytosis and ghost cell formation, increased progressively in male and female Fischer 344 rats administered 250 mg/kg bw 2-butoxyethanol per day by gavage for 1, 2 or 3 days (Ghanayem et al., 2001). Based on available information from his own research and the investigations of others, Ghanayem (1996) hypothesized that the swelling of erythrocytes was due to increased cell membrane permeability to cations and water and noted that such changes in cellular membranes have been observed in the blood of humans who have hereditary spherocytosis, as have stomatocytes, which are associated with altered intracellular levels of sodium and potassium that lead to increased cell water, increased mean cell volume, decreased deformability and increased osmotic fragility. 2-Butoxyacetic acid-induced swelling of rat erythrocytes exposed in vitro continued after removal of 2-butoxyacetic acid and was not reversible, although the rate of swelling decreased (Ghanayem et al., 1989). These authors also investigated the partitioning of 2-butoxyacetic acid between erythrocytes and plasma and determined that the concentration in cells increased over time while that in the plasma remained relatively constant (Ghanayem et al., 1989). In a previous study, Ghanayem and Matthews (1990) showed that administration of calcium channel blockers to rats before exposure to 2-butoxyethanol decreased the erythrocyte swelling and improved the associated haemolytic anaemia; it was speculated that homeo-stasis of intracellular calcium or other cations (whose passage through cellular mem-branes may be affected by such blockers) may be involved in 2-butoxyethanol-induced toxicity (Ghanayem, 1996). Conjugation of 2-butoxyethanol with long-chain fatty acids in the liver has been observed in rats (Kaphalia et al., 1996), although it is not known whether such conjugation would occur when lipids were present in erythrocyte membranes. In addition, Dartsch et al. (1999) reported that the intermediate metabolite, butoxyacetaldehyde, caused depolymerization of intracellular stress fibres that contain actin in mammalian renal epithelial cells, which resulted in morphological alterations in cell shape and volume that were consistent with the hypothesis that cell swelling points to the induction of a necrotic process.

(a) Reproductive toxicity

Very limited information on the potential reproductive toxicity of 2-butoxyethanol is available. In the only investigation of the potential effect of 2-butoxyethanol on reproduc-tive ability identified (i.e. the multigeneration study by Heindel et al. (1990)), adminis-tration of 700–2100 mg/kg bw per day 2-butoxyethanol in the drinking-water to breeding pairs of CD-1 mice throughout mating, pregnancy and lactation did not induce any discernible effects on male reproductive organs or sperm or on the estrous cycle of females. However, fertility was impaired, based on statistically significant reductions in the numbers of litters per pair and live pups per litter after exposure to 1300 and 2100 mg/kg bw per day; these doses were also associated with decreased body-weight gain and drinking-water consumption in the parent mice. Based on the results of a cross-over mating trial, in which exposed males were mated with control females and exposed females were mated with control males, these effects were found to be primarily due to an effect on the treated females. Because of the lack of sufficient pups in the groups treated with 1300 and 2100 mg/kg bw per day, fertility was only examined in the group that was administered 700 mg/kg bw per day. No effects on reproductive ability, as measured by mating and fertility indices, litter size, proportion of live pups or live pup weights, were noted in mice exposed to 2-butoxyethanol in utero, from weaning to sexual maturity or until delivery of a litter.

Acute exposure to 800 ppm [3864 mg/m³] of a saturated vapour of 2-butoxyethanol for 3 h did not result in altered testicular weight in Alpk/Ap rats (Doe, 1984). Similarly, no histopathological effects on the testes, epididymidis or seminal vesicles were observed in Alpk/Ap rats administered a single oral dose of up to 868 mg/kg bw butoxyacetic acid by gavage (Foster et al., 1987).

No effects on testes weight or histopathology were reported in short-term studies in which Fischer 344 (or Fischer 344/N), Sprague-Dawley and Crl:COBS CD (SD)BR rats were administered doses of up to 1000, 506 and 885 mg/kg bw 2-butoxyethanol per day, respectively, and JCL-ICR mice were administered up to 2000 mg/kg bw per day (Nagano et al., 1979, 1984; Grant et al., 1985; Krasavage, 1986; Exon et al., 1991; National Toxicology Program, 1993).

Sperm morphology and vaginal cytology were assessed in Fischer 344/N rats and B6C3F₁ mice that received 2-butoxyethanol in the drinking-water for 13 weeks. In mice, no evidence was found of an adverse effect on the estrous cycle of females administered up to 1306 mg/kg bw per day, while a slight reduction in sperm motility was observed in males that received 694 mg/kg bw per day and absolute left testis weights were slightly reduced at all dose levels (i.e. 553 mg/kg bw per day and above). In the corresponding study in rats, uterine atrophy or decreased sperm concentration were evident, with LOELs of 304 and 281 mg/kg bw per day in females and males, respectively (National Toxicology Program, 1993).

Testicular degeneration and necrosis of the epididymis was noted in a subchronic study in B6C3F₁ mice that were exposed by inhalation to 500 ppm [2415 mg/m³] 2-butoxyethanol, a concentration that was associated with decreased survival and lesions in several organs (National Toxicology Program, 2000). No such effects were noted in the concurrent study in Fischer 344/N rats exposed to up to 500 ppm 2-butoxyethanol.

No effects on male or female reproductive organs were reported in a 2-year bioassay in which Fischer 344/N rats and B6C3F₁ mice were exposed to up to 125 and 250 ppm [604 and 1208 mg/m³] 2-butoxyethanol, respectively (National Toxicology Program, 2000).

(b) Developmental toxicity

In studies in rats and mice, oral administration of 2-butoxyethanol during pregnancy induced fetotoxic and/or embryotoxic effects, but generally only at or above maternally toxic doses. When Fischer 344 rats were administered 0, 30, 100 or 200 mg/kg bw 2-butoxyethanol per day by gavage on days 9–11 of pregnancy (National Toxicology Program, 1989), increased fetal deaths occurred after exposure to 200 mg/kg bw per day. Maternal toxicity (including haemolysis) was evident at doses of 100 mg/kg bw per day or more. The frequency of malformations was not increased at any dose level. Haemato-logical effects (reduced platelet count) were also noted in fetuses of dams that were exposed to 300 mg/kg bw per day on days 11–13 of gestation, although inconsistent changes in mean corpuscular volume were observed at lower doses. The LOELs for deve-lopmental and maternal toxicity were considered to be 200 and 100 mg/kg bw per day, respectively; no effects were noted after exposure to 30 mg/kg bw per day.

Pregnant CD-1 mice were administered 0, 350, 650, 1000, 1500 or 2000 mg/kg bw 2-butoxyethanol per day by gavage on days 8–14 of gestation (Wier et al., 1987). An increase in the number of resorbed embryos was observed after oral administration of 1000 mg/kg bw per day or more. Clinical signs of toxicity were observed in dams (staining of cage papers, lethargy, abnormal breathing, failure to right or mortality) treated with 650 mg/kg bw per day and above. The LOEL for maternal toxicity and the no-observed-adverse-effect level (NOAEL) for developmental toxicity were considered to be 650 mg/kg bw per day. In pups of dams administered 650 or 1000 mg/kg bw per day, no effects on survival or growth as of postnatal day 22 were observed. Administration by gavage of 1180 mg/kg bw per day (only dose tested) to CD-1 mice during days 7–14 of pregnancy resulted in maternal deaths (20%) and reductions in the number of viable litters, but did not affect pup weight or postnatal survival (Schuler et al., 1984; Hardin et al., 1987).

Heindel et al. (1990) observed a slight fetotoxic effect at a dose that was not overtly toxic to the parent animals; in a multigeneration study, a small, but statistically significant reduction in live pup weight (by 4.3%) was observed in the F₁ generation after adminis-tration of 0.5% 2-butoxyethanol in the drinking-water (equivalent to 700 mg/kg bw per day) to male and female CD-1 mice throughout mating, pregnancy and lactation. Greater reductions in live pup weight were noted at higher doses (1 and 2% in the drinking-water; equivalent to 1300 or 2100 mg/kg bw per day) at which overt maternal toxicity (decreased body weight and fluid consumption, increased kidney weight and/or increased mortality) was observed. This effect was attributed to maternal exposure, since it was only observed in pairings in which exposed females were mated with control males. However, although a 4.4% reduction in live pup weight was noted in the F₂ generation, this decrease was not statistically significant (which indicated that the effect did not worsen in successive generations), and 700 mg/kg bw per day was considered by the investigators to be close to the NOAEL for developmental toxicity.

Developmental toxicity in the form of increased numbers of non-viable implants and resorptions and decreased percentage of live fetuses per litter was observed in Fischer 344 rats exposed to 200 ppm [966 mg/m³] 2-butoxyethanol on days 6–15 of pregnancy. Reduced skeletal ossification was evident at doses of 100 ppm [483 mg/m³] or more (concentrations that were also maternally toxic based on weight loss and haematological effects). Therefore, the LOEL for maternal and developmental toxicity was considered to be 100 ppm, since no effects were observed at doses of 25 or 50 ppm [121 or 242 mg/m³] (Tyl et al., 1984). However, no significant developmental effects occurred in Sprague-Dawley rats exposed to 150 or 200 ppm [726 or 966 mg/m³] 2-butoxyethanol on days 7–15 of pregnancy, although haematuria occurred in dams on the first day of exposure at 200 ppm (Nelson et al., 1984). In rabbits exposed to 2-butoxyethanol on days 6–18 of pregnancy, the LOEL for both maternal and developmental toxicity was 200 ppm [966 mg/m³], based on increased mortality, abortions and weight loss and a slight reduction in the number of viable implants per litter; no significant toxicity was noted in dams or fetuses at doses of 100 ppm [483 mg/m³] or less (Tyl et al., 1984).

In a study of Sprague-Dawley rats that received repeated dermal applications (not specified if the site of application was covered) of neat 2-butoxyethanol on days 7–16 of pregnancy, no overt signs of maternal toxicity and no evidence of developmental toxicity were observed at doses of 0.48 mL per day (∼3.6 mmol per day; ∼1920 mg/kg bw per day). However, the higher dose of 1.4 mL per day (∼10.8 mmole per day; ∼5600 mg/kg bw per day) was lethal to the dams (Hardin et al., 1984).

2-Butoxyethanol

2-Butoxyethanol metabolites