Priority Substances List Assessment Report for 2-Ethoxyethanol

2.0 Summary of Information Critical to Assessment of "Toxic" under CEPA 1999 (cont..)

2.4 Effects characterization

2.4.1 Ecotoxicology

2.4.1.1 Terrestrial organisms

No information on the effects of 2-ethoxyethanol on wildlife was identified. Data for experimental animals pertinent to the human health assessment are presented in Section 2.4.3. From the results of inhalation studies presented in this section, the species that were most sensitive to airborne 2-ethoxyethanol were rats and rabbits. Developmental effects (skeletal variations) were induced in rats exposed to 2-ethoxyethanol at a concentration of 50 ppm (184 mg/m³) for 10 days (Doe, 1984). Developmental toxicity (reduced mean number of implantations and number of live fetuses) was also observed in Dutch rabbits exposed to 2-ethoxyethanol in air at a concentration of 50 ppm (184 mg/m³) (Tinston, 1983a).

2.4.1.2 Aquatic organisms

Data on chronic toxicity have been identified only for protozoans, algae and hydra. The most sensitive organisms were microbial populations in waste stabilization ponds, with approximately a 40% inhibition of respirometric activity (i.e., changes in total organic carbon, chemical oxygen demand and 2-ethoxyethanol concentration) at 1 000 000 µg/L in a 5-day study (Davis et al., 1989). Data on acute toxicity have been reported for invertebrates and fish, although in many studies the LC₅₀ for 2-ethoxyethanol was above the highest concentration tested. For example, the 24-hour LC₅₀ for goldfish (Carassius auratus) was >5 000 000 µg/L (Bridie et al., 1979). Hermens et al. (1984) reported a 48-hour IC₅₀ of 7 660 000 µg/L for Daphnia magna.

2.4.2 Abiotic atmospheric effects

Worst-case calculations were made to determine if 2-ethoxyethanol has the potential to contribute to depletion of stratospheric ozone, ground-level ozone formation or climate change (Bunce, 1996).

The Ozone Depletion Potential (ODP) is 0, as 2-ethoxyethanol is not a halogenated compound.

The Photochemical Ozone Creation Potential (POCP) was estimated to be 73 (relative to the reference compound ethene, which has a POCP of 100), based on the following formula:

POCP = (k_{2-ethoxyethanol} / k_ethene) x (M_ethene / M_{2-ethoxyethanol}) x 100

where:

• k_{2-ethoxyethanol} is the rate constant for the reaction of 2-ethoxyethanol with OH radicals (2.0 x 10^-11 cm³/mol per second),
  
  
• k_ethene is the rate constant for the reaction of ethene with OH radicals (8.5 x 10^-12 cm³/mol per second),
  
  
• M_ethene is the molecular weight of ethene (28.1 g/mol), and
  
  
• M_{2-ethoxyethanol} is the molecular weight of 2-ethoxyethanol (90 g/mol).

The Global Warming Potential (GWP) was calculated to be 5.1 x 10^-5 (relative to the reference compound CFC-11, which has a GWP of 1), based on the following formula:

GWP =	(t2-ethoxyethanol / tCFC-11) x (MCFC-11 / M2-ethoxyethanol) x
	(S2-ethoxyethanol / SCFC-11)

where:

• t_{2-ethoxyethanol} is the lifetime of 2-ethoxyethanol (0.002 years),
  
  
• t_CFC-11 is the lifetime of CFC-11 (60 years),
  
  
• M_CFC-11 is the molecular weight of CFC-11 (137.5 g/mol),
  
  
• M_{2-ethoxyethanol} is the molecular weight of 2-ethoxyethanol (90 g/mol),
  
  
• S_{2-ethoxyethanol} is the infrared absorption strength of 2-ethoxyethanol (2389/cm² per atmosphere, default), and
  
  
• S_CFC-11 is the infrared absorption strength of CFC-11 (2389/cm² per atmosphere).

These figures suggest that 2-ethoxyethanol does not contribute to stratospheric ozone depletion, its potential contribution to climate change is negligible and its potential contribution to ground-level ozone formation is moderate. The magnitude of these effects would depend on the concentration of 2-ethoxyethanol in the atmosphere, and concentrations of the substance in air in Canada are estimated to be very low. The contribution of 2-ethoxyethanol to ozone formation is therefore considered negligible compared with those of other more abundant smog-forming substances, such as the reference compound, ethene (Bunce, 1996).

2.4.3 Experimental animals and in vitro

In view of the limited objective of this assessment relative to dose-response (i.e., characterization of lowest effect levels only for critical effects), presentation of data on health effects associated with 2-ethoxyethanol is limited to an overview of the nature of the effects, with emphasis on the lowest identified effect levels from repeated-exposure studies relevant to characterization of margins between estimates of population exposure and levels causing toxic effects; detailed descriptions of study protocols and results are included in the supporting documentation.

2.4.3.1 Kinetics and metabolism

2-Ethoxyethanol is rapidly absorbed in humans and experimental animals exposed via ingestion, inhalation or dermal contact. In humans and laboratory animals, 2-ethoxyethanol is oxidized via alcohol dehydrogenases to the intermediate ethoxyacetaldehyde (EALD) and then rapidly converted via aldehyde dehydrogenases to EAA, the principal and putatively active metabolite, which is eliminated primarily in the urine. In rats, EAA may be conjugated with glycine or be O-deethylated and then further metabolized to carbon dioxide. A secondary pathway in rodents involves microsomal P450 mixed-function oxidases, with deethylation producing acetaldehyde and ethylene glycol.

Although little information is available regarding interspecies differences in the toxicokinetics and metabolism of 2-ethoxyethanol, there is some indication that humans may absorb the substance to a greater extent than do rats (the species most extensively investigated) (Groeseneken et al., 1986, 1987). In addition, although relevant data are very limited, 2-ethoxyethanol may be converted to EAA at a greater rate in humans than in rats, with subsequent renal clearance of the metabolite being slower in humans (Groeseneken et al., 1988); thus, the putatively active metabolite may be present in the blood of humans at higher levels and for longer durations than in rats.

The acetate moiety of 2-ethoxyethanol, 2-ethoxyethyl acetate, which is commonly used in occupational and residential environments, is rapidly hydrolysed to 2-ethoxyethanol via esterases in several tissues in the body (Stott and McKenna, 1985). (For this reason, data on the toxicity of 2-ethoxyethyl acetate have been included in this assessment.)

2.4.3.2 Acute toxicity

2-Ethoxyethanol is of low to moderate acute toxicity in experimental animals following oral exposure, with reported LD₅₀s in various species ranging from 1400 to 5490 mg/kg-bw (Laug et al., 1939; Smyth et al., 1941; Carpenter, 1947; Carpenter et al., 1956; Stenger et al., 1971; Truhaut et al., 1979; Krasavage and Terhaar, 1981; Dow Chemical Company, unpublished data, cited in Clayton and Clayton, 1982; Cheever et al., 1984). However, it is considered to be of low toxicity only following inhalation or dermal exposure, with LC₅₀s (7 or 8 hours) in rats and mice of 1500-2000 ppm (5520-7360 mg/m³) (Werner et al., 1943a; Pozzani et al., 1959; Shell, unpublished data, cited in Tyl et al., 1988) and dermal LD₅₀s of 3314-3930 mg/kg-bw (covered application for 24 hours) in rabbits (Carpenter et al., 1956; Krasavage and Terhaar, 1981; Daughtrey et al., 1984). Target sites of 2-ethoxyethanol-induced acute toxicity include the hematopoietic system, liver, kidneys and stomach. 2-Ethoxyethanol and its acetate did not induce skin sensitization and have only low potential for irritation of skin and eyes (Werner et al., 1943b; Carpenter and Smyth, 1946; Truhaut et al., 1979; Krasavage and Terhaar, 1981; Barbee et al., 1984; Daughtrey et al., 1984; Kennah et al., 1989; Zissu, 1995).

2.4.3.3 Short-term toxicity

2.4.3.3.1 Oral

Based on the few short-term oral studies available, the testes appear to be sensitive target organs in rats, mice and dogs, with histopathological effects (degeneration or atrophy) and/or decreased weights being observed in rats, mice and dogs following repeated doses of 2-ethoxyethanol as low as 919 mg/kg-bw per day (in drinking water), 1000 mg/kg-bw per day (by gavage) and 186 mg/kg-bw per day (in capsules), respectively (Stenger et al., 1971; Nagano et al., 1979, 1984; NTP, 1993). 2-Ethoxyethyl acetate induced similar effects in the testes at gavage doses of 1000 mg/kg-bw per day or more in mice (Nagano et al., 1979, 1984). Reductions in relative thymus weights were also observed in rats administered 357 mg/kg-bw per day or more in drinking water (i.e., doses lower than those that induced testicular effects); no effects on the thymus were observed in mice exposed to much higher doses (NTP, 1993). Hematological effects, consisting of reduced white blood cell counts and packed cell volume, were also observed in mice exposed to 2000 mg/kg-bw per day or more of 2-ethoxyethanol or the acetate by gavage (Nagano et al., 1979, 1984). In the only other short-term oral study in which hematological parameters were investigated, there were slight dose-related decreases in hemoglobin and hematocrit levels in dogs administered 50-200 µL/kg-bw per day (46-186 mg/kg-bw per day) of 2-ethoxyethanol in gelatin capsules for 13 weeks (significance not reported) (Stenger et al., 1971).

2.4.3.3.2 Inhalation

Available data on the toxicity of 2-ethoxyethanol or 2-ethoxyethyl acetate following short-term exposure via inhalation are limited to studies designed primarily to investigate developmental toxicity and two early limited studies in small groups of dogs. Doe (1984) reported changes in red blood cell parameters in pregnant rats exposed to 250 ppm (920 mg/m³) 2-ethoxyethanol for 10 days, while no effects on the blood were observed at 50 ppm (184 mg/m³). Similarly, alterations in hematological parameters (red blood cells, white blood cells and platelets) were observed in pregnant rats exposed to 100 ppm or more of 2-ethoxyethyl acetate (equivalent to 368 mg 2-ethoxyethanol/m³) (Tyl et al., 1988). In studies in rabbits, no effects on the blood were noted in pregnant females exposed to up to 175 ppm (644 mg/m³) 2-ethoxyethanol, while a reduction in hemoglobin concentration was observed only following exposure to the highest concentration of 2-ethoxyethyl acetate tested (i.e., 400 ppm, equivalent to 1473 mg 2-ethoxyethanol/m³) (Doe, 1984).

Alterations in hematological parameters characteristic of anemia (i.e., effects on red blood cells) and an increase in calcium oxalate crystals in the urine were observed in dogs exposed to 840 ppm (3091 mg/m³) 2-ethoxyethanol vapour for 12 weeks (Werner, 1943b), although no such effects were noted in dogs exposed to 600 ppm 2-ethoxyethyl acetate vapour (equivalent to 2210 mg 2-ethoxyethanol/m³) for 6 months (Carpenter, 1947). No histopathological changes were observed in the limited range of organs examined in either study.

2.4.3.4 Subchronic toxicity

2.4.3.4.1 Oral

In the identified subchronic studies in which rats were administered 2-ethoxyethanol by the oral route, the critical targets were the male reproductive organs and the blood. Testicular degeneration was observed in the testes of male F344/N rats administered 2-ethoxyethanol in drinking water for 13 weeks at concentrations equivalent to doses of 400 mg/kg-bw per day or more, while atrophy of the prostate gland was observed at doses of 205 mg/kg-bw per day or more; the severity of these lesions increased with dose. Concentrations of spermatogonia and sperm were also significantly lower in rats administered 205 mg/kg-bw per day or more of 2-ethoxyethanol. In males, signs of mild anemia (reduced red blood cell count and hemoglobin concentration), which was characterized as macrocytic (increased mean cell volume), hypochromic (decreased mean cell hemoglobin concentration) and poorly regenerative, were observed at 792 mg/kg-bw per day and above as early as 1 week after initiation of exposure. The severity of the anemia increased with duration of exposure and was described as marked to moderate after 3 and 13 weeks. Mild thrombocytopenia and leukopenia were also present in males exposed to 400 mg/kg-bw per day or more after 1 week of exposure; however, the thrombocytopenia appeared to be reversible, based on the lack of significant reduction in platelet count, while leukopenia was judged to be moderate after 13 weeks. Female rats also exhibited mild anemia (again characterized as macrocytic, hypochromic and poorly regenerative) after 1 week of exposure to 2-ethoxyethanol, with some parameters being affected at doses as low as 247 mg/kg-bw per day, as well as moderate to marked thrombocytopenia and moderate leukopenia. After 3 and 13 weeks of exposure, the severity of the anemia was considered to have progressed to moderate and was accompanied by moderate thrombocytopenia and marked leukopenia, which progressed to marked leukocytosis. Increased hematopoiesis and hemosiderin pigmentation of the spleen and liver were also noted, but were considered secondary to hematological effects. Alterations in clinical chemistry parameters indicative of general toxicity or liver dysfunction were noted in males and females at ≥205 and ≥466 mg/kg-bw per day, respectively. Based on effects on the thymus, testes, prostate gland and blood, the authors (NTP, 1993) considered the Lowest-Observed-Adverse-Effect Level (LOAEL) in male rats to be 205 mg/kg-bw per day (with a No-Observed-Adverse-Effect Level [NOAEL] of 109 mg/kg-bw per day). The authors considered the NOAEL in female rats to be 466 mg/kg-bw per day; however, in view of the observation of thrombocytopenia at all doses, 122 mg/kg-bw per day could be considered to be the Lowest-Observed-Effect Level (LOEL) in females (although the effect on platelet count appeared to have ameliorated somewhat at this exposure level), with numerous other parameters being significantly different from controls at the next dose.

A similar profile of effects was observed in other subchronic oral studies in different strains of rats. Hematological effects consistent with anemia as well as alterations in white blood cell parameters were observed in rats (strain CR,COBS,CD,BR) administered 900 mg/kg-bw per day of 2-ethoxyethanol by gavage for 6 weeks (Krasavage and Vlaovic, 1982). Reduced hemoglobin and hematocrit were also reported in Wistar rats exposed for 13 weeks to 2-ethoxyethanol by gavage (100 µL/kg-bw per day [93 mg/kg-bw per day] for 59 days followed by exposure to 400 µL/kg-bw per day [372 mg/kg-bw per day] for 30 days) (Stenger et al., 1971). Hemosiderin pigmentation was noted in the spleen of both strains of rats, with the lowest effect level being 186 mg/kg-bw per day in Wistar rats. Effects on male reproductive organs (including reduced testicular weights, atrophy and degeneration) and on sperm parameters (degenerated spermatozoa and hypospermia) were also observed in these strains of rats at doses of 450 mg/kg-bw per day (the lowest dose tested) and above for 6 weeks (Krasavage and Vlaovic, 1982) or 200 µL/kg-bw per day (186 mg/kg-bw per day) and above for 13 weeks, but not at 100 µL/kg-bw per day (93 mg/kg-bw per day) (considered to be the No-Observed-Effect Level [NOEL]) (Stenger et al., 1971). Histopathological changes in the stomach and bone marrow were also noted at 450 mg/kg-bw per day or higher (Krasavage and Vlaovic, 1982).

Data on the effects in mice following subchronic oral exposure to 2-ethoxyethanol are limited to a single study in which B6C3F₁ mice were exposed via drinking water for 13 weeks (NTP, 1993). Based on the results of this study, mice appear to be less sensitive than rats to 2-ethoxyethanol-induced toxic effects. As in rats, the male reproductive system was a target organ in mice, with effects on weight and histopathology of testes observed at 5123 mg/kg-bw per day and above and 7284 mg/kg-bw per day, respectively, while effects on sperm parameters were noted at 5123 mg/kg-bw per day or more. In addition, effects on the estrous cycle were observed in females exposed to 1304 mg/kg-bw per day and above. Although hematological parameters were not examined in mice, hematopoiesis of the spleen was noted at the highest dose in males and at 7255 mg/kg-bw per day and above in females. The incidence of a rather rare lesion, hypertrophy of the X-zone of the adrenal gland, resulting from marked lipid vacuolization, was significantly increased in female mice administered 2725 mg/kg-bw per day or more (and non-significantly increased at 1304 mg/kg-bw per day); this lesion was not observed in any of the subchronic oral studies in rats. Based on this study, the LOELs in male and female mice are considered to be 5123 and 1304 mg/kg-bw per day, with NOELs of 2003 and 722 mg/kg-bw per day, respectively.

2.4.3.4.2 Inhalation

Identified information on the subchronic toxicity of inhaled 2-ethoxyethanol and its acetate is limited to earlier studies in rats and rabbits. Exposure to 25 ppm (92 mg/m³) 2-ethoxyethanol or more for 13 weeks was irritating to the eyes and nose of Sprague-Dawley rats. However, no exposure-related lesions were observed in the extensive range of tissues examined at the highest concentration of 400 ppm (1472 mg/m³) (other exposure groups were not examined), and the only systemic effects noted were reductions in relative weights of the pituitary gland in males and the spleen in females exposed to 400 ppm (1472 mg/m³) and alterations in leukocyte count and blood urea nitrogen in female rats at the highest concentration (Barbee et al., 1984). In male and female Wistar rats exposed to 200 ppm 2-ethoxyethyl acetate (approximately equivalent to 737 mg 2-ethoxyethanol/m³) for 10 months, no hematological effects were noted, and the only histopathological change observed was renal tubular nephritis in males, although only a limited range of tissues was examined (Truhaut et al., 1979).

Exposure to airborne 2-ethoxyethanol (≥25 ppm [92 mg/m³]) was also irritating to the eyes and nose of rabbits. Reduced weight and degeneration of the testes were observed at 400 ppm (1472 mg/m³) (histopathological examinations do not appear to have been conducted in animals exposed to lower concentrations), while anemia was present in both sexes at this concentration (Barbee et al., 1984). As in rats, exposure to 200 ppm 2-ethoxyethyl acetate (equivalent to 737 mg 2-ethoxyethanol/m³) via inhalation resulted in renal tubular nephritis in males; no effects on reproductive organs or blood parameters were reported (Truhaut et al., 1979).

2.4.3.5 Chronic toxicity and carcinogenicity

Although a final version of the only relevant chronic study identified was never published (data analyses were never completed due to problems encountered with the laboratory conducting the study; Eastin, 2000), according to an early account of preliminary results, the testes were the principal target in both rats and mice orally exposed to 2-ethoxyethanol for 2 years (Melnick, 1984).

2.4.3.6 Genotoxicity

The available information on the genotoxicity of 2-ethoxyethanol suggests that 2-ethoxyethanol may have some weak potential, at most, to induce cytogenetic damage, but there is no evidence that it induces mutations. Neither 2-ethoxyethanol nor its acetate was mutagenic in several in vitro assays in Salmonella (Ong, 1980; Shimizu et al., 1985; Zeiger et al., 1985; Guzzie et al., 1986; Slesinski et al., 1988; Hüls AG, 1989; Hoflack et al., 1995) or in a limited number of studies in cultured mammalian cells (Guzzie et al., 1986; Myhr et al., 1986; Slesinski et al., 1988). Mixed or equivocal results have been reported for the induction of chromosomal aberrations, micronuclei or sister chromatid exchange by 2-ethoxyethanol or 2-ethoxyethyl acetate in various mammalian cell lines (Guzzie et al., 1986; Galloway et al., 1987; Slesinski et al., 1988; Villalobos-Pietrini et al., 1989; Elias et al., 1996). 2-Ethoxyethanol did not induce morphological transformation or aneuploidy in vitro, although it did show weak potential to interfere with mitotic division (Elias et al., 1996). While neither of the two principal metabolites of 2-ethoxyethanol, EALD and EAA, was mutagenic in Salmonella (Hoflack et al., 1995), the acetaldehyde consistently tested positive for numerous cytogenetic endpoints in vitro, although results for the acetic acid metabolite were negative or equivocal (Elias et al., 1996).

In the limited in vivo database, there was no evidence of the induction of micronuclei in the bone marrow of mice exposed to 2-ethoxyethanol, 2-ethoxyethyl acetate or EAA (Guzzie et al., 1986; Slesinski et al., 1988; Elias et al., 1996).

2.4.3.7 Developmental toxicity

2.4.3.7.1 Oral

Although only limited information is available on the developmental effects of 2-ethoxyethanol following oral exposure, adverse effects, including increased implantation loss, resorptions and embryo mortality, decreased fetal body weight and various skeletal and cardiovascular abnormalities, were observed in multiple strains of rats, often in the absence of maternal toxicity (Stenger et al., 1971; Goad and Cranmer, 1984; Chester et al., 1986). In only one of the three limited accounts could a NOEL be determined (NOEL = 47 mg/kg-bw per day; LOEL = 94 mg/kg-bw per day) (Stenger et al., 1971). Similar developmental effects were observed at doses lower than those that were maternally toxic in the only identified relevant study in mice; severe malformations (e.g., exencephaly) were observed at higher doses (Wier et al., 1987). Although the doses investigated in mice were higher than those in rats, mice appear to be less sensitive than rats to the developmental toxicity of ingested 2-ethoxyethanol, as only reduced fetal body weight was observed at the lowest dose tested (i.e., 1000 mg/kg-bw per day), whereas increased abnormalities were noted in rats at much lower doses.

2.4.3.7.2 Inhalation

The developmental toxicity of inhaled 2-ethoxyethanol and its acetate has been investigated in rats and rabbits. In many of these studies, fetotoxic effects were observed in multiple strains at concentrations lower than those causing maternal toxicity. In Wistar-derived Alpk/AP rats, the lowest concentration of 2-ethoxyethanol reported to induce developmental effects (skeletal variations) in the absence of maternal toxicity was 50 ppm (184 mg/m³), with a NOEL of 10 ppm (37 mg/m³) (Doe, 1984). In Sprague-Dawley and Fischer 344 rats, exposure to 2-ethoxyethyl acetate during gestation also resulted in increased incidences of skeletal variations at the lowest concentrations tested (130 and 50 ppm, respectively, equivalent to 479 and 184 mg 2-ethoxyethanol/m³) (Nelson et al., 1984; Tyl et al., 1988).

Exposure to 2-ethoxyethanol during pregnancy also induced neurological effects in the developing young, based on behavioural differences, consistent with decreased neuromotor function, and alterations in levels of several neurochemicals (particularly in the cerebrum) observed in Sprague-Dawley rats exposed to 100 ppm (368 mg/m³; the lowest concentration tested) and above (Nelson et al., 1981, 1982a,b).

In Dutch rabbits, Tinston (1983a) observed reduced mean number of implantations and number of live fetuses at 50 ppm (184 mg/m³) 2-ethoxyethanol (the lowest concentration investigated) or more, in the absence of maternal effects. Conversely, Doe (1984) reported no clear effects on these endpoints at concentrations up to 175 ppm (644 mg/m³); however, there were increased incidences of skeletal defects and variations at this exposure level, but not at lower concentrations (10 or 50 ppm [37 or 184 mg/m³]). Developmental effects (increased malformations, anomalies and skeletal variations) were also observed in fetuses of New Zealand white rabbits exposed to 160 ppm (589 mg/m³) 2-ethoxyethanol (the lowest concentration tested) during gestation; slight maternal toxicity was also present at this exposure level. 2-Ethoxyethyl acetate was also developmentally toxic in both these strains of rabbits, with a LOEL of 100 ppm (equivalent to 368 mg 2-ethoxyethanol/m³), although no effects were noted at lower concentrations (25 or 50 ppm as the acetate, equivalent to 92 or 184 mg 2-ethoxyethanol/m³) (Tinston, 1983b; Doe, 1984; Tyl et al., 1988).

2.4.3.7.3 Dermal

Dermally applied 2-ethoxyethanol or its acetate induced developmental effects, including increased resorptions, reduced number of live fetuses per litter, decreased fetal body weights and increased incidence of visceral malformations (predominantly of the cardiovascular system) and skeletal variants, in Sprague-Dawley rats at all doses tested (i.e., ≥4000 mg/kg-bw per day, a dose that was not or only slightly maternally toxic) (Hardin et al., 1982, 1984).

2.4.3.8 Reproductive toxicity

2.4.3.8.1 Oral

The majority of the relevant studies identified have been conducted by the oral route in male rats or mice. Ingested 2-ethoxyethanol, as well as the acetate moiety and the acetic acid metabolite, consistently induced effects on male reproductive organs or sperm parameters in multiple strains of both species. Testicular and epididymal weights were reduced in Long-Evans, Sprague-Dawley, F344/N and CR,COBS,CD,BR rats administered doses of 200 mg/kg-bw per day or more by gavage in water or olive oil or in the drinking water for 4 weeks or longer (Krasavage and Vlaovic, 1982; Oudiz and Zenick, 1986; NTP, 1993; Chung et al., 1999), but not in Long-Evans rats exposed to 150 mg/kg-bw per day by gavage in water for 6 weeks (Hurtt and Zenick, 1986) or Sprague-Dawley rats administered 250 mg/kg-bw per day by gavage in water for 11 days (Foster et al., 1983) (although these effects were noted at greater doses in rats exposed for these durations). Histopathological effects on the testes and spermatocytes were noted following oral exposure to 450 mg/kg-bw per day (the lowest dose tested) or more for 6 weeks (Krasavage and Vlaovic, 1982). Reductions in testicular or epididymal sperm counts or alterations in sperm motility or morphology were noted at doses as low as 150 mg/kg-bw per day (the lowest dose tested) when administered for 6 weeks or longer, with regularly mated males being more sensitive to these effects than non-mated rats (Hurtt and Zenick, 1986). Sperm counts were not assessed in the only study in which lower doses were investigated (i.e., Chung et al., 1999) or in a shorter-term study (11 days) in rats administered 250 mg/kg-bw per day (Foster et al., 1983), although spermatocyte degeneration was observed in the latter study only at 500 mg/kg-bw per day or more. Repeated oral administration of EAA, the predominant metabolite of 2-ethoxyethanol, induced a similar profile of male reproductive effects in rats (Foster et al., 1983, 1987), suggesting that this metabolite may be, at least in part, responsible for these effects.

Reduction in testicular or epididymal weights or alterations in sperm parameters were also observed in mice orally exposed to 2-ethoxyethanol or 2-ethoxyethyl acetate for 5 weeks or longer (Nagano et al., 1979, 1984; Morrissey et al., 1989; NTP, 1993; Chapin and Sloane, 1997), although this species appears to be less sensitive than rats, as the lowest dose associated with male reproductive effects in mice was 1000 mg/kg-bw per day (with a NOEL of 500 mg/kg-bw per day).

Although not as extensively investigated as in males, exposure to 2-ethoxyethanol in the drinking water for 13 weeks induced effects on the estrous cycle in female rats and mice at doses of 804 and 1304 mg/kg-bw per day or more, respectively, with uterine atrophy occurring in rats at higher doses (NTP, 1993).

Two oral studies were identified in which the effects of exposure to 2-ethoxyethanol, 2-ethoxyethyl acetate or EAA on reproductive ability were assessed in mice. In a continuous breeding study, in which both sexes were exposed in the drinking water, all three substances adversely affected reproductive success (in terms of decreased fertility and reductions in numbers and weights of pups), with the LOEL for 2-ethoxyethanol being approximately 1650 mg/kg-bw per day, while no adverse affects were noted at 850 mg/kg-bw per day. Effects were observed at all doses of the EAA metabolite tested (i.e., ≥300 mg/kg-bw per day). The results of cross-over mating trials indicated that exposure of either sex to 2-ethoxyethanol or its acetate adversely affected reproductive ability, while such effects were noted only when females were exposed to 2-ethoxyacetic acid. However, effects on reproductive organs and sperm or estrous cycle parameters were observed at similar doses for all compounds. Continuous exposure in utero and until mating to 1860 mg/kg-bw per day of 2-ethoxyethyl acetate also induced effects on reproductive success and organs and sperm parameters in males of the second generation; however, the authors indicated that it was unclear if the second generation was more sensitive than the first (Morrissey et al., 1989; Chapin and Sloane, 1997). In a secondary account of a similar continuous-breeding study in mice (Gulati et al., 1985), similar effects on reproductive success were observed at doses of 1800 mg/kg-bw per day or more of 2-ethoxyethyl acetate (which were attributed to exposure of females in a cross-over study) as well as effects on sperm and testes in males; in addition, histopathological changes in the testes were observed in the second generation.

2.4.3.8.2 Inhalation

In subchronic studies in experimental animals, reduced testes weight and degeneration of the seminiferous tubules were noted in rabbits exposed to 400 ppm (1472 mg/m³); however, effects on the testes were not observed in similarly exposed rats (Barbee et al., 1984) or in rats or rabbits exposed to higher concentrations of the acetate (Truhaut et al., 1979).

In the only inhalation study on the effects of 2-ethoxyethanol on reproductive ability identified, no effects on mating behaviour or fertility were observed in female rats exposed to up to 649 ppm (2388 mg/m³) for 3 weeks prior to mating with unexposed males (Andrew and Hardin, 1984).

2.4.3.9 Immunological effects

In the two relevant studies identified, there was no evidence that exposure to 2-ethoxyethanol or its acetate induced adverse effects on the immune system in rats or mice (the highest dose tested was 2400 mg/kg-bw per day for 10 days) (Houchens et al., 1984; Smialowicz et al., 1992).

2.4.4 Humans

Several epidemiological studies, designed to investigate the potential effects on the lymphohematopoietic system or on reproduction and development, have been conducted in populations exposed to 2-ethoxyethanol or its acetate in the occupational environment. However, in most of these studies, many of which involved small populations, workers were also exposed to various other substances in the workplace. Although these studies are limited, effects on the blood and, possibly, reproductive effects in men were observed.

In a recent well-conducted cross-sectional study (Kim et al., 1999), effects on white blood cells, suggestive of bone marrow depression, were observed in a group of 57 painters exposed to 2-ethoxyethyl acetate. White blood cell and granulocyte counts were reduced in an exposure-related manner in both the high- and low-exposure groups of workers (statistically significantly lower in those exposed to mean concentrations of 3.03 ppm 2-ethoxyethyl acetate [approximately equivalent to 11 mg 2-ethoxyethanol/m³], although not considered by the authors to be clinically significantly decreased), while a significantly higher proportion of all exposed painters had leukopenia. These effects remained after controlling for several potentially confounding factors. Bone marrow hypoplasia was noted in the three leukopenic men examined. The authors also noted that mean corpuscular volume was increased in the high-exposure group, which the authors hypothesized may be an early indicator of anemia. An increase in the prevalence of anemia was observed in a group of 94 shipyard painters exposed to similar mean concentrations of 2-ethoxyethanol (2.7 ppm [10 mg/m³]), along with several other substances (Welch and Cullen, 1988). Hemoglobin levels had declined since first employment in these workers, but were not related to duration of exposure. Exposed workers also had a slightly higher prevalence of low polymorphonuclear leukocyte counts. Bone marrow hypoplasia was also observed in a survey of seven printers exposed to 2-ethoxyethanol and other substances (Cullen et al., 1983).

Although only three relevant epidemiological investigations have been identified, reduced sperm production was consistently observed in populations occupationally exposed to mean concentrations of 2-ethoxyethanol of 9.9 or 24 mg/m³ (with maximum levels up to 88 mg/m³), along with other substances (Welch et al., 1988; Ratcliffe et al., 1989; Schrader et al., 1996). In a case-control study of 1019 men with a clinical diagnosis of infertility or reduced fertility, there was a significant association between this diagnosis and the detection of EAA in the urine (odds ratio = 3.11) (Veulemans et al., 1993). There was no consistent evidence of effects on male or female reproductive ability in other investigations of men or women exposed to 2-ethoxyethanol, although most of these studies are limited by the mixed exposures of the study populations and the lack of analyses for associations with 2-ethoxyethanol specifically (Beaumont et al., 1995; Schenker et al., 1995; Swan et al., 1995; Correa et al., 1996; Gray et al., 1996; Ha et al., 1996; Schenker, 1996; Swan and Forest, 1996; Chia et al., 1997).