A subchronic dermal exposure study of diethylene glycol monomethyl ether and ethylene glycol monomethyl ether in the male guinea pig

PUNDAMENTAL AND APPLIED TOXICOLOGY 6,339-348

(1986)

A Subchronic Dermal Exposure Study of Diethylene Glycol Monomethyl Ether and Ethylene Glycol Monomethyl Ether in the Male Guinea Pig’** D. W. HOBSON, A. P. D’ADDARIO, R. H. BRUNER, AND D. E. UDDIN Naval A4edical Research Institute, To,xicolog.v Detachment, Wrighl-Patterson

AFB, Ohio 45433

A Subchronic Dermal Exposure Study of Diethylene Glycot Monomethyl Ether and Ethylene Glycol Monomethyl Ether in the Mate Guinea Pig. HOBSON,D. W.. D’ADDARIO, A. P., BRUNER, R. H., .4ND UDDIN, D. E. (1986). Fundam. Appl. Toxicol. 6, 339-348. Diethylene glycot monomethyl ether (DEGME) has been selected as a replacement anti-icing additive for ethylene glycol monomethyl ether (EGME) in Navy jet aircraft fuel. This experiment was performed to determine whether DEGME produced similar toxicity to EGME following dermal exposure. Male guinea pigs were dermatly exposed to 1.OO,0.20, 0.04, or 0 (control) g/kg/day DEGME for 13 weeks, 5 days/week, 6 hr/day. Another group of animals was similarly exposed to 1.OOg/kg/day EGME. Body weights as welt as testicular and splenic weights were reduced as a result of exposure to EGME. DEGME-exposed animals exhibited decreased splenic weight in the high- and mediumdose (1 .OOand 0.20 g/kg/day) exposure groups only. Hematologic changes in EGME-exposed animals included mild anemia with increased erythrocytic mean corpuscular volumes and a tymphopenia with increased neutrophils. Similar hematotogical changes were not observed in any animals exposed to DEGME. Serum creatine kinase activity was increased in animals exposed to EGME, and serum lactate dehydrogenase activity was increased in EGME and 1.OOg/kg/day DEGME-exposed animals. In general, DEGME produced minimal toxicological changes following dennat exposure, whereas the toxicological changes observed following similar exposure to EGME were much more profound. 0 I986 Society ofToxicology.

Ethylene glycol monomethyl ether (EGME; 2-methoxy-ethanol; CAS No. 109-86-4) is a water-miscible solvent used in a wide variety of products including printing inks, textile dyes, leather finishes, epoxy resin coatings, and as an anti-icing additive in Navy jet fuels. EGME has been reported to cause testicular and thymic atrophy, decreased weight gain, and depressions of red blood cell count, hemoglobin concentration, packed cell volume, ’ This work was supported by the Naval Medical Research and Development Command. Research Task MR04 1220 10006. The opinions contained herein are those of the authors and are not to be construed as official or reflecting the views of the Navy Department or the Naval Service at large. The experiments described were conducted in accordance with the principles set forth in the current edition of the Guide for the Care and Use @“Laboratory Animals, Institute of Laboratory Animal Resources, National Research Council. ’ presented in part at the 23rd Annual Meeting of the Society of Toxicology, Atlanta, Georgia, March 1984.

and white blood cell counts in male rats and mice following inhalation or per OSexposure (Nagano et al., 1979; Miller et al., 198 1, 1983a). A recent summary concerning the reproductive toxicity of glycol ethers has been published (Hardin, 1983). Using in vitro techniques, Dugard et a/. (1984) demonstrated that EGME. as well as several other glycol ether compounds, readily penetrates human skin. An irz vivo teratology study conducted by Hardin el a/. (1982) demonstrated that ethylene glycol monoethyl ether (EGEE, 2-ethoxy ethanol; CAS No. I 10-80-5) can penetrate the skin of pregnant rats to the extent of producing significant teratogenic effects. Although EGME and a structurally related alternative fuel additive, diethylene glyco1 monomethyl ether (DEGME; 2,2 methoxy ethoxy ethanol: CAS No. 11 l-77-3) have derma1 exposure potential during fuel-transfer and tank-cleaning operations, toxicolo@cal

339

0272-0590186 $3.00 Copyright 0 I986 by the Society of Toxicology. All rig&?. of reprductim in my form rescrvd.

340

HOBSON

data regarding any potential health risk resulting from dermal exposure to these glycol ether compounds are sparse. The purpose for conducting the present study was twofold: (I) to determine whether EGME produced toxicity in male guinea pigs following subchronic dermal exposure similar to that previously reported for male rats and mice following inhalation or per os exposure and (2) to contrast toxicity resulting from subchronic dermal exposure to EGME to the toxic effects resulting from similar exposure to DEGME using the guinea pig. MATERIALS

AND

METHODS

C/IUF?&/.Y. EGME (Lot 0822BJ. 99% pure) and DEGME (Lot It 14HJ- 99% pure) were purchased from Aldrich Chemical Company. Inc. (Milwaukee, Wise.). ,4nwnols Male Hartley guinea pigs (6-8 weeks of age, 52 l-530 g) were purchased from Hilltop Lab Animals, inc. (Scottdate, Pa.). The animals were individually identified with ear tattoos, randomly assigned to control or exposure groups using a microcomputer-assisted randomization procedure, and allowed to acclimate for 2 weeks prior to exposure. Animals were housed two per cage in potycarbonate cages with hardwood coarse-grade bedding (Beta Chip, Northeastern Products Corp., Warrensburg. N.Y.) in a laminar flow room with controlled temperature (approximately 7O’F). humidity (approximately 40%). and light cycle (I2 hr light and dark). Water and food (Purina Guinea Pig Chow 5025. Ralston Purina Co., St. Louis. MO.) were provided ud lihitum during the exposure period. Each animal was weighed daily prior to dosing and examined for signs of intoxication. Experimenta/ design. The animals were divided into five dermal exposure groups: control (rr = 7, I.00 g/kg/ day. 0.9% saline), EGME (n = 6, I.00 g/kg/day), DEGME (n = 6, I.00 g/kg/day), DEGME (n = 6, 0.20 g/kg/day), DEGME (n = 6,0.04 g/kg/day), and were dosed 5 days/ week for I3 weeks. The doses of DEGME used in this study were selected on the basis that dermal exposure to I .OOg/kg/day. 5 days/week for I3 weeks was considered to be a limit dose greatly in excessofany anticipated human dermal exposure. The remaining two DEGME doses were geometrically spaced doses based on a factor of 5. This spacing was considered to provide an adequate range for evaluation of potential toxic responses elicited by the guinea pig at exposure levels greatly in excessof any anticipated human exposure to the pure material and at two geometrically spaced lower exposure levels. Even the lowest DEGME dose used, 0.4 g/kuday, probably still is in excess ofany routine occupational exposures resulting from fuelhandling operations involving jet fuels containing approximately O.l5%, by volume. DEGME or EGME.

ET AL. The I .OOg/kg/day dermal EGME dose was selected as a contrasting dose level because it was important to compare the two jet fuel additives on an equal dose basis. since DEGME is to replace EGME in the fuel in equal amounts. Additional dose levels of EGME were not considered to be essential for the purpose of this study because this dose was considered to be sufficiently high, with respect to potential human exposures, to determine whether dermal EGME exposure would produce significant toxicity in the male guinea pig relative to its proposed replacement additive, DEGME, and because the dermal penetration rates reported by Dugard et ul. (I 984) for EGME and DEGME indicate that EGME penetrates the skin more readily than DEGME. Test chemicals were applied neat to 2 X 2-cm gauze patches which were affixed to the shaved backs of guinea pigs with hypoallergenic surgical tape (Blenderm, 3M Company, St. Paul, Minn.), and then held in place for 6 hr by a stockinette bandage. Loss ofthe glycol ethers from the application site due to volatilization during the 6-hr exposure period was considered to be negligible due to the occlusive characteristics of the surgical tape/bandage combination used (i.e., the volatilization loss of 0.5 g EGME applied to 2 X 2-cm gauze patches, n = 6, covered with surgical tape in watch glassesheated to 35’C was less than 9% in 6 hr, whereas. uncovered. complete loss of the material due to volatilization occurred after I hr). The volatilization loss of DEGME was considered negligible relative to EGME due to its lower vapor pressure (EGME = 5.3 mm Hg @I25’C; DEGME = 0.18 mm Hg @ 25’C). At the end of the l3-week exposure period each animal was placed in a metabolism cage (fasted, water provided ad Mikun) for the collection of 24-hr urine samples and immediately prior to necropsy. The animals were euthanized via anesthetic overdose (Halothane, Halocarbon Laboratories. Inc., Hackensack. N.J.), whole blood samples were collected from the posterior vena cava, organ weights were recorded, and each animal was examined for gross pathological changes. The following tissues were submitted for histopathological examination: trachea. thyroid, lungs. heart, liver. spleen, thymus. lymph node, kidney. adrenals, salivary gland. nerve, urocyst, stomach, duodenum. jejunum, ileum. colon, testes, seminal vesicle, skin, muscle, and pancreas. All tissues were fixed in neutral buffered Formalin except the right testicles, which were weighed separately and placed in Bouin’s fixative. The tissues were subsequently processed to slides, stained with hematoxylin and eosin, and examined histopathologically. Clinical measurements. Hematological measurements for red blood cell counts (RBC), hemoglobin (Hgb), hematocrit (Hct), mean corpuscular volume (MCV). mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and white blood cell counts (WBC) were made using a Coulter Model S Plus (Coulter Electronics, Hialeah, Fla.). Differential white cell counts were obtained from whole blood smears stained with Wright’s stain. Serum and urine clinical chemistry measurements were obtained using commercially available assaykits (Worthington Diagnostic Systems,Inc., Freehold.

N.J.) adapted for use with a Cobas Bio centrifugal analyzer (Roche Analytical Instruments, Inc., Nutley, N.J.). Serum clinical chemistry parameters measured included alanine aminopeptidase (ALT), albumin, alkaline phosphatase (AP). aspartate aminotransferase (AST), blood urea nitrogen (BUN), creatine kinase (CK). y-glutamyltranspeptidase (GGTP), lactate dehydrogenase (LDH), calcium, cholesterol, creatinine? glucose. and total protein. Isoenzyme analysis was not performed. Commercially prepared control sera (Decision, Beckman Instruments, Inc., Brea, Calif.) were used to monitor the quality of clinical chemical assays. Serum testosterone was measured by radioimmunoassay (Immunochem. Carson, Calif.). Urine clinical chemical parameters measured were alkaline phosphatase, calcium, creatinine, alanine aminotransferase, and y-glutamyltranspeptidase (AP, ALT, and GGTP were used as enzyme markers to detect renal tubular epithelial cell damage). Prior to the determination of urinary calcium, urine samples were acidified to pH I .O with hydrochloric acid to release oxalate or phosphate-bound calcium. Urine specific gravity (Hand Protometer, National Instrument Co., Inc.. Baltimore, Md.) and pH (Model 801A Digital Ionalyzer, Orion Research, Inc., Cambridge, Mass.) were also recorded. Creatinine clearance (endogenous) was calculated from the corresponding serum creatinine, urinary creatinine. 24-hr urine volume, and surface area values obtained for individual animals. Surface areas were estimated on the basis of body weight using the method described by Hong et ul. (1977) for the guinea pig. Statistical methods. Serum and urinary clinical chemistry values, terminal body weights, organ weights, and hematological values were evaluated using one-way analysis of variance (ANOVA). Differences between treatment groups were analyzed using Duncan’s multiple-range test (Duncan, 1955). Daily body weight data was evaluated

using two-way analysis of variance, where dose and experimental day were the variables tested.

RESULTS Body Weight The mean body weights for each treatment group during the 13-week exposure period are shown in Fig. 1. Only EGME-treated animals exhibited a statistically significant (JJ < 0.01) decrease in weight gain relative to control animals. Organ Weights The mean weights of the testes and spleens from guinea pigs treated with EGME (1 .OOg/ kg/day) were significantly decreased (Table 1) when compared with controls. Animals in the two highest DEGME treatment groups (1 .OO and 0.20 g/kg/day) also showed significant decreases in mean splenic weight. In all cases, the decrease was also significant when expressed as organ weight per 100 grams of body weight. Clinical Chemistry Significant elevations in the mean values for serum creatine kinase (CK) and serum lactate

FIG. I. Mean body weights for guinea pigs dermally exposed to control (circle, I .OOg/kg/day, 0.9% saline), EGME (triangle, 1.00 g/kg/day), DEGME (solid square, 1.00 g/kg/day), DEGME (half-solid square 0.20 g/kg/day), and DEGME (open square 0.04 g/kg/day) for 13 weeks.

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HOBSON

ET

TABLE SUMMARY

OF TERMINAL

BODY

WEIGHT

EXPOSED

I

AND ORGAN

TO EGME

AL,

WEIGHT

OR DEGME Experimental

Weight parameter measured

(g) (gflO0 g body WI)

Pancreas (g) Pancreas (g/ IO0 g M

Prostate (g) Prostate (g/ IO0 g body wt)

Testes (g) Testes (g/ 100 g body wt)

group val~es’.~ DEGME

910.0

Seminal vesicles (g) Seminal vesicles (g/l00 body wt)

PIGS DERMALLY

P3.20 g/Wday)

1

t&

FOR GUINEA

Control

il

Liver Liver

DATA

FOR 90 DAYS

5 (87.8)

760.6

6 (125.9)*

863.3

(64.4)

DEGME (0.04 gjkgIday)

6 922.8

(76.3)

6 935.7

(90.7)

3.38 (0.27)

2.50

(0.35)

2.92 (0.43)

3.06 (0.29)

3.22 (0.42)

0.37 (.029)

0.33

(0.03)

0.34 (0.04)

0.33 (0.03)

0.35 (0.05)

30.03 (I .80) 3.3 1 (0.20)

26.34 3.46

(5.53) (0.39)

26.54 (3.51) 2.87 (0.23)

26.89 (4.38) 2.87 (0.38)

2.95 (0.89)

I.75

(0.75)

3.26 (0.47)

2.94 (0.80)

3.19 (0.74)

0.32 (0.08)

0.23

(0.09)

0.38 (0.07)

0.32 (0.08)

0.34 (0.06)

I.38 (0.25) 0. I5 (0.04)

0.82 0. I I

(0.26)* (0.02)*

I ,02 (0.19)* 0.12 (0.02)*

0.99 (o.l5)* 0.11 (o.ol)*

I.27 (0.39) 0. I4 (0.04)

2.02 (0.58)

I.07 IO (0.42)

I .66 (0.67)

I .75 (0.28)

1.84 (0.12)

0.22 (0.06)

0. I5

(0.08)

0. I9 (0.08)

0. I9 (0.02)

0.20 (0.02)

2.86 ( I ,O6)

I .69

(0.63)

3.07 (0.29)

2.80 (1.31)

3.23 (0.46)

0.31 (0.10)

0.23

(0.09)

0.36 (0.05)

0.31 (0.18)

0.36 (0.05)

2.60 (0.32) 0.29 (0.04)

0.62 0.08

(0.28)** (0.03)**

2.25 (0.26) 0.26 (0.03)

2.46 (0.14) 0.27 (0.03)

2.66 (0.36) 0.28 (0.03)

27.1 I (2.37) (n = 5) 3.19 (0.02) (n = 5)

g

a Values are mean (standard deviation). ’ g/l00 = g of tissue per IOQ g body weight. * Significantly different from control values (p c 0.05). ** Significantly different from control values (p c 0.0 I )

dehydrogenase (LDH) were evident in EGME ( 1.OOg/kg/day)-treated animals (Table 2). The highest DEGME treatment groups ( 1.OOg/kg/ day) also showed a significant mean increase in serum LDH. Hematology

Guinea pigs dermally exposed to EGME (1 .OO g/kg/day) for 13 weeks exhibited decreased RBC counts and increased MCV which were determined to be statistically significant (Table 3). A slight decrease in MCHC was also observed in DEGME (1 .OOg/kg/day)treated animals. Differential white cell counts indicated that a significant lymphopenia with neutrophilia was evident in EGME (1 .OO g/

kg/day)-dosed guinea pigs relative to control animals, Urinalysis

Guinea pigs treated with EGME (1 .OO g/ kg/day) and at all levels of DEGME exposure ( 1.OO,0.20, and 0.04 g/kg/day) exhibited significantly increased urinary calcium excretion (Table 4). No other significant changes were observed in the urine parameters measured. Gron Pathology

One EGME-treated animal became moribund by Day 50. Findings at necropsy were

EGME,

DEGME\

SUBCHRONIC

DERMAL TABLE

EXPOSURE

2

SLJMMARYOFTERMINAL SERUM CLINICALCHEMISTRYDATAFOR EXPOSEDTOEGMEORDEGMEFOR~ODAYS

EGME

serum paranwer measured

Cmtrol

PI

ALT

(W/liter)* Albumin (g/dl) AP (tU/liW AST (IU/liW BUN (mg/dl) Calcium (mg/dl) Choleswol (mg/dl) Creatmine (mg/dl) CK (W/liter) GGTP (W/liter) LDH (IU/liW Testos&erone (rig/ml) Total protein (g/dl)

49.3 3.3 71.1 30.9 25.7 8.8 60.4 I.1 103.6 7.0 53.7 5.84 7.08

I (18.1) (0.2) (15.9) (7.8) (3.9) (0.5) (22.8) (0.2) (52.1) (3.7) (29.5) (3.45) (0.69) (n = 6)

CI .oo d%kWl

71.2 3.3 65.4 33.4 31.6 8.7 25.4 I.0 322.2 9.6 144.2 4.20 6.74

5 (33.3) (0.3) (5.9) ( I I .3) (7.5) (0.9) (16.6) (3) (l64.5)** (2.3) (52.8): (2.53) (0.58)

DEGME t I .OO g/kudayI

62.2 3.7 76.5 33.5 25.8 9.2 43.2 I.1 129.7 9.3 134.5 6.89 6.3X

6 (30.7) (0.3) (12.81 (4.3) (5.0) (1.4) (16.7) (0.2) (87.6) (4.6) ( ! 00.4)’ (2. I I j (1.69)

GUINEAPIGSDERMALLY

DEGME (0.04 g/kg/day)

DEGME

CO.20 g/kg/day) 6S.? 3.6 70.2 34.8 23.7 9.2 45.3 I.1 73.8 5.8 68,X 5,07 6.90

0 Values are mea” (standard deviation). ’ 4LT = alanine amillopeplidase: AP = alkaline phosphatasc; AST = aspartate aminotransferase: = creative kinase; GGTP = gamma-~utamyltrans~ptida~; LDH = lactate dehydrogenase. * Significantly different from control values (p < 0.05). ** Significantly different from control values (p < 0.01).

inconclusive, but a severe decrease in red blood cells ( I .60 X 106/mm3) with greatly elevated MCH (84.3 pg) and MCHC (over working range) values were observed. In this animal, the surface of the glandular portion of the stomach had multiple brown ulcerations ranging from pinpoint size to approximately 0. I cm in diameter. At 13 weeks, the only significant treatmentrelated lesion observed was severe testicular atrophy in all animals treated with EGME. Several animals in each treatment group exhibited small, irregular, white, subcapsular foci on at least one lobe of the liver. This finding was not thought to be treatment related and was attributed to focal coagulative necrosis of the liver, a commonly observed lesion of unknown etiology in the guinea pig. (Wagner, 1976).

343

STUD\

6 (44.7) (0.3) (15.2) (8.3) (2.2) (0.4) (14.3) (0.1) (41.9) (3.9) (31.1) (2.71) (0.24)

BUN

47.4 3.4 71.6 33.3 25.0 8.8 47.9 I.0 99.0 IO.8 55.4 3.12 6.58

6 (26. I) (0.2) (12.3) (8.7) (3.1) (0.4) (19.7) (0.2) (57.9) (3.7) (n = 5) (33.5) (2.24) (0.74)

= blood urea nitrogen:

CK

treated animals (Fig. 2). These degenerative changes were characterized by complete loss of spermatogenic cells while Sertoli cells and interstitial (Leydig) cells remained largely unaffected. Testicular lesions were not recorded in DEGME-treated animals (Fig. 3). The only significant histopathologic change noted in the DEGME-exposed animals occurred in the liver and consisted of mild, periportal, hepatocellular fatty change. Although these findings were regarded as evidence for mild, reversible DEGME-induced hepatotoxicity, it was noted that fatty deposits did not occur in a distinctly dose-related fashion. No morphological changes indicative of toxic insult to the lymphoid system were noted in the lymphoid tissues examined (spleens, thymuses, and lymph nodes). The incidence of testicular and hepatic findings is shown in Table 5.

Histopatholog) Testicular lesions, consisting of moderateto-severe segmental degeneration of the seminiferous tubules, were observed in all EGME-

DISCUSSION The results obtained from this study demonstrate that guinea pigs dermally exposed to

344

HOBSON

ET AL.

TABLE 3 SUMMARY OF TERMINAL HEMATOLOGICAL DATA FOR GUINEA PIGS DERMALLY EXPOSED TO EGME OR DEGME FOR 90 DAYS Experimental group values0 Hematological parameter measured

Control

,, RBC (X106/mm3)b

1

5

5.94 (0.22)

5.35 (0.72)*

H!$

k/d~)

Hct (7%) MCV (pm3) TVI~I

m

MCHC (9%) WBC (X 103/mm3) Basophils’ Eosinophils’ Lymphocytes’ Monocytes’ Neutrophils‘

EGME C1.NI dMW

DEGME t 1.OOg/kg/day)

DEGME

W20 dWdad

6 6.09

6 (0.36)

6.05

(0.25)

DEGME (0.04 g/kg/day) 6 5.76 (0.22)

16.5 47.3

(0.6) (1.9)

15.5 45.1

(1.5) (5.2)

16.6 49.2

(1.1)

16.2 (1.2)

(3.1)

48.7

(2.6)

16.2 46.6

(0.6) (1.6)

79.4 27.8

(3.2) (1.2)

84.5 29.2

(3.6)* (1.6)

80.7 27.3

(1.2) (0.6)

so.4 26.8

(2.8) (1.9)

80.9 28.1

(1.7) (0.6)

34.9 7.7

(0.7) (3.4)

34.5 6.3

(1.0)

33.8 7.6

(0.6)* (1.2)

33.3 7.9

(1.8) (3.2)

34.7 7.4

(0.4) (2.0)

0 Ku 0.1 (0.2)

(1.1) 0 (0) 0.1 (0.1)

5.6 0.3

(2.9) (0.2)

2.8 0.2

1.7

(0.7)

3.2

cl m 0.1 (0.1)

0 0

(0) co.11

0 (0) 0.1 (0.1)

(I.])* (0.3)

5.2

0.2 (0.1)

4.5 0.5

(1.8) (0.4)

4.5 0.2

(0.7) (0.2)

(0.5)*

2.1

2.9

(1.1)

2.6

(1.4)

(1.5) (0.7)

” Values are given as mean (standard deviation). * RBC = red blood cells: Hgb = hemoglobin: Hct = hematocrit: MCV = mean corpuscular volume: MCH = mean corpuscular hemoglobin: MCHC = mean corpuscular hemoglobin concentration: WBC = white blood cells. ’ Values are given as absolute numbers X 103/mm3. * Significantly different from control values (p < 0.05).

TABLE 4 SUMMARY OF URINE CLINICAL CHEMISTRY DATA FORGUINEA PIGS DERMALLY EXPOSED ‘TO EGME OR DEGME FOR 90 DAYS Experimental group values’ Urine parameter measured n 24-hr volume (ml) PH Specific gravity (g/ml) AP (RJ/day)’ Calcium (mg/day) Creatinine (mg/day) ALT (RI/day) GGTP (KJ/day) Creatinine clearance (ml/min/m’)

Control 7 28.7 8.7 I.029 0.12 I.4

(6.0) (0.6)

21.2

5 (6.1)

7.8

(1.3)

(0.006) (0.09) (0.6)

l.O30(0.008) 0.17 (0.06) 3.3 (l.l)* 16.9 (2.6)

20.8 0.09 0.64

(8.1) (0.07) (0.15)

15.9

(7.0)

0.09 0.38 15.5

4 21.5 8.0 1.033

(3.1) (0.8) (0.005)

0.16 4.2 19.0

(0.09) (2.2)* (1.6)

(0.05) (0.15)

0.14 0.41

(0.09) (0.19)

(6.6)

9.2

(3.2)

21.3

6 (5.6)

(0.8) (0.008)

8.7

(0.4)

0.13 3.8 II.7 0.09

(0.06) (1.9)* (5.6) (0.05)

0.12

(0.07)

3.9 14.2 0.16

t l.o)*

0.43

(0.20)

0.52

(0.12)

8.6

(3.3)

11.5

(4.8)

17.6 8.0 I.027

5 (8.4)

a Values are given as mean (standard deviation). ’ AP = alkaline phosphatase: ALT = alanine aminotransferase; GGTP = y-glutamyltranspeptidase * Significantly different from control values (p < 0.05).

I.026 (0.004)

(5.8) (0.07)

EGME. DEGME,

SUBCHRONIC

DERMAL

EXPOSURE

STUDY

345

FIG. 2, Section of testes showing the effect of EGME (I .OOg/kg/day) applied dermaily to male guinea pigs. 5 days/week for 13 weeks. H & E, X400.

EG ME (1 .OOg/kg/day) for 13 weeks exhibit dec reased body, testicular, and splenic weights; test.icular atrophy and degeneration; lympho-

penia with elevated neutrophils; and decreased red blood cell counts. These effects are consistent with the findings of previous studies

346

HOBSON

ET AL

FIG. 3. Section of testes from a male guinea pig dermally exposed to DEGME (I .OOg/kg/day, 5 days/ w ,eek for 13 weeks) showing essentially normal morphology. H & E. X400.

with r.ats, rabbits, and mice subchronically exposed I to EGME via the oral or inhalation route (Nagano et ul., 1979: Miller et uf., 1981, 1983: Q. In contrast. dermal exposure to

DEGME (1 .OO,0.20, or 0.04 g/kg/day) fo br 13 weeks does not result in the production of toxicological changes similar to those seen with EGME in the guinea pig with the excepItion

EGME,

DEGME.

SUBCHRONIC

DERMAL TABLE

INCIDENCE

Experimental

EXPOSURE

5

OF HISTOPATHOL~~ICAL CHANGES OBSERVED IN GUINEA TO DEGME OR EGME FOR 13 WEEKS

group

Segmental degeneration of seminiferous tubules

347

STUDY

PIGS DERMALLY

Mild, periportal. hepatocellular fatty change

EXPOSED

Focal coagulation necrosis of the liver

Control EGME (1 .OO g/kg/day) DEGME (1 .OO g/kg/day) DEGME (020 g/kg/day) DEGME

(0.4 g/kg/day)

of decreased splenic weight, which was observed in 1.00 and 0.20 g/kg/day DEGMEtreated animals. increased serum LDH activity seen after exposure to EGME and DEGME ( 1.OOg/kg/ day) may be related to the effects of EGME on hematopoiesis reported in previous studies (Werner et ul., 1943: Cullen et ul., 1983; Miller et u/., 198 1) or increased erythrocyte membrane fragility caused directly by EGME. Miller et al. (198 1) have reported, however, that red blood cell osmotic fragility of male and female rats exposed to 1000 ppm EGME vapor (6 hr/day, for 9 days during an 1 l-day period) was not significantly different from that of control animals. Since LDH isoenzyme analysis was not performed, it is difficult to attribute the increases observed in serum LDH to particular tissue source. Serum CK activity was elevated in animals exposed to EGME, no source of tissue damage or other source of increased serum levels of this enzyme could be determined, other than the possibility that this finding might be related to the testicular atrophy observed in this treatment group. Elevated urinary calcium levels were observed following exposure to both EGME and DEGME. The reason for this is not readily apparent as there was no evidence of increased resorption of bone, renal mineralization, or renal damage. It seems likely, however, that excretion of acid metabolites of EGME such as methoxyacetic acid (Miller et cd., 1983b; Moss et al., 1985) or possibly methoxyethoxy-

acetic acid from DEGME via the urine may result in the formation of calcium salts. The formation of these calcium salts would then lead to increased urinary calcium levels by decreasing the number of calcium ions available for tubular reabsorption by the kidney. It is already known that diglycolic acids and triglycolic acids are calcium chelators which tend to form stable, soluble, calcium complexes (Ancillotti et ul., 1977). Herold et ul. (1982) have observed that topical application of polyethylene glycol to rabbits results in the formation of mono- and diglycolic acid metabolites which chelate calcium and produce increased, compensatory total serum calcium concentrations with normal or decreased ionized serum calcium levels. Since we observed hypercalciuria, but not hypercalcemia, in the glycol ether-exposed animals, we conclude either that our level of exposure was sufficient only to increase the excretion of calcium into the urine without significantly affecting the serum calcium pool or that the rate of glycol ether metabolism is sufficiently high in the kidney to the extent that hypercalciuria results from sequestration of calcium ions by acid metabolites of the glycol ethers produced in the kidney and excreted via the urine. The significant lymphopenia and concomitant observation of decreased splenic weight suggest that EGME may be toxic to lymphoid organs. House et al. (1985) reported, however, that EGME or its principal metabolite, methoxyacetic acid, did not produce significant alterations in immune function or host resis-

348

HOBSON

tance to Listeria rnonocytogenes in mice exposed via gavage to doses of 0.25, 0.50, and 1.O mg/kg of body weight ( 10 doses over a 2week period). They did observe a reduction in thymus weight in mice exposed 10 the high and intermediate doses of each chemical. No explanation for this result was given, but it was concluded that thymic atrophy produced by the glycol ethers should not necessarily be interpreted as being indicative of a decrease in immune function or host resistance. There appears to be a need for further research concerning the mechanism of lymphoreticular toxicity of the glycol ethers. Dermal exposure of male guinea pigs to EGME (1 .OO gfkg/day, 5 days/week) for 90 days produces testicular lesions identical to those reported to occur following exposure via other routes in rats, mice, and rabbits. The structurally related glycol ether compound, DEGME, does not appear to produce testicular lesions when administered at dose levels as high as 1.00 gfkg/day, 5 days/week, for 90 days. These results, therefore. would tend to support the use of DEGME as a replacement for EGME in jet aircraft fuels as well as in other applications where such replacement is feasible and a potential for dermal exposure exists. ACKNOWLEDGMENTS

ET AL, SCOTT,R. C. ( 1984). Absorption of glycol ethers through human skin ill vim. Emirm. Hcmlth Perspect. 57, 193197.

DLJNCAX D. B. (1955). Multiple range and multiple F tests. f3wnmics 11, l-42. HARDN, B, D, (1983). Reproductive toxicity ofthe glycol ethers. Tm~ico/ogy 27, 9 l-102. HARDIN, B, D., NIEME~ER.R. W,. SMITH, R, W.%KLJCZUK, M, H., MATH~NOS.P. R.? AND WEAVER, T. F, ( 1982). Teratogenicity of 2-ethoxyethanol by dermal application. Drug Chem. To.\-ico[. 5, 277-294. HEROLD. D. A., RODEHEAVER, G. T., BELLAMY, W. T., FITTON, L. A.. BRUNS,D. E,, AND EDUCH~R. F. ( 1982). Toxicity of topical polyethylene glycol, Tmico/. .+p/ Phurmao~l.

65, 329-335.

HONG. C. C., EDIGER? R. D,. RAETZ, R,, AND DJURICKOVIC, S. (1977). Measurement of guinea pig body surface area. Luh .4nim. sci. 27, 474-476. HOUSE, R. V.? LAUER, L. D,. MURRAY, M. J.. WARD. E. C.. AND DEAN, J. H. (1985). Immunological studies in B6C3Fl mice following exposure to ethylene glycol monomethyl ether and its principal metabolite methoxyacetic acid. To.~iw/. .-tppl. Pharmacol. 77, 358-362. MILLER, R, R., AYRES, J. A., CALHOUN, L. L.%YOUNG, J. T,, AND MCKENNA, M. J. (I98 I), Comparative shortterm inhalation toxicity of ethylene glycol monoethyl ether and propylene glycol monomethyl ether in rats and mice. To.yicol. ‘4~~1. Phaimaco!. 61, 368-377. MILLER. R. R.. AYRES, J. A.? AND YOUNG, J. T. (1983a). Ethylene glycol monomethyl ether. I. Subchronic vapor inhalation study with rats and rabbits. Fundam App/. Tmicol.

3, 49-54.

MILLER, R, R.. HERMANN, E. A., LANGVARDT, P. W.. MCKESNA. M. J., AND SCHWETZ,B. A. (1983b). Comparative metabolism and disposition of ethylene glycol monomethyl ether and propylene glycol monomethyl ether in male rats. Toxicol. Appi. Pharrnacol. 67, 229237.

The authors thank Dr. T. Hickey, J. Stewart, L. Whalen, D. Morris, S. Perkins, S. Barre& C, Stewart* and D. Henn for their contributions toward the completion of this work.

Moss, E. J., THOMAS, L. V.. COOK, M. W., WALTERS, D. G.. FOSTER,P. M. D.. CREASY. D. M., AND GRAY, T. J, B. (1985). The role of metabolism in 2-methoxyethanol-induced testicular toxicity. ToxicoI. App/.

REFERENCES

NAGANO, K., NAKAYAMA, E.. KOYANO, M., OOBAYASKI, H.. ADACHI, H.%AND YAMADA, T. (1979). Mouse testicular atrophy induced by ethylene glycol monoalkyl ethers. Japan J. Ind. Heuhh 21, 29-35. WAGNER, J. E. (1976). Miscellaneous disease conditions of guinea pigs, In k3iobg.v qffhe Guitlea Pig (J. E. Wagner and P. J. Manning, eds.), pp. 227-234. Academic Press, New York. WERNER, H. W., MITCHELL, J. L,? MILLER, J. W.. AND VON OETT’ENGEN.W. F. (I 943). The acute toxicity of vapors of several monoalkyl ethers of ethylene glycol. J. fnd. ffy~g. Tmicol. 25, 157-163.

ANCILLOTTI, E,, BOSCHI,G., PEREGO,G., AND ZAZZE~A, A, ( 1977). Calcium binding capacity of carboxylic acids with an ethereal function. J. C’&?n. %c. Da/fan Tram 9,901-905.

CULLEN, M. R.. RADDO, T,, WALDRON, J. A., SPARER, J., AND WELCH, L, S. (1983). Bone marrow injury in lithographers exposed to glycol ethers and organic solvents used in multicolor offset and ultraviolet curing printing processes.Arch. Environ. Heallh 38, 347-354. DUGARD, P. H.. WALKER, M., MAWDSLEY, S. J., AND

Pharmacol

79, 480-489.