Short-term Oral Toxicity of Pentyl Ether, 1,4-Diethoxybutane, and 1,6-Dimethoxyhexane in Male Rats

Raymond Poon*,1, Marc Rigden*, Ih Chu* and Victor E. Valli{dagger}

* Environmental Health Science Bureau, Health Canada, Ottawa, Ontario, Canada and {dagger} College of Veterinary Medicine, University of Illinois, Urbana, Illinois 61801

Received July 16, 2003; accepted September 13, 2003

ABSTRACT

Pentyl ether (PE) and two newly synthesized polyoxy ethers, 1,4-diethoxybutane (DEB) and 1,6-dimethoxyhexane (DMH), have been proposed as candidate diesel fuel additives. To characterize and compare their toxicity and to provide information for risk assessment, a 4-week oral study was conducted on these compounds. Male Sprague-Dawley rats (288 ± 20 g) were divided into groups of seven animals each, and were administered by gavage low (2 mg/kg body weight), medium (20 mg/kg body weight), or high (200 mg/kg body weight) doses of PE, DEB, or DMH, respectively, 5 days/week for 4 weeks. Animals in the control group received the vehicle (corn oil, 1 ml/100 g body weight) only. At the end of the exposure period, relative testis and thymus weights were reduced by 30 and 46%, respectively, in animals treated with the high dose of DMH. Significant reductions in serum lactate dehydrogenase (LDH), serum uric acid, and blood platelet counts were also observed in the high dose of DMH. Serum corticosterone was significantly depressed in the high doses of PE and DEB and in the low dose of DMH. Serum thiobarbituric acid–reactive substances (TBARS) were decreased (p < 0.05) in all DMH treatment groups and in the medium and high dose PE and DEB groups, while liver TBARS were unaffected by treatment. In the liver, increased glutathione (GSH) level and glutathione-S-transferases activity were detected in the high dose DMH group. Urinary ascorbic acid levels were markedly increased in animals receiving the high doses of PE, DEB, and DMH. Urinary formic acid was increased by 13 times in the high dose PE and DEB groups. Testes of all animals receiving the high dose of DMH showed a moderate to marked degree of degeneration of the seminiferous tubules, including a mild degree of vacuolation. At the same time, the epididymis of these animals had substantially reduced sperm density with prominent presence of spermatid giant cells. Mild histological changes were seen in the liver at all dose levels for all three chemicals. Thyroid effects were also observed in the high dose PE and DEB groups and in the medium and high dose DMH groups. It was concluded that DMH is the most toxic of the three ethers tested, with testicular, epidiymal, and thymic effects being the most prominent at 200 mg/kg. Other significant changes included depressed platelet counts and serum biochemical changes. Increased production of formic acid, an ocular toxin, from PE and DEB treatments may also be of toxicological concern.

Key Words: diesel fuel additives; aliphatic ethers; testicular effects; epididymal effects; thymic effects; formic acid.

Cetane, also known as n-hexadecane (C16H34), is a colorless, oily hydrocarbon and a reference fuel used to determine the ignition quality of diesel fuels. Cetane enhancers are important diesel fuel additives. Their presence in diesel fuels improves combustion quality and reduces hydrocarbon and carbon monoxide emissions in the exhaust (Heaton et al., 1993Go). Combustion quality is improved when ignition delay is shorter and cetane number is higher. Cetane number is a measure of the ignition quality of a diesel fuel that represents the percentage by volume of cetane in a mixture of {alpha}-methylnapthalene that gives the same ignition delay as the fuel being tested. Because of their high cetane number and miscibility, many aliphatic ethers, including pentyl ether (PE) and polyoxyethers 1,4-diethoxybutane (DEB) and 1,6-dimethoxyhexane (DMH), have been considered as candidate diesel fuel additives.

In the development of new fuel additives, it is important that the toxicity of these ethers be investigated and their potential health risks assessed at an early stage. Recently, the acute toxicity of a candidate additive, dimethoxymethane (methylal), was studied in rats, and its stability and toxicity were characterized (Poon et al., 2000Go). A review of the literature reveals that toxicity data is minimal for PE and nonexistent for DEB and DMH. The intravenous LD50 for PE was reported to be 164 mg/kg (Di Paolo, 1978Go) in mouse. In this report, we studied and compared the systemic toxicity and histopathological effects of PE, DEB, and DMH on male rats following a short-term exposure. Because at low doses the effects may be subclinical in nature, sensitive biomarkers such as those for enzyme activities, hepatic effects (urinary ascorbic acid), and oxidative stress (TBARS, GSH) were also studied. In the studies of methyl tertiary butyl ether (MTBE), an ether oxygenate previously added to some gasoline, the formation of formaldehyde and its potential effects received much attention (Casanova and Heck, 1997Go; Hutcheon et al., 1996Go; Williams-Hill et al., 1999Go). Therefore, the measurement of urinary formic acid, an end metabolite of formaldehyde, was included in the present study. Because the test compounds are moderately volatile (boiling points of 163–188°C), it is expected some of the ingested compounds will be exhaled. Accordingly, biochemical markers of pulmonary effects were part of this study. An oral route of administration was chosen to emulate the exposure scenario of intake through potential contamination of drinking water (Stern and Tardiff, 1997Go) and accidental ingestion of fuels (Litovitz et al., 1999Go).

MATERIALS AND METHODS

Chemicals.
Pentyl ether (PE) was purchased from Sigma Aldrich (Oakville, Ontario, Canada); 1,6-dimethoxyhexane (DMH) and 1,4-diethoxybutane (DEB) were synthesized by Wychem Ltd (Suffolk, England). The purity of these three compounds was >99% as measured by gas chromatography. All other chemicals were of reagent grades and obtained from Fisher Scientific (Ottawa, Ontario, Canada) or Sigma Chemical Co. (St. Louis, MO).

Animal treatment.
Seven week old male Sprague-Dawley rats (Charles River Laboratories, St. Constant, Quebec, Canada) were divided into 10 groups of 7 animals each. They were housed in individual Health Guard® cages (Research Equipment Co., Bryant, TX) and were given food and water ad libitum. After 1 week of acclimatization, the groups were administered, by gavage, each of the test substances (PE, DEB, and DMH) in corn oil at one of the following doses: 2 mg/kg body weight (low), 20 mg/kg body weight (medium), or 200 mg/kg body weight (high). The oral administration was performed daily, 5 days/week, for 4 consecutive weeks. Control animals were administered corn oil (1.0 ml/100 g body weight) only. Body weights and food consumption were measured weekly. Cage-side observations were made weekly. The battery of observations consisted of the following: posture, clonic movement, gait score, piloerection, pitosis, lacrimation, salivation, vocalization, and ease of removal from home cage.

Overnight urine was collected from individual animals at the end of the fourth week. A portion of the urine was stored at -80°C for the determination of protein by a Lowry microprotein determination kit (Sigma Chemical Co.) and N-acetylglucosaminidase (NAGA) activity (Poon et al., 1995Go). The remaining urine was preserved in 0.4 N HCl/10 mM EDTA for the determination of ascorbic acid by an HPLC method (Poon et al., 1994Go). All urine analyte concentrations were normalized against creatinine, which was measured enzymatically using a Roche creatinine assay kit (Roche Diagnostics, Quebec, Canada).

At termination, the animals were anesthetized with inhaled Isoflurane® (Baxter Corp., Toronto, Canada). Blood was withdrawn from the abdominal aorta for hematological analysis and serum clinical chemistry. The right lung was clamped and the left lung was instilled with saline (17.5 ml/kg body weight) according to the method of Hatch et al. (1986)Go. The lavage fluid was centrifuged at 700 x g for 15 min and the cell-free bronchoalveolar lavage fluid (BALF) was stored at -70°C pending the analysis for protein and NAGA, using the methods described above for urine. The brain, heart, thymus, liver, kidneys, spleen, and testis were excised and weighed. A 2 g piece of liver was removed and homogenized in 3 ml of 0.05 M Tris/0.15% KCl buffer, pH 7.4. Part of the homogenate was centrifuged at 9000 x g for 20 min to obtain the S9 fraction. Both the homogenates and S9 fractions were stored at -80°C until use. The liver, kidneys, thymus, thyroid, and epididymis were preserved in phosphate-buffered formalin. Testes were fixed in Bouin’s solution, washed in 70% ethanol, and preserved in phosphate-buffered formalin. The fixed tissues were dehydrated with graded alcohol, cleared, and impregnated in paraffin. The paraffin blocks were sectioned to 5 µm thickness and stained with hematoxylin and eosin for microscopic examination.

Hematological and biochemical analysis.
A Technicon H1E hematology analyzer (Bayer, Toronto, Ontario, Canada) was used for the determination of the following hematological parameters: erythrocyte count, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, platelet count, white blood cell counts, and percentages of lymphocytes. A Technicon RA-XT analyzer was used to obtain the following serum chemistry parameters: aspartate aminotransferase activity, alkaline phosphatase activity, total protein, albumin, inorganic phosphate, urea nitrogen, creatinine, glucose, and cholesterol. The traditional lactate dehydrogenase (LDH) activity as well as the LDH-X activity were measured in serum using lactic acid and DL{alpha}-hydroxycaproic acid, as the substrate, respectively (Blanco et al., 1976Go). Serum testosterone was determined using a radioimmunoassay kit (ImmunChem, ICN Pharmaceutical Inc., Costa Mesa, CA), while serum corticosterone was measured using an HPLC procedure (Wong et al., 1994Go).

Reduced glutathione (GSH) was measured in liver homogenates using a colorimetric kit method (Calbiochem, La Jolla, CA). Glutathione-S-transferase (GST) activity was measured in the liver S9 fractions (Habig et al., 1974Go). Thiobarbituric acid–reactive substances (TBARS) in the liver homogenates and serum were determined by a fluorescence method (Yagi, 1982Go).

Statistical analysis.
One-way ANOVA and Duncan’s multiple range test were used to identify groups that were significantly different from the control at the p < 0.05 level.

RESULTS

Food consumption and body weight gain of animals treated with PE, DEB, or DMH did not differ significantly from the control animals (Table 1Go). None of the treated animals exhibited clinical signs of toxicity. Animals receiving the high dose of DMH (200 mg/kg) had significantly smaller testes and thymus gland, with the average weights of these organs relative to body weight decreased by 30 and 46%, respectively, compared to the control group (p < 0.05; Table 1Go). The relative weights of the brain, heart, liver, spleen, and kidneys were unaffected by treatment.


View this table:
[in this window]
[in a new window]
 
TABLE 1 Food Consumption, Body Weights, and Relative Organ Weights Following 4-Week Oral Administration of PE, DEB, and DMH
 
Serum uric acid was significantly depressed in animals receiving the high dose of DMH (Table 2Go). Serum lactate dehydrogenase activity, measured using the traditional substrate lactate was also significantly reduced in the high dose DMH group. However, when assayed using DL{alpha}-hydroxycaproic acid as the substrate, serum LDH-X activity was not detected in the control or any of the treatment groups (data not shown). Serum aspartate aminotransferase activities and testosterone levels were unaffected by treatment. No treatment-related changes were observed in serum glucose, cholesterol, total protein, albumin, inorganic phosphate, creatinine, urea nitrogen, and alkaline phosphatase (data not shown). Serum corticosterone levels were significantly depressed in the high dose PE and DEB groups and in the low dose DMH group (Fig. 1Go).


View this table:
[in this window]
[in a new window]
 
TABLE 2 Changes in Serum Chemistry and Hematology Following 4 Week Oral Administration of PE, DEB, and DMH
 


View larger version (14K):
[in this window]
[in a new window]
 
FIG. 1. Serum corticosterone in male rats following 4-week oral administration of PE, DEB, and DMH. Mean of seven animals per group with the horizontal bar denoting 1 SD. *p < 0.05 compared to control.

 
A significant decrease in platelet counts was observed in the high dose DMH group (Table 2Go). There were no significant treatment-related changes in red blood cell, white blood cell, or lymphocyte counts.

Serum TBARS levels were decreased (p < 0.05) in all of the DMH treatment groups and in groups receiving the medium and high doses of PE and DEB (Table 3Go). No significant changes in TBARS were observed in the liver.


View this table:
[in this window]
[in a new window]
 
TABLE 3 Thiobarbituric Acid–Reactive Substances (TBARS) Levels in the Liver and Serum of Male Rats Following 4 Week Oral Administration of PE, DEB, and DMH
 
Elevations in GST activity and GSH levels (p < 0.05) were found only in the liver of animals receiving the high dose of DMH (Fig. 2Go).



View larger version (19K):
[in this window]
[in a new window]
 
FIG. 2. Hepatic GSH and GST levels in male rats following 4-week oral administration of PE, DEB, and DMH. Mean of seven animals per group with the horizontal bar denoting 1 SD. *p < 0.05 compared to control.

 
Urinary ascorbic acid levels in animals treated with high doses of PE, DEB, and DMH were increased by 7, 4.9, and 6.5 times that of control, respectively (Fig. 3Go). Formic acid levels in the urine of animals treated with high doses of PE and DEB were markedly increased by ~13 times that of control (Fig. 4Go). Although the mean urinary formic acid concentration in the high dose DMH animals was five-fold that of the control, the difference was not statistically significant due to the wide data dispersion. None of the treated animals showed any increase in the urinary NAGA activity and protein levels (data not shown). Also, no treatment-related increases in NAGA activity and protein levels were detected in the cell-free bronchoalveolar fluids (data not shown).



View larger version (13K):
[in this window]
[in a new window]
 
FIG. 3. Urinary ascorbic acid levels in male rats following 4-week oral administration of PE, DEB, and DMH. Mean of seven animals per group with the horizontal bar denoting 1 SD. *p < 0.05 compared to control.

 


View larger version (13K):
[in this window]
[in a new window]
 
FIG. 4. Urinary formic acid in male rats following 4-week oral administration of PE, DEB, and DMH. Mean of seven animals per group with the horizontal bar denoting 1 SD. *p < 0.05 compared to control.

 
The most pronounced histological changes were found in the testes and epididymides of all seven animals receiving the high dose of DMH (Table 4Go). Testicular changes consisted of a moderate to marked degree of degeneration of seminiferous tubules characterized by the loss of seminiferous epithelium and developing spermatids, increased presence of spermatid giant cells in the lumen, and vacuolation of the epithelial cells (Fig. 5Go). Changes in the epididymis consisted of a moderate degree of reduction in sperm density and the presence of spermatid giant cells among other spermatid debris (Fig. 6Go).


View this table:
[in this window]
[in a new window]
 
TABLE 4 Histopathlogical Changes in the Liver, Thyroid, Testicles, Epididymis, and Kidneys Following 4 Week Oral Administration of PE, DEB, and DMH
 


View larger version (116K):
[in this window]
[in a new window]
 
FIG. 5. (A) Testis from a rat in the control group; the capsule (left) and the seminiferous tubules are normal with sperm at varying stages of maturation. (B) Testis from a rat in the high dose DMH group. There is complete loss of seminiferous epithelium in a tubule in the upper right with reduced diameter and several others with loss of developing spermatids. There is an intraepithelial vacuole (upper left) and several spermatid giant cells in the tubule in the upper center. The tubule in the upper left has lost all but the germinal layer of cells and those in the lumen have been sloughed from a more proximal area.. Hematoxylin and eosin stain x130.

 


View larger version (97K):
[in this window]
[in a new window]
 
FIG. 6. (A) Epididymis from a rat in the control group with a normal sperm density and morphology. (B) Epididymis from a rat in the high dose DMH group. There is a marked reduction in sperm density with numerous abnormal sperm and spermatid debris and giant cells. The epithelium is normal and has an increased number of intraepithelial nuclei, likely lymphocytes and abnormal spermatids. Hematoxylin and eosin stain x260.

 
Morphological changes in the thyroid glands were detected in animals receiving the medium and high doses of DMH (Fig. 7Go) and the high doses of DEB and PE. The changes consisted of a mild reduction in follicle size accompanied by a minimal degree of reduction of colloid density (Table 4Go). In the liver, a mild to moderate degree of perivenous homogeneity was found in the animals treated with all three ethers at all dose levels. In the high dose DMH group, the homogeneity was extended to the midzone (Table 4Go). In five of seven animals in the high dose PE group, peripherized basophilia in the perivenous area was also observed. In the renal proximal tubules, mild degrees of cytoplasmic inclusions and intratubular shedding of cytoplasm were commonly observed in the control animals but were accentuated in all treated animals (Table 4Go). Although thymic atrophy was clearly present in the high dose DMH group, the thymus morphology in this treatment group and in all other treatment groups appeared normal (results not shown).



View larger version (92K):
[in this window]
[in a new window]
 
FIG. 7. (A) Thyroid gland from a rat in the control group; the epithelium is of uniform depth with oval nuclei and abundant colloid of uniform density. (B) Thyroid gland from a rat in the high dose DMH group. There is a marked and uniform reduction in follicle size with mild vesiculation of epithelial nuclei and a moderate increase in epithelial height with marked cytoplasmic vacuolation and loss of colloid density. Hematoxylin and eosin stain x560.

 
DISCUSSION

Among the three aliphatic ethers studied, DMH at 200 mg/kg produced the most conspicuous adverse effects of testicular and thymic atrophy. The histopathological basis for the observed testicular atrophy is clearly related to degenerative changes occurring in the seminiferous tubules consisting of loss of epithelium, loss of developing spermatids, and reduced tubular diameter. The markedly reduced sperm density observed in the epididymides of these animals can be interpreted as a functional manifestation of the lesions in the seminiferous tubules resulting in a decline in sperm production (Ku et al., 1993Go). Although the reduction in epididymal sperm density was severe, a multigenerational study is required in the future to evaluate the impact of DMH on fertility (Chapin et al., 1997Go). A limitation of the present study is that the staining technique precluded a stage-specific screening for affected cell types (Hess, 1990Go). However, the prominent presence of spermatid giant cells in the epididymides provided supporting evidence that degenerative changes were occurring in the seminiferous tubules and affecting spermatogenesis (Holstein and Eckmann, 1986Go; Morton et al., 1986Go; Somkuti et al., 1991Go). A wide variety of testicular toxicants induced the appearance of multinucleated spermatid giant cells (Anderson et al., 1992Go; Dodd et al., 1994Go; Working et al., 1985Go), often accompanied by a reduction of sperm density in the epididymis (Fail et al., 1990Go; Somkuti et al., 1991Go).

The absence of LDH-X, a testicular isoyme released into the circulation by damaged testes (Reader et al., 1991Go), provided a biochemical indication that there were no acute testicular injuries. However, this does not preclude the possibility that testicular injuries had occurred at earlier time points. The lack of treatment-related changes in serum testosterone suggested that interstitial cells, the major site of testosterone release, were unaffected by all three ethers.

There are two industrial solvents with structures that bear some similarity to that of DEB and DMH: 1,2-dimethoxyethane and 1,2-diethoxyethane, also known as ethylene glycol dimethyl ether and ethylene glycol diethyl ether, respectively. Both compounds have been shown to be embryotoxic and developmental toxins (George et al., 1992Go; Hardin and Eisenmann, 1987Go; Leonhardt et al., 1991Go; Nagano et al., 1984Go). Nagano et al. (1984)Go demonstrated that mice treated with 1,2-dimethoxyethane at oral doses of 250 mg/kg or higher for 5 weeks had decreased testicular weight, atrophy of the seminiferous tubules with markedly reduced sperm density, and generally normal Sertoli and Leydig cells. In the present study, DMH-induced testicular atrophy was also accompanied by reduced sperm density in the epididymides. It is interesting to note that another structurally related compound, bis(2-methoxyethyl) ether (diglyme), also induced testicular atrophy, degeneration of spermatocytes, and spermatid giant cells following 20 daily doses at 684 mg/kg (Cheever et al., 1989Go). 2-Methoxyacetic acid, which is known to produce testicular and thymic atrophy (Miller et al., 1982Go; Moss et al., 1985Go), has been proposed to be the metabolite responsible for the toxic effects observed in bis(2-methoxyethyl) ether– and 1,2-dimethoxy ethane-treated animals (Daniel et al., 1991Go; Hardin and Eisenmann, 1987Go). Given the observed effects of DMH on the testis, epididymis, and thymus, it can be postulated that 2-methoxyacetic acid may be the predominant metabolite responsible. It can also be postulated that DEB was without effect at the present dose levels because one of its major metabolites, ethoxyacetic acid, is known to be less toxic than methoxyacetic acid (Foster et al., 1987Go; Smialowicz et al., 1992Go).

Miller and coworkers have shown that 2-methoxyacetic acid induced a reduction in bone marrow cellularity (Miller et al., 1982Go). They (Miller et al., 1984Go) also demonstrated that 2-methoxyethanol (ethylene glycol monomethyl ether), which was metabolized to 2-methoxyacetic acid (Medinsky et al., 1990Go), caused a decrease in platelet counts in rats and rabbits. It can be postulated that the DMH-associated depression in platelet counts may also be mediated by 2-methoxyacetic acid as a metabolite.

Rodents responded to a wide variety of hydrocarbon hepatotoxins by increasing the excretion of ascorbic acid (Burns et al., 1960Go; Poon et al., 1994Go). Typically, organochlorine-type hepatotoxins stimulated ascorbic acid excretion at intake levels of 1–5 mg/kg/day (Chu et al., 1998Go; Poon et al., 1999Go). The fact that all three ethers stimulated ascorbic acid excretion at 200 mg/kg indicated that they were comparatively much less potent as a hepatotoxin. Indeed, considering as a whole the reversible nature of the histopathological changes, the absence of hepatomegaly, and the normal serum aspartate aminotransferase activity, it can be concluded that the hepatic effects were adaptive and reversible in nature.

Changes in the thyroid were reversible in nature; the minimal reduction in colloid density suggested that the functional aspects of the thyroid may not be adversely affected.

Although DMH induced frank thymic atrophy, architectural and cytologic abnormalities were not detected at the histological level. Thymic atrophy in rodents has been reported to be correlated with increased serum corticosterone (Cuff et al., 1996Go; Gorski et al., 1988Go) or directly induced by the glucocorticoid analog dexamethasone (Lundberg, 1991Go; Sun et al., 1992Go). In the present study, serum corticosterone did not appear to be a cause of thymic atrophy because its level decreased rather than increased in the treated animals. Further studies focusing on the effects of DMH on the phenotypes and cellular responses of thymocytes will be needed to understand the mechanisms of toxicity.

Urinary protein and NAGA are sensitive biomarkers of injuries to proximal tubular cells. The absence of increases in these two parameters, together with a lack of treatment-related increases in kidney weights, indicated that the three aliphatic ethers are not likely to be nephrotoxic. Cytoplasmic inclusion of hyaline droplets in the proximal tubular cells is a change that is specific to the males of the rodent species (Halder et al., 1985Go; Short et al., 1989Go). Therefore, the accentuation of this change and the associated cytoplasmic shedding, mainly in the high dose DMH group, may not have toxicological implications in humans.

Protein and NAGA levels in cell-free bronchoalveolar lavage fluids are also sensitive biomarkers of early injuries in the lung (Henderson et al., 1979Go; Poon et al., 1995Go). The lack of significant increases of these biomarkers in the treated animals suggested that the aliphatic ethers were not pulmonary toxicants.

A biochemical effect unique to DMH is the increased liver GSH and GST activity. These increases suggested that there was an enhancement in the liver’s capacity to metabolize high doses of DMH via the mercapturic acid detoxification pathway.

None of the aliphatic ethers induced oxidative stress as judged by the absence of increased TBARS in the liver and serum. It is not clear at this point whether reduced serum TBARS signifies that the aliphatic ethers, DMH in particular, possess some antioxidant properties. The marked stimulation of urinary excretion of formic acid in of PE- and DEB-treated rats is a novel observation. Formic acid is an endogenous metabolite (Kavet and Nauss, 1990Go) found in the urine, normally at the range of 1.2 to 17.5 mg/g creatinine (Trieberg and Schaller, 1980Go). The magnitude of increase in urinary formic acid in the high dose PE and DEB groups is comparable to that found in urine of rats following intraperitoneal injection of 100 mg/kg methanol (Kornbrust and Bus, 1982Go). It is not known if the increase is related to a perturbation in one-carbon metabolism, increased lipid peroxidation, changes in renal excretion, or the metabolism of the aliphatic ethers themselves. Some small molecular weight hydrocarbons are known to be metabolized to formaldehyde through oxidative demethylation (Cederbaum and Cohen, 1980Go; Kukielka and Cederbaum, 1995Go). Formic acid was considered the proximal toxicant responsible for adverse effects associated with acute methanol poisoning, and humans and other primates are known to be more susceptible (Kavet and Nauss, 1990Go; McMartin et al., 1980Go). It remain to be seen whether acute exposure to aliphatic ethers in diesel fuels can raise the formic acid to levels that can be considered to be toxic in humans.

Returning to the primary focus of this study, i.e., the comparative toxicity of candidate diesel additives, DMH at 200 mg/kg clearly produced the most severe adverse effects. The most prominent of these are thymic and testicular atrophy, degeneration of seminiferous tubules, and reduced epididymal sperm density, and they may be related to metabolites such as 2-methoxyacetic acid. Other significant effects included decreased platelet counts and serum uric acid. All three ethers at 200 mg/kg produced mild and reversible histological changes in the liver and thyroid gland. The presence of high levels of formic acid, an ocular toxin, in animals dosed with PE and DEB may be of health concern during acute poisoning.

ACKNOWLEDGMENTS

The authors wish to thank A. Yagminas for his expert advice, and B. Nadeau, S. Masson, Y. Dirieh, and T. Odorizzi for their excellent technical assistance. This work was supported in part by the Federal Programme on Energy Resource and Development.

NOTES

1 To whom correspondence should be addressed at Health Canada, Environmental Health Center, locator No. 0803 B, Tunney’s Pasture, Ottawa, Ontario, Canada K1A 0L2. Fax: (613) 957-8800. E-mail: raymond_poon{at}hc-sc.gc.ca. Back

REFERENCES

Anderson, M. B., Pedigo, N. G., Katz, R. P., and George, W. J. (1992). Histopathology of testes from mice chronically treated with cobalt. Reprod. Toxicol. 6, 41–50.[CrossRef][ISI][Medline]

Blanco, A., Burgos, C., Gerez de Burgos, N. M., and Montamat, E. E. (1976). Properties of the testicular lactate dehydrogenase isoenzyme. Biochem. J. 153, 165–172.[ISI][Medline]

Burns, J. J., Conney, A. H., Dayton, P. G., Evans, S., Martin, G. R., and Taller, D. (1960). Observations on the drug induced synthesis of D-glucuronic acid, L-gulonic acid, and L-ascorbic acid in rats. J. Pharmacol. Exp. Ther. 129, 132–138.[ISI][Medline]

Casanova, M., and Heck, H. A. (1997). Lack of evidence for the involvement of formaldehyde in the hepatocarcinogenicity of methyl tertiary-butyl ether in CD-1 mice. Chem. Biol. Interact. 105, 131–143.[CrossRef][ISI][Medline]

Cederbaum, A. I., and Cohen, G. (1980). Oxidative demethylation of t-butyl alcohol by rat liver microsomes. Biochem. Biophys. Res. Commun. 97, 730–736.[ISI][Medline]

Chapin, R. E., Sloane, R. A., and Haseman, J. K. (1997). The relationships among reproductive endpoints in Swiss mice, using the reproductive assessment by continuous breeding database. Fundam. Appl. Toxicol. 38, 129–142.[CrossRef][ISI][Medline]

Cheever, K. L., Weigel, W. W., Richards, D. E., Lal, J. B., and Plotnik, H. B. (1989). Testicular effects of bis(2-methoxyethyl) ether in the adult male rat. Toxicol. Ind. Health 5, 1099–1109.[ISI][Medline]

Chu, I., Poon, R., Yagminas, A., Lecavalier, P., Håkansson, H., Valli, V. E., Kennedy, S. W., Bergman, Å, Seegal, R. F., and Feeley, R. (1998). Subchronic toxicity of PCB 105 (2,3,3',4,4'-pentachlorobiphenyl) in rats. J. Appl. Toxicol. 18, 285–292.[CrossRef][ISI][Medline]

Cuff, C. F., Zhao, W., Nukui, T., Schafer, R., and Barnett, J. B. (1996). 3,4-Dichloropropionanilide-induced atrophy of the thymus: Mechanism of toxicity and recovery. Fundam. Appl. Toxicol. 33, 83–90.[CrossRef][ISI][Medline]

Daniel, F. B., Cheever, K. L., Begley, K. B., Richards, D. E., Weigel, W. W., and Eisenmann, C. J. (1991). Bis(2-methoxyethyl) ether: Metabolism and embryonic disposition of a developmental toxicant in the pregnant CD-1 mouse. Fundam. Appl. Toxicol. 16, 567–575.[ISI][Medline]

Di Paolo, T. (1978). Molecular connectivity in quantitative structure-activity relationship study of anesthetic and toxic activity of aliphatic hydrocarbons, ethers, and ketones. J. Pharm. Sci. 67, 566–568.[ISI][Medline]

Dodd, D. E., Stuart, B. O., Rothenberg, S. J., Kershew, M., Mann, P. C., James, J. T., and Lam, C. E. (1994). Acute, 2-week, and 13-week inhalation studies on dimethylethoxysilane vapor on Fischer 344 rats. Inhalat. Toxicol. 6, 151–166.[ISI][Medline]

Fail, P. A., George, J. D., Grizzle, T. B., Heindel, J. J., and Chapin, R. E. (1990). Final report on the reproductive toxicity of boric acid (CAS No. 10043-35-3) in CD-1-Swiss mice. NTIS Technical Report (NTP-90-105).

Foster, P. M. D., Lloyd, S. C., and Blackburn, D. M. (1987). Comparison of the in vivo and in vitro testicular effects produced by methoxy-, ethoxy, and N-butoxy acetic acids in the rat. Toxicology 43, 17–30.[CrossRef][ISI][Medline]

George, J. D., Price, C. J., Marr, M. C., Kimmel, C. A., Schwetz, B. A., and Morrissey, R. E. (1992). The developmental toxicity of ethylene glycol diethyl ethers in mice and rabbits. Fundam. Appl. Toxicol. 19, 15–25.[ISI][Medline]

Gorski, J. R., Muzi, G., Weber, L. W., Pereira, D. W., Iatropoulos, M. J., and Rozman, K. (1988). Elevated plasma corticosterone levels and histopathology of the adrenals and thymuses in 2,3,7,8-tetrachlorodibenzo-p-dioxin treated rats. Toxicology 53, 19–32.[CrossRef][ISI][Medline]

Habig, W. H., Pabst, M. J., and Jakoby, W. B. (1974). Glutathione S-transferase. The first enzymatic step in mercapturic acid formation. J. Biol. Chem. 25, 7130–7139.

Halder, C. A., Holdswoth, C. E., Cockrell, B. Y., and Piccirillo, V. J. (1985). Hydrocarbon nephropathy in male rats: Identification of the nephrotoxic components of unleaded gasoline. Toxicol. Ind. Health. 1, 67–87.[Medline]

Hardin, B. D., and Eisenmann, C. J. (1987). Relative potency of four ethylene glycol ethers for induction of paw malformations in CD-1 mouse. Teratology 35, 321–328.[ISI][Medline]

Hatch, G. E., Slade, R., Stead, A. G., and Graham, J. A. (1986). Species comparison of acute inhalation toxicity of ozone and phosgene. J. Toxicol. Environ. Health 19, 43–53.[ISI][Medline]

Heaton, D. M., Martin, B., Bertoli, C., and Giavazzi, F. (1993). Fuel composition effects on diesel engine emissions – a joint European study. Proc. 2nd Int. MechE Seminar on Fuels for Automotive and Industrial Diesel Engines, pp. 23–33.

Henderson, R. F., Rebar, A. H., and Denicola, D. B. (1979). Early damage indicators in the lung. IV. Biochemical and cytological response of the lung to lavage with metal salts. Toxicol. Appl. Pharmacol. 51, 129–135.[ISI][Medline]

Hess, R. A. (1990). Quantitative and qualitative characteristics of the stages and transitions in the cycle of the rat seminiferous epithelium: Light-microscopic observation of perfusion-fixed and plastic embedded testes. Biol. Reprod. 43, 525–542.[Abstract]

Holstein, A. F., and Eckmann, C. (1986). Multinucleated spermatocytes and spermatids in human seminiferous tubules. Andrologia 18, 5–16.[ISI][Medline]

Hutcheon, D. E., Arnold, J. D., Hove, W. T., and Boyle, J. (1996). Disposition, metabolism, and toxicity of methyl tert butyl ether, an oxygenate for reformulated gasoline. J. Toxicol. Environ. Health 47, 453–464.[CrossRef][ISI][Medline]

Kavet, R., and Nauss, K. M. (1990). The toxicity of inhaled methanol. Crit. Rev. Toxicol. 21, 21–50.[ISI][Medline]

Kornbrust, D. J., and Bus, J. E. (1982). Metabolism of methyl chloride to formic acid in rats. Toxicol. Appl. Pharmacol. 65, 135–143.[ISI][Medline]

Ku, W. E., Chapin, R. E., Wine, R. N., and Gladen, B. C. (1993). Testicular toxicity of boric acid (BA): Relationship of dose to lesion development and recovery in the F334 rat. Reprod. Toxicol. 7, 305–319.[CrossRef][ISI][Medline]

Kukielka, E., and Cederbaum, A. I. (1995). Increased oxidation of ethylene glycol to formaldehyde by microsomes after ethanol treatment: Role of oxygen radicals and cytochrome P450. Toxicol. Lett. 78, 9–15.[ISI][Medline]

Leonhardt, D. E., Coleman, L. W., and Bradshaw, W. S. (1991). Perinatal toxicity of ethylene glycol dmethyl ether. Reprod. Toxicol. 5, 157–162.[CrossRef][ISI][Medline]

Litovitz, T. L., Klein-Schwartz, W., Caravati, E. M., Youniss, J., Crouch, B., and Lee, S. (1999). 1998 Annual report of the American Association of Poison Control Centers toxic exposure surveillance system. Am. J. Emerg. Med. 17, 435–487.[ISI][Medline]

Lundberg, K. (1991). Dexamethasone and 2,3,7,8-tetrachlorodibenzo-p-dioxin can induce thymic atrophy by different mechanism in mice. Biochem. Biophys. Res. Commun. 178, 16–23.[ISI][Medline]

McMartin, K. E., Ambre, J. J., and Tephly, T. R. (1980). Methanol poisoning in human subjects. Role of formic acid accumulation in metabolic acidosis. Am. J. Med. 68, 414–418.[ISI][Medline]

Medinsky, M. A., Singh, G., Bechtold, W. E., Bond, J. A., Sabourin, P. J., Birnbaum, L. S., and Henderson, R. F. (1990). Disposition of three glycol ethers administered in drinking water to male F344/N rats. Toxicol. Appl. Pharmacol. 102, 443–455.[ISI][Medline]

Miller, R. R., Carreon, R. E., Young, J. T., and McKenna, M. J. (1982). Toxicity of methoxyacetic acid in rats. Fundam. Appl. Toxicol. 2, 158–160.[Medline]

Miller, R. R., Hermann, E. A., Young, J. T., Landry, T. D., and Calhoun, L. L. (1984). Ethylene glycol monomethyl ether and propylene glycol monomethyl ether: Metabolism, disposition, and subchronic inhalation toxicity studies. Environ. Health Perspect. 57, 233–239.[ISI][Medline]

Morton, D., Weisbrode, S. E., Wyder, W. E., Maurer, J. K., and Capen, C. C. (1986). Spermatid giant cells, tubular hypospermatogenesis, spermatogonial swelling, cytoplasmic vacuoles, and tubular dilation in the testes of normal rabbits. Vet. Pathol. 23, 176–183.[Abstract]

Moss, E. J., Thomas, L. V., Cook, M. W., Walters, D. G., Foster, P. M. D, Creasy, D. M., and Gray, T. J. (1985). The role of metabolism in 2-methoxyethanol-induced testicular toxicity. Toxicol. Appl. Pharmacol. 79, 480–489.[ISI][Medline]

Nagano, K., Nakayama, E., Oobayashi, H., Nishizawa, T., Okuda, H., and Yamazaki, K. (1984). Experimental studies on toxicity of ethylene glycol alkyl ethers in Japan. Environ. Health Perspect. 57, 75–84.[ISI][Medline]

Poon, R., Chu, I., Lecavalier, P., Bergman, A., and Villeneuve, D. C. (1994). Urinary ascorbic acid—HPLC determination and application as a noninvasive biomarker of hepatic response. J. Biochem. Toxicol. 9, 297–304.[ISI][Medline]

Poon, R., Duc, V., and Vincent, R. (1995). N-acetyl-ß-D-glucosaminidase activity in bronchoalveolar lavage fluid: Characterization and response to ozone exposure. Inhalation Toxicol. 7, 1195–1206.[ISI]

Poon, R., Lecavalier, P., Chu, I., Yagminas, A., and Nadeau, B. (1999). Effects of bis(4-chlorophenyl) sulfone on rats following 28-day dietary exposure. J. Toxicol. Environ. Health, Part A 56, 185–198.

Poon, R., Moir, D., Elwin, J., Nadeau, B., Singh, A., Yagminas, A., and Chu, I. (2000). A study of the acid lability and acute toxicity of dimethoxymethane in rats. Int. J. Toxicol. 19, 179–185.[CrossRef][ISI]

Reader, S. C., Shingles, C., and Stonard, M. D. (1991). Acute testicular toxicity of 1,3-dinitrobenzene and ethylene glycol monomethyl ether in the rat: evaluation of biochemical effect markers and hormonal responses. Fundam. Appl. Toxicol. 16, 61–70.[ISI][Medline]

Short, B. G., Burnett, V. L., and Swenberg, J. A. (1989). Elevated proliferation of proximal tubule cells and localization of accumulated {alpha}-globulin in F344 rats during chronic exposure to unleaded gasoline or 2,2,4-trimethylpentane. Toxicol. Appl. Pharmacol. 101, 414–431.[ISI][Medline]

Smialowicz, R. J., Williams, W. C., Riddle, M. M., Andrews, D. L., Luebke, R. W., and Copeland, C. B. (1992). Comparative immunosuppression of various glycol ethers orally administered to Fischer 344 rats. Fundam. Appl. Toxicol. 18, 621–627.[ISI][Medline]

Somkuti, S. G., Lapadula, D. M., Chapin, R. E., and Abou-Donia, M. B. (1991). Light and electron microscopic evidence of tri-o-cresyl phosphate (TOCP)-mediated testicular toxicity in Fischer 344 rats. Toxicol. Appl. Pharmacol. 107, 35–46.[ISI][Medline]

Stern, B. R., and Tardiff, R. G. (1997). Risk characterization of methyl tertiary butyl ether (MTBE) in tap water. Risk Anal. 17, 727–743.[ISI][Medline]

Sun, X. M., Dinsdale, D., Snowden, R. T., Cohen, G. M., and Skilleter, D. N. (1992). Characterization of apoptosis in thymocytes isolated from dexamethasone-treated rats. Biochem Pharmacol. 44, 2131–2137.[CrossRef][ISI][Medline]

Trieberg, G., and Schaller, K.-H. (1980). A simple and reliable enzymatic assay for the determination of formic acid in urine. Clin. Chim. Acta 108, 355–360.[CrossRef][ISI][Medline]

Williams-Hill, D., Spears, C. P., Prakash, S., Olah, G. A., Shamma, T., Moin, T., Kim, L.Y., and Hill, C. K. (1999). Mutagenicity studies of methyl-tert-butylether using the Ames tester strain TA102. Mutat. Res. 446, 15–21.[ISI][Medline]

Wong, Y. N., Chien, B. M., and D’mello, A. P. (1994). Analysis of corticosterone in rat plasma by high-performance liquid chromatography. J. Chromatogr. B Biomed. Appl. 661, 211–218.[CrossRef][Medline]

Working, P. K., Bus, J. S., Hamm, T. E., Jr. (1985). Reproductive effects of inhaled methylene chloride in the male Fischer 344 rat. II. Spermatogonial toxicity and sperm quality. Toxicol. Appl. Pharmacol. 77, 144–157.[ISI][Medline]

Yagi, K. (1982). Assay for serum lipid peroxide level and its clinical significance. In Lipidperoxide in Biology and Medicine (K. Yagi, Ed.), pp. 232–242. Academic Press, New York.