Ethylene glycol monoethyl ether [MAK Value Documentation, 2008]

[110-80-5]

Supplement 2008

MAK value (2007)	2 ml/m^3 ≙ 7.6 mg/m^3
Peak limitation (2001)	Category II, excursion factor 8
Absorption through the skin (1980)	H
Sensitization	–
Carcinogenicity	–
Prenatal toxicity (1994)	Pregnancy risk group B
Germ cell mutagenicity	–
BAT value (1992)	50 mg ethoxyacetic acid/l urine
Chemical name	2-ethoxyethanol

Documentation of the toxicity of ethylene glycol monoethyl ether (from here onwards referred to as EGEE) began in 1983 (documentation “2-Ethoxyethanol” 1983; only available in German) one supplement on the toxicokinetics and reproductive toxicity of EGEE and EGEE acetate was adopted in 1994 (documentation “2-Ethoxyethanol, 2-Ethoxyethyl acetate” 1998, a translation of the 1994 German); a grouped documentation on limitation of exposure peaks was evaluated in a supplement in 2001 (documentation “Ethylene glycol monoethyl ether” 2001, to be published simultaneously with this document). This supplement presents the studies with EGEE and EGEE acetate that pertain to the relevant end points and have been published since that time. The data must be evaluated together since EGEE acetate rapidly hydrolyzes to EGEE, which forms the critical metabolite ethoxyacetic acid. The MAK value also applies to the sum of the concentrations of the two substances in the air.

A BUA report (BUA 1995), IUCLID data sets (ECB 2000 a, 2000 b), a review of the Cosmetic Ingredient Review Expert Panel (Johnson 2002), an ECETOC report (ECETOC 2005) and other reviews are available on EGEE and EGEE acetate.

Metabolism and Toxicokinetics

Dermal absorption

EGEE is well absorped dermally, and has already been described in the 1994 supplement (documentation “2-Ethoxyethanol, 2-Ethoxyethyl acetate” 1998). The dermal absorption of EGEE in vitro (Barber et al. 1992; Lockley et al. 2002; Wilkinson and Williams 2002) and in vivo (see below) was also established in further studies. Since esterification of the alcohol group caused no reduction in the absorption rate through human skin in vitro (Dugard et al. 1984), a high dermal absorption is also assumed for EGEE acetate.

A mean permeability coefficient of 19 ± 6 cm/h for EGEE vapour and a dermal flux of 0.7 ± 0.3 mg/cm² and hour for liquid EGEE were found in 5 volunteers who had been exposed to vaporous and liquid EGEE, respectively. This confirms that both vaporous and liquid EGEE are readily absorped by the skin. When the whole body was exposed to vapours of EGEE, about 42% of the total uptake of EGEE was estimated to be absorbed via the skin. Dermal absorption resulting from 60-minute contact of both hands and forearms (about 2000 cm²) with liquid EGEE would exceed the intake by inhalation of EGEE after 8-hour exposure to 5 ml/m³ by a factor of 20 (Kezic et al. 1997).

After occlusive dermal application of undiluted ¹⁴C-EGEE to the rat skin, 25% of the applied dose was excreted within 24 hours, 15% being excreted in the urine, 6% being exhaled as CO₂ and 1.2% appearing in the faeces; 1.3% remained in the body. Free EGEE, ethoxyacetic acid, ethoxyacetyl glycine and ethylene glycol were detected in the urine (Lockley et al. 2002).

Metabolism

In vitro studies showed that EGEE is also oxidized to ethoxyacetic acid by the isoenzymes alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) in the rat skin (Lockley et al. 2005).

Biomonitoring

BAT value documentations are available for EGEE and EGEE acetate; a BAT value of 50 mg ethoxyacetic acid/l urine was established in each case (Henschler and Lehnert 1993 a, 1993 b).

The metabolite ethoxyacetic acid is responsible for the toxicity of EGEE and EGEE acetate. Since EGEE and EGEE acetate are readily absorbed through the skin and ethoxyacetic acid accumulates in the body because of its long half-life of 21 to 24 hours (Groeseneken et al. 1986 b), the previously available studies in volunteers and exposed workers sometimes revealed very different correlations between the concentrations of EGEE and EGEE acetate in the air and the excretion of ethoxyacetic acid in the urine (see Table 1). It has meanwhile been possible to calculate a correlation in a PBPK model based on experimental studies. In this compartment-based toxicokinetic model, exposure of workers was simulated for 8 hours per day on 5 days per week until a steady state was reached. Physical activity of 12 hours (8 hours of work and 4 hours of leisure) was assumed followed by a 12-hour phase of rest. A value of 110 µmol ethoxyacetic acid/mmol creatinine (95th percentile) in biological material corresponding to about 120 mg/l urine was calculated for 8-hour exposure to EGEE at 18 mg/m³ (about 5 ml/m³) (Truchon et al. 2006; see Table 1).

Table 1. Studies of a correlation between the concentrations of EGEE and EGEE acetate in the air and the concentration of ethoxyacetic acid in urine

Cohort		Air	Urine	References
		EGEE or EGEE A (ml/m^3)	Ethoxyacetic acid (mg/l)
1) assuming 1.2 g creatinine/l urine Key: EGEE: ethylene glycol monoethyl ether EGEE A: ethylene glycol monoethyl ether acetate; POS: post-shift; PRS: pre-shift
volunteers (n = 5), 4-h exposure	without physical activity	EGEE:  2.7	3.2	Groeseneken et al. 1986 a, b
		EGEE:  5.3	6.0
		EGEE: 10.7	8.7
	30 watts	EGEE:  5.3	11.8
	60 watts	EGEE:  5.3	17.4
volunteers (n = 5), 4-h exposure	without physical activity	EGEE A: 2.6	2.2	Groeseneken et al. 1987 a, 1987 b
		EGEE A: 5.1	4.0
		EGEE A: 9.8	6.5
	30 watts	EGEE A: 5.1	7.3
	60 watts	EGEE A: 5.1	13.7
female workers (n = 5)		EGEE + EGEE A: 5.0	180 ± 42^1)	Veulemans et al. 1987
workers (n = 30)		EGEE A: 12 (2.9–34)	1.3–32^1)	Lowry et al. 1993
workers in the production of varnishes (n = 12)		EGEE: 2.8	128.5 (PRS); 167.8 (POS)	Angerer et al. 1990
		EGEE A: 2.7
worker (formulation; n = 19)	Friday	EGEE: 1.9 EGEE A: 2.2	105.3 (POS)	Angerer et al. 1991
	Monday	EGEE: 2.0 EGEE A: 0.4	37.8 (PRS)
	Tuesday	EGEE: 1.4 EGEE A: 0.1	35.9 (POS)
worker (production; n = 19)	Monday	EGEE: 2.9 EGEE A: 0.5	53.2 (PRS)	Söhnlein et al. 1993
	Tuesday	EGEE: 2.1 EGEE A: 0.1	53.8 (POS)
worker (screen printing; n = 19)		EGEE A: 0.9	8.3	Johanson et al. 1989
PBPK model (exposure 8 hr/d, 5 d/week, 50 watts for 12 h)		EGEE: 5.0	120^1) (95th percentile)	Truchon et al. 2006

Effects in Humans

Allergenic effects

In a patch test, 20 patients with confirmed or suspected contact allergy to cosmetic products were tested with 2% EGEE in petrolatum. No signs of irritation were observed, but individual test conditions were not reported (Johnson 2002).

Haematological effects

Two studies that are only available as abstracts were carried out among workers of two factories in Beijing who had been exposed to high concentrations of EGEE (no other details); impaired sperm parameters (see below) as well as a decrease in the erythrocyte count, haemoglobin, haematocrit and leukocyte count were reported. The liver function was not impaired (Wang et al. 2003, 2004 b).

An increased incidence of haematological effects (anaemia and granulocytopenia) was observed in a group of 94 workers (shipyard painters) who had been exposed to various substances including EGEE and ethylene glycol monomethyl ether at mean concentrations of 2.6 and 0.8 ml/m³, respectively, and maximum concentrations of 21.5 and 5.6 ml/m³, respectively. These effects were not found in the group of 55 controls (other shipyard workers) (Welch and Cullen 1988). Additional dermal absorption is assumed but cannot be assessed since no biomonitoring was mentioned for the measurement of 2-ethoxyacetic acid or 2-methoxyacetic acid concentrations in urine. Therefore, the result of this study can only be used as evidence that ethylene glycol monomethyl ether and EGEE cause haematological effects, but no quantitative assessment can be made.

A group of 32 female workers who had been exposed to EGEE revealed urinary concentrations of ethoxyacetic acid of 120.87 mg/g creatinine (geometric mean; about 144 mg/l urine). In the control group of 20 female workers without exposure to EGEE, the urinary excretion of ethoxyacetic acid was 2.71 mg/g creatinine (3.2 mg/l urine). Average erythrocyte counts and haemoglobin levels were normal in both groups. However, 2 women in the exposed group had erythrocyte counts and haemoglobin concentrations that were somewhat lower than the standard levels (Wang et al. 2004 a). Since there is no information about the level of ethoxyacetic acid exposure of these two women and whether menses had any influence, these data cannot be used for the present assessment.

An increased incidence of haematological effects was also observed in a group of 18 shipyard workers (painters) exposed to high concentrations of a solvent mixture containing a geometric mean of 3.0 ml EGEE acetate/m³ (maximum: 18.3 ml/m³). The leukocyte count was statistically significantly reduced. However, according to the authors, the reduction was not clinically significant. The geometric mean concentrations of ethoxyacetic acid in the urine of these workers were 9.2 mg/g creatinine (11.0 mg/l urine) with a geometric standard deviation of 5.5 mg/g creatinine (6.7 mg/l urine) and a maximum of 227 mg/g creatinine (272 mg/l urine). A high exposure group consisted of both workers who applied paint in spray form and wore respirators and workers who mixed paint for example and wore no respirators. In a group of 12 workers with low exposure, the leukocyte count was slightly, but not statistically significantly reduced. In this group, the concentration of EGEE acetate in the air was 1.8 ml/m³ (geometric mean; maximum: 8.1 ml/m³), the urinary ethoxyacetic acid concentration was 0.6 mg/g creatinine (0.7 mg/l urine) and the highest concentration was maximally 15 mg/g creatinine (18 mg/l urine). Co-exposure included xylene (28 ml/m³; maximum: about 250 ml/m³) and toluene (12 ml/m³; maximum: about 155 ml/m³) (Kim et al. 1999). The maximum urinary ethoxyacetic acid concentrations of 272 mg/l urine among the workers with higher exposure suggest that individual workers were exposed to a relatively high level. However, since it must be assumed that there were also individual workers in the high exposure group who were exposed to a very low level since preventive measures had been taken and since no correlation was specified between individual exposures and haematological effects, this study is not suitable for making quantitative assessments about the level of exposure at which haematological effects occur.

A group of 29 male and female workers who used EGEE acetate as the primary cleaning and printing solvent was examined for haematological effects and compared with 56 controls with low or no exposure. The geometric mean of the concentration of EGEE acetate in the air was 7.41 ml/m³ (range: 1.35–15.5 ml/m³), but it had been 45.51 ml/m³ (workplaces of the male workers) and 38.82 ml/m³ (workplaces of the female workers) 4 months before the study. The current mean exposure of the 12 female workers at the manual printing machines was significantly higher (geometric mean: 9.34 ml/m³) and longer (8 h/d) than that of the 17 male workers (geometric mean: 4.87 ml/m³; 2.4 h/d) at the automatic printing machines. Haemoglobin and haematocrit levels of the female workers were significantly lower than those in the control group, and there was a concentration-response relationship. No difference was found between the male workers and the control group (Loh et al. 2003). Additional dermal absorption must be assumed for the female workers since they wore no protective gloves; respirators were provided for the male workers. Since no biomonitoring was carried out, no quantitative assessment can be made.

Fertility

There are several studies among workers in the semiconductor industry who were exposed to ethylene-based glycol ethers and their acetates (ethylene glycol monomethyl ether and ethylene glycol monomethyl ether acetate; EGEE and EGEE acetate) and to propylene-based glycol ethers, xylene and n-butyl acetate (Gray et al. 1996; Ha et al. 1996; Lamm et al. 1996; Schenker 1996; Schenker et al. 1995; Swan et al. 1995). Since all studies assessed the exposure to ethylene-based glycol ethers together, no distinction can be made from exposure to ethylene glycol monomethyl ether and ethylene glycol monomethyl ether acetate, whose developmental toxicity (teratogenicity) and adverse effect on fertility were more pronounced in animal studies than those of EGEE and EGEE acetate (Nelson et al. 1984). Nor were any concentrations of glycol ethers in the air or biomonitoring studies mentioned. Therefore, these studies cannot be used for the present assessment.

Male fertility

Evidence of a spermatotoxic effect was provided for EGEE and EGEE acetate among exposed workers.

Exposure of 73 workers employed as painters in a shipyard included EGEE (mean: 2.6 ml/m³; median: 1.2 ml/m³; maximum: 21.5 ml/m³) and ethylene glycol monomethyl ether (mean: 0.8 ml/m³; median: 0.44 ml/m³). The results of a sperm analysis indicated that exposure to glycol ethers had an effect on the sperm count. Although the mean for the sperm count/ejaculation (158 × 10⁶/ml) was not significantly different for the exposed group from that of the control group (211 × 10⁶/ml), according to the authors, there were biologically important differences in the incidence of oligospermia (13.5%; control: 5%) and azoospermia (5%; normal population: 1%) (Welch et al. 1988; see documentation “2-Ethoxyethanol, 2-Ethoxyethyl acetate” 1998, a translation of the 1994 German). Additional dermal absorption is assumed but cannot be assessed since no biomonitoring was mentioned for the measurement of ethoxyacetic acid or methoxyacetic acid concentrations in the urine. No quantitative assessment can therefore be made as to which exposures lead to changes in sperm parameters.

In a cross-sectional study, which was used for deriving the MAK value in 1994, the average sperm count per ejaculate was statistically significantly lower among 37 workers exposed to EGEE than that of 38 controls after consideration of confounders such as abstinence, sample age, subjects' age, tobacco consumption, alcohol and caffeine use, urogenital disorders, fever and other illnesses. No significant differences in semen volume and concentration, semen pH, viability, motility and velocity, semen morphology or testicular volume were observed. The proportion of men with oligozoospermia was higher in the exposed group (16.2%) than in the control group (10.5%), but this difference was not statistically significant. The currently measured exposure concentrations of EGEE ranged from “not detectable” to 24 ml/m³ with a geometric mean of 6.6 ml/m³. However, exposure to EGEE had been reduced in 2 of 3 buildings 2 to 3 weeks before the study: There was a reduction from 16.9 to 3.0 ml/m³ in building A; the airborne levels of 10.7 and 14.9 ml/m³ remained the same in building B, and in building C there was a reduction from about 16.9 to 2.4 ml/m³ as in building A. Measurements of the urinary metabolite ethoxyacetic acid during the study yielded concentrations between “not detectable” and 163 mg/g creatinine (about 196 mg/l urine assuming 1.2 g creatinine/l urine). In a subgroup of 10 workers with relatively high current urinary ethoxyacetic acid concentrations of 85 ± 31.3 mg/g creatinine (about 100 mg/l urine assuming 1.2 g creatinine/l urine), a regression analysis revealed no differences in the semen parameters of workers with lower current exposure as compared with non-exposed workers (Ratcliffe et al. 1989). The number of 10 workers in this group may have been too small to detect effects. It is also problematical that the workers were divided into exposure or control group based on the urinary ethoxyacetic acid concentrations measured during the study. Effects due to previous exposure could thus not be ruled out in workers of the control group. Possible differences might thus have been less marked. Since the reduction in exposure in two of the three buildings occurred within the period of an average spermatogenic cycle of 70 days, effects on the sperm would still have been observed at the time of the study irrespective of the currently measured exposure concentrations in the air or urine. Because of the limitations of this study, effects at 85 ± 31.3 mg ethoxyacetic acid/g creatinine (about 100 mg/l urine) cannot be ruled out. In spite of its limitations, the study shows that effects on sperm parameters result at EGEE concentrations of up to about 17 ml/m³.

On account of the small number of exposed workers in the two studies described above (Ratcliffe et al. 1989; Welch et al. 1988) and their resulting lower statistical validity, the two studies were evaluated together. The assessment revealed significant effects on sperm concentration and semen volume (Schrader et al. 1996). However, no quantitative statements were made about effect levels of EGEE concentrations in the air or ethoxyacetic acid concentrations in the urine.

In a case-control study, 1019 patients with reproductive disorders who were diagnosed infertile or subfertile on the basis of their spermiograms (“cases”) were compared with 475 patients with reproductive disorders who were assessed as normally fertile on the basis of their spermiograms (“controls”). Ethoxyacetic acid was measured in the urine of 39 of the 1019 “cases” (3.8%) at a significantly higher rate than in the urine of 6 of the 475 “controls” (1.3%) at concentrations of 1.3 to 71 mg/l. Methoxyacetic acid was found in only one “case” and two “controls”. Oligozoospermia was found in 43 of the 45 persons with evidence of ethoxyacetic acid in the urine (“cases” and “controls”); 11 of them revealed azoospermia. There was no significant correlation between the concentrations of ethoxyacetic acid determined in the urine and various sperm quality parameters. In the opinion of the authors, the lack of such a correlation could be explained by the expected latency between exposure and the occurrence of observable effects. In addition, the frequency and duration of exposure and the period between last exposure and measurement of the ethoxyacetic acid concentration in the urine was inadequately characterized (Veulemans et al. 1993; see (documentation “2-Ethoxyethanol, 2-Ethoxyethyl acetate” 1998, a translation of the 1994 German).

In two studies, which were however only published as abstracts, significantly reduced sperm counts were observed among workers of two factories in Beijing who had been exposed to high concentrations of EGEE (no other details). Progressive motility and the proportion of sperm with normal morphology were significantly lower than in the control group. Haematological parameters were also decreased (see “Haematological effects”). There were no differences in sex hormones in the blood, luteinizing hormone, follicle-stimulating hormone, prolactin or oestradiol (Wang et al. 2003, 2004 b).

Female fertility

Various studies are available on the fertility of women who were employed in the semiconductor industry or in chip production. Increased relative risks of spontaneous abortions and lower fertility were reported (Chen et al. 2002; Correa et al. 1996; Gray et al. 1996; Hsieh et al. 2005; Lamm et al. 1996; Schenker 1996; Schenker et al. 1995; Swan and Forest 1996; Swan et al. 1995). These studies cannot be used for the present assessment because, as mentioned before, ethylene-based glycol ethers were assessed together in these studies, no distinction was made between ethylene glycol monomethyl ether and ethylene glycol monomethyl ether acetate and the glycol ether concentrations were not measured in the air, nor was biomonitoring performed.

The menstrual cycles of 52 female workers in the LCD manufacturing industry were compared with those of 55 female workers from other areas of the factory. No significant differences were observed with regard to the period of the menstrual cycle, the duration of the menses and the amount of flow even after adjustment. However, at 0.51 ml/m³ (geometric mean), the exposure concentration of EGEE acetate was relatively low. The urinary excretion of ethoxyacetic acid was 0.12 mg/g creatinine (geometric mean) at the start of shift and 0.16 mg/g creatinine at the end of shift, and therefore also very low (Chia et al. 1997).

Developmental toxicity

Although several studies were carried out to examine the effect of occupational exposure to glycol ethers on the occurrence of congenital malformations, none of them allow conclusions to be drawn about a possible developmental toxicity of EGEE since exposure to individual glycol ethers was not recorded.

In a Europe-wide case-control study, a total of 984 cases of one or several major congenital malformations were recorded between 1989 and 1992 including 222 induced abortions, 42 stillbirths and 720 children who were found to have malformations during their first week of life. One or two controls (live offspring without malformations from the same hospital) were selected for each case. An overall association was observed between exposure to glycol ethers (in general) and all malformations (OR: 1.4; 95% CI: 1.1–1.9). There was a particular association between exposure to glycol ethers and neural tube defects (OR: 1.94; 95% CI: 1.16–3.24), multiple anomalies (OR: 2.00; 95% CI: 1.24–3.23) and cleft lips (OR: 2.03; 95% CI: 1.11–3.73). The OR for cleft lips increased with the level of exposure (Cordier et al. 1997; Ha et al. 1996).

The same working group carried out a case-control study with a similar design in the Slovak Republic. The study comprised 107 cases (live or stillborn children and therapeutic abortions) with severe malformations and 131 children without malformations as controls. After exposure to glycol ethers, the adjusted OR of 2.3 (95% CI: 0.7–7.0) was increased, although not significantly, for all congenital malformations (Cordier et al. 2001).

In a case-control study comprising 538 children with special malformations (anencephaly, spina bifida, craniorachischisis and iniencephaly) and 539 controls, the mothers were exposed to 74 chemicals including glycol ethers during pregnancy. Maternal exposure to glycol ethers showed no association with the malformations (Shaw et al. 1999).

PBPK models

Physiologically based pharmacokinetic (PBPK) models were developed for EGEE and EGEE acetate for humans and for pregnant rats. PBPK models of EGEE acetate (Gargas et al. 2000; Hays et al. 1999), data of a study on the developmental toxicity of EGEE in rats (Doe 1984; NOAEC: 50 ml/m³) and data from a volunteer study with 4-hour exposure to EGEE or EGEE acetate (Groeseneken et al. 1986 a, 1986 b, 1987 a, 1987 b) were used for this purpose. The models considered five compartments, rapid hydrolysis of EGEE acetate to EGEE, metabolization of EGEE to ethoxyacetic acid and urinary excretion of ethoxyacetic acid. Based on the NOAEC of 50 ml/m³ for developmental toxicity in rats, the physiological parameters of the rat were compared with those of a pregnant woman and a NAEC was calculated from these for humans. NAECs of 25 ml/m³ were derived for both substances. The authors suggested an 8-hour limit value of 2 ml/m³, taking into account safety factors (2.5 for interspecies extrapolation, 3.16 for inter-individual variability and 1.8 for pharmacokinetic intra-species differences) (Sweeney et al. 2001).

Genotoxicity

Two groups of 19 workers exposed to EGEE (2.9 ml/m³), EGEE acetate (0.5 ml/m³) and ethylene glycol monobutyl ether (0.5 ml/m³) had urinary ethoxyacetic acid and butoxyacetic acid concentrations of 53.2 and 0.2 mg/1 urineon Monday before the shift and respectively, of 53.8 and 16.4 mg/1 urine on Tuesday after the shift. The numbers of SCEs and micronuclei were not increased in lymphocytes that had been obtained from blood samples collected on Tuesday after the shift (Söhnlein et al. 1993).

Animal Experiments and in vitro Studies

Subacute, subchronic and chronic toxicity

Haematopoietic and lymphatic organs, the testicular seminiferous epithelium, kidneys and liver are the main target organs of the toxicity of EGEE (BUA 1995). The results of relevant studies on the inhalation and ingestion of EGEE and EGEE acetate are summarized in Table 2 and Table 3, respectively.

Inhalation

In 13-week inhalation studies carried out with EGEE using a sufficient scope of examination (body weight gain, haematological and clinicochemical parameters and ophthalmological and histopathological examinations), no alterations were observed in rats up to 400 ml/m³ (NOAEC). Changes in the blood count and in the testes were observed in rabbits at 400 ml/m³; the NOAEC for EGEE is 100 ml/m³ (Barbee et al. 1984; Table 2).

Table 2. Studies with repeated inhalation and ingestion of EGEE in rats, mice, rabbits and dogs

Species, strain, No. of animals per group	Exposure	Findings	References
inhalation
rat, Sprague Dawley, 15 ♂ and 15 ♀	13 weeks, 0, 25, 100, 400 ml/m^3, 6 h/d, 5 d/week	400 ml/m^3 : NOAEC	Barbee et al. 1984
rabbit, white New Zealand, 10 ♂ and 10 ♀	13 weeks, 0, 25, 100, 400 ml/m^3, 6 h/d, 5 d/week	100 ml/m^3 : NOAEC 400 ml/m^3 : b.w. gain ; haemoglobin, haematocrit and erythrocyte count ; testicular changes	Barbee et al. 1984
ingestion
rat, F344, 30 ♂	13 weeks, 0, 109, 205, 400, 792, 2240 mg/kg b.w. and d, drinking water	109 mg/kg b.w.: NOAEL at 205 mg/kg b.w and above.: b.w. gain ; thrombocytopenia; absol. and rel. thymus weights ; atrophy of prostate at 400 mg/kg b.w. and above: testicular atrophy; haematopoiesis in the spleen; anaemia; total protein at 792 mg/kg b.w. and above: leukopenia; leukocytosis; thymic atrophy; haemosiderin deposits and haematopoiesis in the liver; bone marrow hyperplasia; albumin concentration ; rel. and absol. testis weights , epididymis weight ; aspermia 2240 mg/kg b.w.: mortality (5/10); haemosiderin deposits in the spleen	NTP 1993
rat, F344, 30 ♀	13 weeks, 0, 122, 247, 466, 804, 2061 mg/kg b.w. and d, drinking water	466 mg/kg b.w.: NOAEL at 804 mg/kg b.w. and above: b.w. gain ; absol. and rel. thymus weights ; leukopenia; leukocytosis; thymic atrophy; haemosiderin deposits in the liver; albumin concentration ; length of oestrous cycle 2061 mg/kg b.w.: mortality (7/10); haemosiderin deposits in the spleen	NTP 1993
rat, 10; no other details	13 weeks, 52–1890 mg/kg b.w. and d, drinking water	210 mg/kg b.w.: NOAEL at 740 mg/kg b.w. and above: b.w. gain ; water consumption ; changes of liver and kidney weights; histopathological changes in the liver, kidney, spleen or testes (no other details) 1890 mg/kg b.w.: mortality	Smyth et al. 1951
rat, Wistar, 5 ♂ and 5 ♀	13 weeks, 0, 46, 93, 186 mg/kg b.w. and d; from day 56. 0, 46, 372, 743 mg/kg b.w. and d; no other details	93 mg/kg b.w.: NOAEL 93/372 mg/kg b.w.: haemoglobin and haematocrit at 186 mg/kg b.w. and above: haemosiderin deposits in the spleen; testicular changes (oedematous and disintegrating interstitium; absence of advanced maturation stages of spermatogonia and spermacytes)	Stenger et al. 1971
rat, F344, 50 ♂ and 50 ♀	103 weeks, 0, 500, 1000, 2000 mg/kg b.w. and d, 5 d/week, gavage	500 mg/kg b.w.: ♂ and ♀: b.w. gain ; incidence of animals with enlarged spleens and pituitary changes ; ♂: testicular atrophy; enlarged adrenal; ♀: subcutaneous tissue in the mamma 1000 mg/kg b.w.: ♂: mortality 2000 mg/kg b.w.: ♂ and ♀: mortality ; gastric ulcerations; exposure terminated after 17–18 weeks; ♂: testicular atrophy (no other details) histopathological examinations of the testes of animals of the high exposure group only	Melnick 1984
mouse, JCL-ICR, 5 ♂	5 weeks, 0, 500, 1000, 2000, 4000 mg/kg b.w. and d, 5 d/week, gavage	500 mg/kg b.w.: NOAEL at 1000 mg/kg b.w. and above: testicular atrophy; absol. and rel. testis weights 2000 mg/kg b.w.: leukocyte count 4000 mg/kg b.w.: 100% mortality	Nagano et al. 1979
mouse, B6C3F1, 10 ♂ and 10 ♀	13 weeks, 0, 587, 971, 2003, 5123, 7284 mg/kg b.w. and d, drinking water	2003 mg/kg b.w.: NOAEL at 5123 mg/kg b.w. and above: b.w. gain , absol. testis weight 7284 mg/kg b.w.: testicular degeneration; haematopoiesis in the spleen	NTP 1993
mouse, B6C3F1, 10 ♀	13 weeks, 0, 722, 1304, 2725, 7255, 11 172 mg/kg b.w. and d, drinking water	1304 mg/kg b.w.: NOAEL at 2725 mg/kg b.w. and above: adrenal hypertrophy 7255 mg/kg b.w.: b.w. gain ; haematopoiesis in the spleen	NTP 1993
mouse, B6C3F1, 50 ♂ and 50 ♀	103 weeks, 0, 500, 1000, 2000 mg/kg b.w. and d, 5 d/week, gavage	500 mg/kg b.w.: ♂ and ♀: b.w. gain 1000 mg/kg b.w.: ♂: testicular atrophy 2000 mg/kg b.w.: ♂ and ♀: mortality ; exposure terminated after 17–18 weeks; ♂: gastric ulcerations; testicular atrophy (no other details) histopathological examinations of the testes of animals of the high exposure group only	Melnick 1984
dog, beagle, 3 ♂ and 3 ♀	13 weeks, 0, 46, 93, 186 mg/kg b.w. and d, 7 d/week, gelatin capsule	46 mg/kg b.w.: NOAEL at 93 mg/kg b.w. and above: haemoglobin and haematocrit 186 mg/kg b.w.: slight kidney changes (3/6); testicular changes (e.g. absence of the advanced maturation stages of the seminiferous epithelium)	Stenger et al. 1971

Inhalation studies carried out with EGEE acetate in rats and rabbits for 10 months revealed nephritis in male rats and slight kidney changes in rabbits at the only tested concentration of 200 ml/m³ (Truhaut et al. 1979; Table 3). No effects were observed in an inhalation study with up to 24-week exposure carried out in dogs at the only investigated concentration of 600 ml/m³ (ECB 2000 b; Table 3).

Ingestion

Male rats proved to be more sensitive to the haematotoxic effect of EGEE than female rats. In a 13-week study, the NOAEL for male and female rats was 109 and 466 mg/kg body weight and day, respectively. Doses of 205 mg/kg body weight and day and above led to thrombocytopenia in male animals, reduced absolute and relative thymus weights and reduced body weight gains; doses at 400 mg/kg body weight and day and above induced testicular atrophy and anaemia. Doses of 792 mg/kg body weight and day in males and 804 mg/kg body weight in females revealed leukopenia, leukocytosis, thymic atrophy, haemosiderin deposits in the liver and a reduced albumin concentration (NTP 1993; Table 2).

In studies with 5-week administration of EGEE or EGEE acetate in mice, a NOAEL of 500 mg/kg body weight and day was obtained for both substances; testicular atrophy was observed from 1000 mg/kg body weight and day (Nagano et al. 1979; Table 2 and Table 3). In 13-week studies with EGEE in mice, a NOAEL of 1304 mg/kg body weight and day and 2003 mg/kg body weight and day was obtained for males and females, respectively, the females thus being more sensitive than males. Adrenal hypertrophy occurred in females from 2725 mg/kg body weight and day, and reductions in body weight gains and absolute testicular weights were observed in males from 5123 mg/kg body weight and day. Doses of more than 7000 mg/kg body weight and day led to increased haematopoiesis in the spleen of males and females and to testicular degeneration in males (NTP 1993; Table 2). In a 103-week study, testicular atrophy was observed in male mice at a concentration of EGEE from as low as 1000 mg/kg body weight (Melnick 1984; Table 2).

Table 3. Studies with repeated inhalation and ingestion of EGEE acetate in rats, rabbits, mice and dogs

Species, strain, No. of animals per group	Exposure	Findings	References
N.A.D. = no abnormality detected
inhalation
rat, Wistar, 10 ♂ and 10 ♀	10 months, 0, 200 ml/m^3, 4 h/d, 5 d/week	200 ml/m^3 : ♂: nephritis with degenerations of the epithelium; ♀: NOAEC N.A.D.: body weight gain, haematology, urinalysis and gross pathology	Truhaut et al. 1979
rabbit, white New Zealand, 10 ♂ and 10 ♀	10 months, 0, 200 ml/m^3, 4 h/d, 5 d/week	200 ml/m^3 : ♂, ♀: slight kidney changes N.A.D.: body weight gain, haematology, urinalysis and gross pathology	Truhaut et al. 1979
dog, no other details	24 weeks, 0, 600 ml/m^3, 7 h/d, 5 d/week	600 ml/m^3 : no effects	ECB 2000 b
ingestion
mouse, JCL-ICR, 5 ♂	5 weeks, 0, 500, 1000, 2000, 4000 mg/kg b.w. and d, 5 d/week, gavage	500 mg/kg b.w.: no effects (NOAEL) at 1000 mg/kg b.w. and above: testicular atrophy; absol. and rel. testis weights 2000 mg/kg b.w.: leukocyte count , reduced number of spermatozoa, spermatids and spermatocytes 4000 mg/kg b.w.: 100% mortality	Nagano et al. 1979

A NOAEL of 46 mg/kg body weight and day for EGEE showed that dogs were the most sensitive species investigated. Haemoglobin and haematocrit were reduced from 93 mg/kg body weight and day, and slight kidney changes were observed in males and females, and testicular changes in males, at 186 mg/kg body weight (Stenger et al. 1971; Table 2).

Local effects on skin and mucous membranes

Skin

The 24-hour occlusive application of 0.5 ml EGEE to the clipped intact dorsal skin of rabbits led to slight irritation (0.6 of 23). In a different study, EGEE was assessed as non-irritating after application of the same concentration of 0.5 ml to the shaved intact dorsal skin of rabbits for 4 hours (no data on occlusion). A further study demonstrated that the open dermal application of 500 mg EGEE (about 0.5 ml) led to slight irritation (BUA 1995).

After occlusive application of EGEE acetate to the intact and abraded rabbit skin, only slight irritation was also observed after 24 hours (1 of 4); after 72 hours, there was hardly any irritation (0.08 of 8). EGEE acetate caused slight irritation in two further studies, whereas the substance was described as non-irritating to the rabbit skin in another test procedure (BUA 1995).

Eyes

The instillation of 0.1 ml EGEE into the conjunctival sac of rabbits revealed moderate irritation (median 20.8 of 110). Weak irritation (maximally 3 of 10) was also observed after instillation of 0.005 to 0.5 ml (BUA 1995).

After instillation of 0.1 ml of a 30% solution and of undiluted EGEE acetate into the conjunctival sac of rabbits, slight irritation (3 and 15 of 110, respectively) was recorded 24 hours after application. EGEE acetate was assessed as non-irritating in two other Draize tests in rabbits (BUA 1995).

Allergenic effects

EGEE showed no sensitizing potential in a maximization test carried out according to Magnusson and Kligman (BUA 1995).

In a further maximization test carried out according to Magnusson and Kligman in a total of 30 Dunkin-Hartley guinea pigs, EGEE and EGEE acetate revealed no sensitization either (Johnson 2002).

Reproductive toxicity

Fertility

The toxic effect of EGEE and EGEE acetate on the reproductive tract has been well documented in animal studies. For example, reduced testis weights and a slight degeneration of the seminiferous tubules were observed in a 13-week inhalation study with EGEE in rabbits from 400 ml/m³. The NOAEC was 100 ml/m³ (Barbee et al. 1984). Testicular atrophy was observed from 400 mg/kg body weight and day in studies in rats that were given EGEE orally for 13 weeks (administration in the drinking water; NTP 1993) or from 186 mg/kg body weight and day (administration by gavage; Stenger et al. 1971). The NOAELs in the two 13-week studies were 109 and 93 mg/kg body weight and day (see Table 2). In a further study, EGEE was administered orally by gavage to 5-week-old juvenile rats and 9-week-old adult rats over a period of 4 weeks at doses of 0, 50, 100, 200 and 400 mg/kg body weight and day. Slightly increased relative testis and epididymis weights were observed in juvenile rats even at the lowest dose of 50 mg/kg body weight and day and above, although they were not related to the dose and were not statistically significant, whereas these weights were statistically significantly reduced in adult rats at 400 mg/kg body weight and day. The cytometric analysis of testicular cell populations revealed reduced proportions of mature (elongated spermatids) and immature haploid cells (round and elongated spermatids) and increased proportions of diploid (spermatogonia, secondary spermatocytes and somatic cell tissue) and tetraploid cells (primary spermatocytes) only in adult rats at 400 mg/kg body weight and day (Yoon et al. 2001). Administration of EGEE to 8-week-old rats for 4 weeks led to reduced body weight gains from 200 mg/kg body weight and day and to reduced epididymis weights from as low as 100 mg/kg body weight and day. Histopathological testicular changes were observed at 200 and above mg/kg body weight and day (Yoon et al. 2003).

When EGEE was administered to 12-week-old Sprague Dawley rats for 5 weeks at doses of 0, 100, 300 and 600 mg/kg body weight and day, significantly reduced sperm motility was observed at the high dose level (Wang et al. 2006).

The up to 7-week administration of EGEE to 10- to 15-week-old rats at doses of 0, 250 and 500 mg/kg body weight and day led to reduced body weight gains at 250 mg/kg body weight and day and to body weight losses at 500 mg/kg body weight and day. From 250 mg/kg body weight and day, the relative and absolute testis and epididymis weights were reduced, and there were changes in sperm parameters such as a reduced sperm count and impaired motility. After 7-week administration of 500 mg/kg body weight and day, the sperm count was considerably decreased (34 × 10⁶/g testis as compared with the control of 883 × 10⁶/g), and the sperm were no longer motile (Horimoto et al. 2000; Isobe et al. 1996).

In a one-generation study in CD-1 mice with continuous mating, groups of 40 males and 40 females were given 0, 0.5, 1.0 and 2% EGEE in the drinking water for 14 weeks, corresponding to 0, 760, 1500 and 2600 mg/kg body weight and day. All offspring were examined immediately after birth. Fertility was impaired from 1500 mg/kg body weight and day. Reduced absolute testis and epididymis weights and morphological sperm changes as well as a considerably reduced number of live offspring and reduced body weights of the offspring were observed. No dam became pregnant at 2600 mg/kg body weight and day, and the oestrus cycle was prolonged (Lamb et al. 1984, 1997).

EGEE acetate was also examined in a one-generation study in CD-1 mice with continuous mating by the NTP 1993. The animals were given the substance in the drinking water for 18 months. The doses corresponded to 0, 900, 1800 and 3000 mg/kg body weight and day. The number of litters and the number of live offspring was reduced at 1800 mg/kg body weight and above; the results of cross-mating experiments showed that this was due mainly to the exposure of females. Effects on sperm parameters, testis weights and the incidence of abnormal sperm were less pronounced than the effects on female fertility (no other details) (ECB 2000 b).

Developmental toxicity

Studies of the developmental toxicity of EGEE and EGEE acetate are summarized in Table 4 and Table 5, respectively. Since these are often older studies from the 1970s and 1980s, prior to the standardization of the terminology used for skeletal and visceral changes, the terminology of the publications is adopted.

Prenatal developmental toxicity

The most important studies of prenatal developmental toxicity have already been described in the 1994 supplement (documentation “2-Ethoxyethanol, 2-Ethoxyethyl acetate” 1998, a translation of the 1994 German).

In a developmental toxicity study relevant for the present assessment that was carried out in groups of 24 Wistar rats and 24 Dutch rabbits with inhalation exposure of the animals to EGEE concentrations of 0, 10, 50 and 250 ml/m³ (rats) and 0, 10, 50 and 175 ml/m³ (rabbits) for 6 hours a day from days 6 to 15 (rats) and days 6 to 18 (rabbits) of gestation, the NOAEC for developmental toxicity was 50 ml/m³ for both species (Doe 1984; Table 4). Rats revealed significantly increased incidences of externally visible and visceral anomalies (“minor defects”; 18.4%; control: 11.7%) and of skeletal anomalies (“minor defects”; 97.5%; control: 46.3%) at 250 ml/m³. Malformations (“major defects”) were found in only one animal with fusion of the right kidney or ureter with the left kidney and a rudimentary right kidney. Maternal effects with decreases of haemoglobin, haematocrit and mean corpuscular volume of the erythrocytes also only occurred at 250 ml/m³. In rabbits, no maternal effects were observed at 175 ml/m³, but there was an increased incidence of skeletal anomalies (“minor defects”; 64.5%; control: 32.5%) and skeletal variations (79.1%; control: 51.5%) in foetuses (Doe 1984; Table 4).

In a developmental toxicity study carried out with EGEE in white New Zealand rabbits with exposure from days 1 to 18 of gestation, an increased incidence of resorptions, malformations and anomalies (e.g. ventral wall defects and fusion of aorta with pulmonary artery) and variations (e.g. extra ribs and variations of sternebrae) were found in the maternally toxic range with clearly reduced feed consumption, but only slightly reduced body weight gains. The higher concentration of 617 ml/m³ led to maternal mortality and total resorptions (Andrew and Hardin 1984; Table 4).

In prenatal developmental toxicity studies carried out with EGEE acetate in F344 rats and white New Zealand rabbits, the NOAEC was also 50 ml/m³. In rats, there was an increased incidence of variations from 100 ml/m³, skeletal malformations (uni- or bilateral cervical ribs) from 200 ml/m³ and externally visible, visceral and skeletal malformations at 300 ml/m³. In rabbits, the incidence of variations (reduced ossification) was increased from 100 ml/m³, and externally visible tail and heart malformations as well as an increased incidence of 14th ribs were even observed from 200 ml/m³ (Tyl et al. 1988; Table 5).

Table 4. Studies of pre- and postnatal developmental toxicity of EGEE

Species, strain, No. of animals per group	Exposure	Findings	References
Key: GD: day of gestation; PND: postnatal day; SD: Sprague Dawley; n.s.: not specified
Prenatal developmental toxicity
inhalation
rat, Wistar, 29–38 ♀	3 weeks before mating/GDs 1–19, 0/0, 150/0, 649/0, 0/202, 150/202, 0/767, 649/767 ml/m^3, 7 h/d, 5 d/week, exam on GD 21	150/0 and 649/0 ml/m^3 : dams and foetuses: no effects 0/202 and 150/202 ml/m^3 : dams: no effects; foetuses: foetal weight , crown-rump length , anomalies (“minor”: altered morphology of ribs and cardiovascular defects) and variations (extra ribs and retarded ossification) 0/767 and 649/767 ml/m^3 : dams: b.w. gain ; foetuses: total resorptions	Andrew and Hardin 1984
rat, Wistar, 24 ♀	GDs 6–15, 0, 10, 50, 250 ml/m^3, 6 h/d, exam on GD 21	50 ml/m^3 : NOAEC 250 ml/m^3 : dams: anaemia; foetuses: external and visceral defects (“minor”: mainly dilated renal pelvis) and skeletal defects (“minor”)	Doe 1984
rabbit, Dutch, 24 ♀	GDs 6–18, 0, 10, 50, 175 ml/m^3, 6 h/d, exam on GD 29	50 ml/m^3 : NOAEC 175 ml/m^3 : dams: no effects; foetuses: skeletal defects (“minor”) and variations	Doe 1984
rabbit, white New Zealand, 29–38 ♀	GDs 1–18, 0, 160, 617 ml/m^3, 7 h/d, exam on GD 29	160 ml/m^3 : dams: feed consumption distinctly , rel. liver weight ; foetuses: number of live foetuses , number of resorptions , malformations , anomalies and variations 617 ml/m^3 : dams: mortality , b.w. and rel. kidney weight ; foetuses: total resorptions	Andrew and Hardin 1984
ingestion
rat, SD, n.s.	GDs 7–15, 0, 200 mg/kg b.w. and d, gavage, exam on GD 20	200 mg/kg b.w.: dams: b.w. gain ; foetuses: foetomortality , foetal weight and skeletal and cardiovascular (24%) anomalies	Goad and Cranmer 1984
rat, n.s., 8–19 ♀	GDs 7–17, 0, 210–550 mg/kg b.w. and d, drinking water, exam on GD 21	210–270 mg/kg b.w.: 31% embryomortality 270–400 mg/kg b.w.: 69% embryomortality and foetal weight ; no malformations 400–550 mg/kg b.w.: 100% embryomortality	Chester et al. 1986
rat, Wistar at least 20 ♀	GDs 1–21, 0, 12, 23, 47, 93, 186, 372 mg/kg b.w. and d, gavage, exam on GD 22	23 mg/kg b.w.: NOAEL 47 mg/kg b.w.: total proportion of foetuses with skeletal findings (5.3%; control: 2.7%) at 93 mg/kg b.w.and above: total proportion of foetuses with skeletal findings (21.1%; control: 2.7%) and foetal weight 186 mg/kg b.w.: total proportion of foetuses with skeletal findings (88.8%; control: 2.7%; mainly incompletely ossified calvaria, aplasia of hyoid, thoracic and lumbar schisis, partial aplasia of the sternum and wavy ribs) and proportion of live foetuses ; no severe malformations	Stenger et al. 1971
mouse, CD-1, 6 ♀	GDs 8–14, 0, 1000, 1800, 2600, 3400, 4200 mg/kg b.w. and d, exam on GD 22	1000 mg/kg b.w.: foetuses: foetal weight 1800 mg/kg b.w.: dams: b.w. gain ; foetuses: resorptions and malformations (fused, small or absent digits of front paws, exencephaly and cleft palates) 3400 mg/kg b.w.: dams: mortality 4200 mg/kg b.w.: 100% resorption rate	Wier et al. 1987
subcutaneous injection
rat, Wistar at least 20 ♀	GDs 1–21, 0, 23, 47, 93 mg/kg b.w. and d, exam on GD 22	23 mg/kg b.w.: NOAEL 47 mg/kg b.w.: total proportion of foetuses with skeletal aberrations (6.2%; control: 3.6%) at 93 mg/kg b.w.and above: total proportion of foetuses with skeletal aberrations (27.8%; control: 3.6%)	Stenger et al. 1971
mouse, Swiss, 22 ♀	GDs 1–18, 0, 47, 93 mg/kg b.w. and d, exam on GD 19	93 mg/kg b.w.: NOAEL	Stenger et al. 1971
rabbit, Gelbsilber, 15 ♀ expos. and 8 ♀ contr.	GDs 7–16, 0, 23 mg/kg b.w. and d, exam on GD 29	23 mg/kg b.w.: NOAEL	Stenger et al. 1971
dermal
rat, SD, 18 ♀	GDs 7–16, 4 × daily 0, 0.25, 0.5 ml/application (about 0, 1, 2 ml/day), about 0, 3445, 6889 mg/kg b.w. and d, exam on GD 21	3445 mg/kg b.w.: dams: b.w. gain ; foetuses: 50% total resorptions, foetal weight , visceral (heart, kidney, brain and eyes) and skeletal malformations and variations 6889 mg/kg b.w.: dams: ataxia and absol. liver weight ; foetuses: 100% total resorptions	Hardin et al. 1982, 1984
Postnatal developmental toxicity
inhalation
rat, SD, 59 ♀ (main study)	GDs 7–13 or GDs 14–20, range-finding study: 0, 100, 200, 300, 600, 900, 1200 ml/m^3, 4 h/d; pre- and perinatal examinations for mortality	at 200 ml/m^3 and above: offspring: mortality (25%) 900 ml/m^3 : 100% resorption rate	Nelson and Brightwell 1984; Nelson et al. 1981
	main study: 0, 100 ml/m^3, 4 h/d; postnatal examinations	100 ml/m^3 : dams: slightly prolonged gestation; offspring: impaired performance in a rotarod test and prolonged latency in an open field test (expos. on GDs 7–13), less activity in a running wheel, poorer results in shock avoidance conditioning (expos. on GDs 14–20) and neurochemical alterations in the brain on PND 21
ingestion
mouse, CD-1, 20 ♀	GDs 8–14, 0, 800, 1200 mg/kg b.w. and d, exam on PNDs 1, 8, 15, 22	at 800 mg/kg b.w. and above: offspring: litter size , birth weights and visible tail deformities 1200 mg/kg b.w.: dams: b.w. gain ; offspring: postnatal survival rate	Wier et al. 1987
mouse, CD-1, 50 ♀	GDs 7–14, 0, 3605 mg/kg b.w. and d, postnatal examinations	3605 mg/kg b.w.: dams: 10% mortality; offspring: 100% mortality	Schuler et al. 1984

Table 5. Studies of prenatal developmental toxicity of EGEEacetate

Species, strain, No. of animals per group	Exposure	Findings	References
Key: GD: day of gestation; SD: Sprague Dawley; n.s.: not specified
inhalation
rat, F344, 30 ♀	GDs 6–15, 0, 50, 100, 200, 300 ml/m^3, 6 h/d, exam on GD 21	50 ml/m^3 : NOAEC at 100 ml/m^3 and above : dams: haematological effects; foetuses: variations at 200 ml/m^3 and above: dams: feed consumption and b.w. gain ; foetuses: total resorptions , foetal weight , skeletal malformations (cervical ribs uni- or bilaterally) 300 mg/kg b.w.: foetuses: external, visceral and skeletal malformations	Tyl et al. 1988
rat, SD, 9–20 ♀	GDs 7–15, 0, 130, 390, 600 ml/m^3, 7 h/d, exam on GD 20	130 ml/m^3 : foetuses: foetal weight at 390 ml/m^3 and above: foetuses: resorptions , foetal weight , visceral variations (cranial) and malformations (mainly cardiac malformations), skeletal malformations (wavy and fused 14th ribs) and variations (mainly reduced ossification) 600 ml/m^3 : foetuses: total resorptions; dams: n.s.	Nelson et al. 1984
rabbit, Dutch, 23–24 ♀	GDs 6–18, 0, 25, 100, 400 ml/m^3, 6 h/d, exam on GD 29	25 ml/m^3 : NOAEC 100 ml/m^3 : foetuses: foetal weight , skeletal variations and defects (no “major”) 400 ml/m^3 : dams: feed consumption and b.w. gain and haemoglobin ; foetuses: postimplantation losses , external and visceral defects (“minor”), skeletal variations and defects (“minor” and “major”)	Johnson 2002; Doe 1984
rabbit, white New Zealand, 24 ♀	GDs 6–18, 0, 50, 100, 200, 300 ml/m^3, 6 h/d, exam on GD 29	50 ml/m^3 : NOAEC at 100 ml/m^3 and above: dams: b.w. gain , clinical signs and platelet count ; foetuses: foetal weight and increased incidence of variations (reduced ossification) at 200 ml/m^3 and above: foetuses: number of live foetuses , external malformations (tail malformations), visceral malformations (heart malformations), skeletal malformations (14th ribs) and variations	Tyl et al. 1988
ingestion
mouse, CD-1, 20 ♀ and 20 ♂	multi-generation study, 0, 900, 1800, 3000 mg/kg b.w. and d, drinking water	900 mg/kg b.w.: NOAEL 1800 mg/kg b.w.: testicular atrophy (F2 animals) 3000 mg/kg b.w.: number of live offspring/litter and number of litters/pair	BUA 1995
dermal
rat, SD, 17–18 ♀	GDs 7–16, 0, 6000 mg/kg b.w. and d, exam on GD 21	6000 mg/kg b.w.: dams: b.w. gain ; foetuses: total resorptions , number of live foetuses/litter , foetal weight , cardiovascular malformations, skeletal variations and retarded ossifications	Hardin et al. 1984

After both ingestion and subcutaneous injection of EGEE (see Table 4) to rats for the whole period of gestation, the proportion of foetuses with skeletal aberrations was increased in relation to the dose at 47 mg/kg body weight and day and above (Stenger et al. 1971). Cardiovascular anomalies were observed from 200 mg/kg body weight and day (Goad and Cranmer 1984). A NOAEL of 23 mg/kg body weight and day was found for both ingestion and subcutaneous injection (Stenger et al. 1971). In mice, ingestion of the lowest dose of 1000 mg/kg body weight and day from days 8 to 14 of gestation led to reduced foetal weight, and 1800 mg/kg body weight and day caused an increased incidence of resorptions and malformations (Wier et al. 1987).

The dermal application of EGEE at 3445 mg/kg body weight and day and of EGEE acetate at 6000 mg/kg body weight from days 7 to 16 of gestation led to foetal mortality, reduced foetal weight, and visceral and skeletal anomalies. Heart malformations were mainly observed. Lower doses were not used; EGEE was lethal for the foetuses at 6889 mg/kg body weight and day. This dose led to ataxia and reduced liver weights in the dams (Hardin et al. 1982, 1984; Table 4 and Table 5).

Postnatal developmental toxicity

When pregnant rats were exposed to EGEE by inhalation at 100 ml/m³ from days 7 to 13 or days 14 to 20 of gestation, impaired performance in a rotarod test, prolonged latency in an open field test, less activity in a running wheel, poorer results in shock avoidance conditioning and neurochemical alterations in the brain were found in the pups as compared with the control group (Nelson and Brightwell 1984; Nelson et al. 1981; Table 4). Since only one concentration was used, the relevance of the findings for possible concentration-response relationships cannot be assessed, nor can a final assessment be made.

Offspring of pregnant mice that inhaled EGEE at a concentration of 800 ml/m³ from days 8 to 14 of gestation revealed reduced litter sizes and birth weights and visible tail deformities. The postnatal survival rate was decreased at 1200 mg/kg body weight and day (Wier et al. 1987; Table 4).

Genotoxicity

In vitro

EGEE revealed no mutagenicity in S. typhimurium or E. coli both in the presence and in the absence of metabolic activation. In several sister chromatid and chromosome aberration tests carried out with EGEE in CHO cells, increased rates of sister chromatid exchanges and of chromosome aberrations were observed without metabolic activation only from very high concentrations of 3179 µg/ml and 6830 µg/ml, respectively. The clastogenicity in CHO cells was reduced or eliminated when metabolic activation had been added to the in vitro test system. No genotoxicity was observed in an HPRT test in CHO cells, whereas significantly increased mutation rates were obtained at some concentrations in the mouse lymphoma test with metabolic activation (BUA 1995). However, since these could not be reproduced (NTP 1993) and were in the range of the control values of the other test series, this finding is not regarded as relevant for the present assessment. Nor was EGEE acetate genotoxic in the Salmonella mutagenicity assay, in a test for sister chromatid exchanges in CHO cells or in the HPRT test (ECB 2000 b).

In vivo

In vivo, no genotoxicity was detected with EGEE in Drosophila melanogaster (SLRL test) or mice (micronucleus test) (BUA 1995).

EGEE acetate was also negative in a micronucleus test in mice (ECB 2000 b).

Carcinogenicity

Groups of 50 male and 50 female F344 rats and B6C3F1 mice were given EGEE by gavage on 5 days per week for 103 weeks at doses of 0, 500, 1000 and 2000 mg/kg body weight and day. Since mortality was markedly increased (mainly because of gastric ulcerations) in rats and mice in the high dose group, all animals of this dose group were sacrificed after 18 weeks. A dose-related reduction of body weight gain was observed in rats of the low and middle dose groups. Gross-pathological examinations revealed enlarged adrenals and testicular atrophy in male rats of the low and middle dose groups. Testicular atrophy was found in male mice from 1000 mg/kg body weight and day (Melnick 1984). Since no comprehensive histopathological examinations were carried out, no conclusions can be drawn with regard to carcinogenicity.

No carcinogenicity studies are available for 2-ethoxyethyl acetate.

Manifesto (MAK value, classification)

Since EGEE is readily absorbed through the skin and the toxic metabolite ethoxyacetic acid accumulates during the working week, systemic exposure to ethoxyacetic acid is decisive for toxicity and therefore the basis for deriving the MAK value. In 1992, the BAT value for EGEE was established at 50 mg ethoxyacetic acid/l urine (Henschler and Lehnert 1993 a). In 1994, the MAK value for EGEE was established on the basis of the findings obtained by Ratcliffe et al. 1989 since effects on sperm parameters could not be ruled out in the group of workers who excreted 85 ± 31.3 mg 2-ethoxyacetic acid/g creatinine (about 100 mg/l urine). A MAK value of 5 ml/m³ was established assuming that this concentration corresponded to ethoxyacetic acid concentrations of 10 to 35 mg/l in the urine at the end of a working week. However, a new PBPK model shows that the concentration of EGEE of 5 ml/m³ after inhalation exposure alone corresponds to about 120 mg ethoxyacetic acid/l urine (95th percentile) at the end of a working week. Since effects on sperm may occur at about 100 mg ethoxyacetic acid/l urine and the BAT value is 50 mg ethoxyacetic acid/l urine, the MAK value has been lowered to 2 ml/m³; this correlates with the BAT value of 50 mg ethoxyacetic acid/l urine according to the PBPK model. In 2001, Peak Limitation Category II with an excursion factor of 8 was established for EGEE by analogy to other short-chain glycol ethers since the metabolite ethoxyacetic acid responsible for the critical systemic toxicity has a very long half-life and irritation is expected only above 50 ml/m³. This excursion factor has been retained.

EGEE was not mutagenic in vitro, but showed clastogenicity at high concentrations. No genotoxicity was observed in vivo in a micronucleus test in mice. No classification in any of the germ cell mutagen categories is therefore required.

In a carcinogenicity study, rats and mice were exposed for 2 years, and no tumours were observed by gross pathology. Since no comprehensive histopathological examinations were carried out, no conclusions can be drawn with regard to carcinogenicity. However, there is no evidence to justify a classification of EGEE in one of the Carcinogen Categories.

A NOAEC of 50 ml/m³ was obtained for developmental toxicity in rats and rabbits. Visceral and skeletal defects and variations, and sometimes malformations as well, were observed in rats from 250 ml/m³. The incidence of skeletal defects and variations was increased in Dutch rabbits after 6-hour exposure to EGEE at 175 ml/m³ daily from days 6 to 18 of gestation; an increased incidence of malformations was observed at 160 ml/m³ after 7-hour exposure of white New Zealand rabbits from days 1 to 18 of gestation. Although the MAK value for EGEE has been lowered from 5 to 2 ml/m³, the margin between the NOAEC of 50 ml/m³, or between 160 ml/m³, i.e. the concentration which led to an increased incidence of malformations in rabbits, and the MAK value is not high enough for a classification in Pregnancy Risk Group C. Therefore, EGEE remains in pregnancy risk group B. The high absorption of EGEE and EGEE acetate through the skin and the accumulation of ethoxyacetic acid in humans has been taken into account here.

Since EGEE is readily absorbed through the skin, the designation “H” has been retained and is justified.

EGEE showed no sensitizing potential in a maximization test carried out in guinea pigs. No other studies or effects in humans are available. The substance is therefore not designated with “Sa” or “Sh”.

2

1. 1

   MAK value for the sum of the concentrations of ethylene glycol monoethyl ether and ethylene glycol monoethyl ether acetate in the air

2. 2

   completed 14.12.2006