Environmental Health Perspectives

Vol. 57, pp. 69-74, 1984

Developmental Toxicity of Four Glycol Ethers

Applied Cutaneously to Rats

by Bryan D. Hardin,* Phillip T. Goadt and JeAnne R. Burg*

Previous NIOSH studies demonstrated the embryo- and fetotoxicity and teratogenicity of ethylene glycol

monoethyl ether (EGEE) applied to the shaved skin of pregnant rats. In the present study ethylene glycol

monoethyl ether acetate (EGEEA), ethylene glycol monobutyl ether (EGBE), and diethylene glycol

monoethyl ether (diEGEE) were tested in the same experimental model, using distilled water as the

negative control and EGEE as a positive control. Water or undiluted glycols were applied four times daily

on days 7 to 16 of gestation to the shaved interscapular skin with an automatic pipetter. Volumes of EGEE

(0.25 mL), EGEEA (0.35 mL), and diEGEE (0.35 mL) were approximately equimolar (2.6 mmole per

treatment). EGBE at 0.35 mL four times daily (approximately 2.7 mmole per treatment) killed 10 of 11

treated rats, and was subsequently tested at 0.12 mL (0.9 mmole) per treatment. EGEE- and EGEEAtreated rats showed a reduction in body weight relative to water controls that was associated with

completely resorbed litters and significantly fewer live fetuses per litter. Fetal body weights were also

significantly reduced in those groups. Visceral malformations and skeletal variations were significantly

increased in EGEE and EGEEA groups over the negative control group. No embryotoxic, fetotoxic, or

teratogenic effects were detected in the EGBE- or diEGEE-treated litters.

Introduction

The alkyl ether derivatives of ethylene and diethylene

gylcol are an important class of solvents with numerous

applications in consumer products as well as in industrial processes. Several members of this chemical family

have recently been shown to be potent reproductive

toxins in several animal species, as reviewed in detail

elsewhere (1,2). Teratogenicity has been reported for

ethylene glycol monomethyl ether (EGME) in mice

(3) and rats (4); for ethylene glycol dimethyl ether

(EGdiME) in mice (5); for ethylene glycol monoethyl

ether (EGEE) in rabbits (6) and rats (6-8); and for

ethylene glycol monoethyl ether acetate (EGEEA) in

rats (4). Evidence of testicular toxicity has been reported for EGME in mice (9-11), rats (10-12), and

rabbits (12); for ethylene glycol monomethyl ether

acetate (EGMEA) in mice (9); for EGEE in mice (9),

rats (8), and dogs (8); for EGEEA in mice (9); and for

diethylene glycol dimethyl ether (diEGdiME) in mice

and rats (13). In contrast, ethylene glycol monobutyl

ether (EGBE) and diethylene glycol monoethyl ether

(diEGEE) both have been tested once by inhalation

exposure for teratogenicity (4) with negative results.

EGBE and ethylene glycol monophenyl ether (EGPhE)

*Department of Health and Human Services, U. S. Public Health

Service, Centers for Disease Control, National Institute for Occupational Safety and Health, Division of Biomedical and Behavioral

Science, 4676 Columbia Parkway, Cincinnati, OH 45226.

tIntox Laboratories, Inc. 914 Barber Street, Little Rock, AR 72203.

have been tested for testicular toxicity with negative

results (9).

The glycol ethers do not have especially high vapor

pressures (14), but they penetrate the skin readily and

are commonly used in applications that are conducive to

skin contact. Hardin et al. (7) applied undiluted EGEE

to the skin of pregnant rats to investigate its potential

for developmental toxicity by this route of exposure.

When 0.5 mL was applied four times daily on days 7 to

16 of gestation, all pregnant females had completely

resorbed litters. Treatment with 0.25 mL on the same

schedule induced significant embryotoxic, fetotoxic, and

teratogenic effects (7). The cardiovascular system was

the primary target for visceral malformations, as had

been previously reported in rats (4,6) and rabbits (6)

exposed to EGEE by inhalation. The present study was

conducted to evaluate the reproductive and developmental toxicity of three other alkyl glycol ethers in the same

2utaneous exposure model.

Methods and Materials

Purified glycol ethers were purchased from Fisher

Scientific: EGEE, Catalog No. E-180, Lot 70107;

EGEEA, Catalog No. E-181, Lot 794501; EGBE,

Catalog No. E-179, Lot 703549; and diEGEE, Catalog

No. D-51, Lot 792796. Time-mated primigravida SPF

Sprague-Dawley rats were purchased from Charles

River Breeding Laboratories, Wilmington, MA. Rats

were shipped on day 4 and received on day 5 of gestation
HARDIN, GOAD AND BURG

(sperm = day 1), when they were weighed and marked

for individual identification by toe clipping. The interscapular region was shaved with electric clippers on day

5 or 6, and rats were formally randomized by weight to

one of five groups. All rats were singly housed throughout the study in suspended stainless steel cages in a

ventilated cage rack (Hazelton Systems, Aberdeen,

MD) with free access to food and water. These cages are

designed such that room air was drawn through the

wire mesh floors of each cage and into an overhead

plenum from which the exhaust air was ducted directly

to a fume hood. Light was on a 12-hr light-dark cycle.

Rats were received in two separate shipments in

consecutive weeks and the experiment was conducted in

two replicates. Tieatment volumes were selected based

on results of the earlier study of the teratogenicity of

EGEE when applied to the skin of rats (7). A volume of

EGEE (0.25 mL four times daily) that was teratogenic

in that study was used as the positive control. Approximately equimolar volumes (0.35 mL four times daily, for

a total daily dose of 1.4 mL) of EGEEA, EGBE, and

diEGEE were employed initially. Because the rats

treated with 0.35 mL of EGBE showed marked toxic

effects, the treatment volume was reduced in the

second replicate to 0.12 mL EGBE four times daily.

Undiluted glycols or water (control) were applied to the

shaved interscapular region using an automatic pipetter

(Finpipette) four times daily at 2.5-hr intervals on days

7 to 16 of gestation.

In addition to the randomization body weights (day 5

of gestation), body weights were recorded on the first

and sixth days of treatment and on the first and fifth

post-treatment days (gestation days 7, 12, 17 and 21,

respectively). On day 21, females were killed by CO2

asphyxiation and gravid uterus weights were recorded.

Fetuses were weighed and examined for external

malformations. Alternate fetuses were preserved in

Bouin's fluid or 70% ethanol for subsequent visceral

examination by the Wilson (15) method or skeletal

examination according to Staples and Schnell (16).

Uteri from apparently nonpregnant females were placed

in 10% sodium sulfide to reveal early termination of

pregnancy

All maternal and fetal body weights, maternal organ

weights were analyzed by analysis of variance techniques. If significant (p < 0.05) differences between

groups were detected, a posteriori Waller-Duncan

K-ratio t-tests were conducted to describe in more

detail the source of variation. Implantation data (total

and numbers of live and dead) were analyzed using the

Kruskal-Wallis test. Frequencies of malformations and

of variations were analyzed by Fisher-Irwin exact test

of the numbers of affected litters. Because repeated

contrasts were made with the water control group, the

level of significance was set at p < 0.01. All procedures

were carried out using SAS 82.3 and BMDP statistical

packages on the Parklawn Computer Center, Rockville, MD.

Results

Toxic signs were not noted in EGEE-, EGEEA-, or

diEGEE-treated rats. However, red-stained urine was

observed after only two treatments with 0.35 mL

EGBE. By the end of the first treatment day, EGBEtreated rats produced burgundy-colored urine. Continued treatment with EGBE led to ataxia progressing to

moderate to marked inactivity and rough haircoats with

dark stains around the muzzle and ano-genital area.

Several rats' tails blackened distally and were gradually

eaten away as the apparent necrosis progressed. Deaths

occurred on the third to seventh days of treatment (days

Table 1. Gestational body weights (mean ± SD) of pregnant rats in various treatment groups.

Treatment group

EGEE EGEEA EGBEa diEGEE Water

(0.25 mL) (0.35 mL) (0.12 mL) (0.35 mL) (control)

Group size 18 18 9 13 17

Body weight, g

Day 5 188 ± 8 192 ± 9 192 ± 10 193 ± 7 189 ± 7

Day 7 210 ± 7 212 ± 12 219 ± 12 213 ± 10 210 ± 7

Day 12 239 ± 10 240 ± 14 239 ± 13 242 ± 12 246 ± 11

Day 17 262 ± 15 256 ± 17 268 ± 16 269 ± 17 278 ± 14

Day 21 284 ± 32 268 ± 29 336 ± 30 323 ± 31 331 ± 22

Body weight gain, g

Day 5-7 22 ± 8 20 ± 7 27 ± 6 19 ± 8 21 ± 5

Day 5-12* 51 ± 9xy 48 ± 9Y 48 ± 12Y 49 ± 7x,y 57 ± lOx

Day 5-17t 74 ± 13Y 65 ± 12z 77 ± 18Y 76 ± 13Y 89 ± 11x

Day 5-21t 97 ± 29Y 76 ± 27Z 144 ± 31x 130 ± 27x 142 ± 19x

Gravid uterus weight, gt 23 ± 21Y 9 ± 12z 59 ± 22X 65 ± 20X 60 ± 15x

Extragestational body weight, gb 261 ± 18 259 ± 24 276 ± 18 258 ± 18 271 ± 15

Extragestational body weight gain, g* 74 ± 15x y 67 ± 20v 85 ± 18X 65 + 16Y 82 ± 15x

aReplicate 2 only. All other groups are combined data for replicates 1 and 2.

bDay 21 body weight minus gravid uterus weight. cExtragestational body weight minus day 5 body weight.

*Significant difference (p < 0.05). Groups with the same superscript do not differ from each other by a posteriori test.

tSignificant difference (p < 0.001). Groups with the same superscript do not differ from each other by a posteriori test.

70
DEVELOPMENTAL TOXICITY OF CUTANEOUSLY APPLIED GLYCOLS

9-13 of gestation). In replicate 1, only one of 11 rats

treated with 0.35 mL EGBE survived. In the second

replicate, the EGBE treatment volume was reduced to

0.12 mL four times daily (0.48 mL EGBE/day) and no

toxic signs were noted.

Body weights of pregnant females are summarized in

Table 1, along with gravid uterus and extragestational

body weight (day 21 body weight minus gravid uterus

weight). Day 5 (randomization) body weights did not

differ between replicates and body weight data were

therefore combined across replicates for analysis. Analysis of variance confirmed that body weights on day 5 did

not differ across treatment groups. Body weights on

days 7, 12, 17, and 21 were analyzed by multivariate

analysis of the change relative to day 5. Treatment with

EGEE and EGEEA profoundly reduced maternal body

weight gain, and at days 17 and 21 body weight gain in

the EGEEA group was significantly lower than in the

EGEE positive controls. Gravid uterus weights were

also markedly reduced in those groups, accounting for

much of the difference in body weight. Extragestational

body weights did not differ significantly (p < 0.10),

but extragestational body weight gain was significantly

different across groups (p < 0.05). Extragestational

weight gain in EGEEA and diEGEE groups was

significantly less than in the water controls by a

posteriori test.

Data in Table 2 reveal that, relative to the water

controls, both EGEE and EGEEA treatments were

strongly embryotoxic as reflected in significantly higher

frequencies of completely resorbed litters and increased

numbers of dead implants per litter. The number of

dead implants per litter was also significantly higher in

EGEEA- than in the positive control EGEE-treated

litters. Considering only litters with at least one live

fetus, there was a significant reduction in the number of

live fetuses per litter in the EGEE- and EGEEAtreated groups. Similarly, the body weights of live

fetuses in these two groups were significantly reduced

relative to the water-treated controls. No evidence of

embryo- or fetotoxicity was noted in the EGBE and

diEGEE groups. On gross examination of fetuses, the

only malformations noted were three fetuses from

EGEE-treated litters with acaudia and imperforate

anus (Table 2).

Discussion

The results of this study confirmed the previously

reported embryo- and fetotoxicity and teratogenicity of

EGEE applied to the skin of pregnant rats. As was

previously reported, cardiovascular defects were the

predominant visceral malformation. A wide spectrum of

skeletal variants was also seen both in the present and

the earlier skin teratology evaluation of EGEE. Vertebral malformations and reduced skeletal ossification

also occurred with increased frequency in EGEEtreated litters but were not statistically significant. The

only gross malformations noted in the study were three

EGEE-treated fetuses with acaudia and imperforate

anus, a defect also seen in the previous evaluation of

cutaneously applied EGEE.

The spectrum and frequency of malformations and

variations in the EGEEA-treated litters was remarkably similar to that described for the EGEE-treated

group. Statistically significant increases in cardiovascular malformations and skeletal variations were detected.

Similar responses to EGEE and EGEEA are not

unexpected since EGEEA should be rapidly hydrolyzed

in vivo to EGEE and acetate. At an equimolar dose, in

fact, EGEEA in this study caused even more severe

maternal, embryo, and fetal toxicity than did EGEE.

This was reflected in significantly reduced in body

weight gain (Table 1) and increased dead implants per

litter (Table 2) in EGEEA-treated litters relative to the

EGEE litters.

Neither EGBE nor diEGEE was associated with

embryo- or fetotoxicity, and the incidence of visceral

and skeletal defects was not increased in either group.

Table 2. Observations at necropsy.

TIeatment group

EGEE EGEEA EGBEa diEGEE Water

(0.25 mL) (0.35 mL) (0.12 mL) (0.35 mL) (control)

Group size 18 18 9 13 17

No. (%) litters totally resorbed 7*(38.9) 13*(72.2) 0 0 0

No. implants per litterb 11.6 ± 2.9 11.7 ± 2.7 11.2 ± 4.3 11.8 ± 1.7 11.6 ± 2.5

No. dead implants per litter"t 7.7 ± 5.1y 10.6 ± 3.3x 1.2 ± 1.0Z 1.2 ± 1.2z 1.2 ± 1.2z

No. live fetuses per litter with

live fetuses"t 6.5 ± 4.1y 4.2 ± 3.4Y 10.0 ± 3.8x 10.6 ± 2.3x 10.4 ± 2.6X

(no. litters) (11) (5) (9) (13) (17)

Fetal body weight, gbt 3.0 ± 0.6y 2.6 ± 1.0v 4.2 ± 0.7x 3.9 ± 0.8x 3.9 ± 0.6x

Gross malformations

litters (fetuses) 3(3)C 0 0 0 0

aReplicate 2 only. All other groups are combined data for replicates 1 and 2.

bMean ± SD. cAcaudia and imperforate anus.

*Differs significantly (p < 0.01) from water controls.

tSignificant differences (p < 0.001). Groups with the same superscript do not differ from each other (p > 0.05) by a posteriori test.

71
HARDIN, GOAD AND BURG

EGBE is a potent hemolytic agent in rodents, as

reflected in the rapid onset of red-stained urine (within

5 hr of first dosing) and subsequent death of rats

receiving 0.35-mL doses. Equimolar volumes of EGEE,

EGEEA and diEGEE caused no mortality and no

clinical signs of toxicity. The extragestational body

weight gain (Table 1) of diEGEE-treated rats was

significantly reduced, to a degree comparable to the

reduction the EGEEA-treated rats. Thus, while diEGEE

was not embryo- or fetotoxic, it was maternally toxic

under these test conditions.

In summary, the results of this study confirm that

EGEE can be absorbed in embryotoxic, fetotoxic, and

teratogenic quantities through the intact skin of pregnant rats. EGEEA was shown to share these properties

and to be at least as active as EGEE by this route. It

has been previously reported that inhalation exposure

of pregnant rats to EGBE resulted in no developmental

toxicity, and none was detected here at approximately

one-third the molar dose used for EGEE and EGEEA.

A treatment volume of EGBE equimolar to the EGEE

and EGEEA volume was strongly hemolytic and ultimately lethal, demonstrating that EGBE also penetrates the skin readily. There also was no measurable

developmental toxicity in rats treated with diEGEE,

despite administration at a dose causing slight maternal

toxicity. These data demonstrate that reproductive and

developmental toxicity, like other manifestations of

glycol ether toxicity, are possible following cutaneous

exposure.

Table 3. Visceral malformations and variations: number of litters (fetuses) affected.

Treatment Group

EGEE EGEEA EGBEa diEGEE Water

(0.25 mL) (0.35 mL) (0.12 mL) (0.35 mL) (control)

Tbtal examined 10(34) 3(10) 8(45) 13(70) 17(87)

Malformations

Cardiovascular: total 8(9)t 3(5)t 0 0 0

Right aorta 4(4)b 2(2) 0 0 0

Double aorta 1(1) 1(1) 0 0 0

Right ductus 3(3)b 1(1) 0 0 0

Ventricular septal defect 5(5) 3(5) 0 0 0

Abnormal subelavian 1(1) 0 0 0 0

Renal: total 7(8)* 1(2) 0 2(3) 3(3)

Hydronephrosis 4(4) 1(1) 0 1(2) 0

Hydroureter 4(4) 0 0 1(2) 3(3)

Ectopic kidney 1(2) 1(1) 0 1(1) 0

Hypoplastic kidney 0 1(1) 0 0 0

Fused kidneys 1(1) 0 0 0 0

Brain: total 4(4)* 1(2) 0 2(3) 0

Hydrocephalus 2(2) 1(2) 0 2(3) 0

Hemorrhage 2(2) 0 0 0 0

Eye: total 2(2) 2(2) 0 0 0

Microophthalmia 1(1) 0 0 0 0

Abnormal optic nerve 1(1) 0 0 0 0

Folded retina 2(2) 2(2) 0 0 0

Tobtal malformations 10(21)t 3(8) 0(0) 3(5) 3(3)

Variations

Cardiovascular

Missing innominate 2(2) 1(1) 0 0 0

Renal: total 5(7) 0 4(5) 6(13) 10(15)

Dilated renal pelvis 2(3) 0 0 3(6) 6(7)

Dilated ureter 4(5) 0 4(5) 4(9) 7(10)

Tbtal variations 5(8) 1(1) 4(5) 6(13) 10(15)

Testicular defects

Males examined 21 6 23 40 39

Testicular: total 3(3) 1(1) 1(1) 4(4) 1(1)

Ectopic (slight) 2(2) 1(1) 1(1) 4(4) 1(1)

Undescended 1(1) 0 0 0 0

Agenesis 1(1) 0 0 0 0

aReplicate 2 only. All other groups are combined data for replicates 1 and 2.

bIncludes one fetus with situs inversus.

*Differs significantly from water controls (p < 0.01) by two-tail Fisher-Irwin exact test of litters affected. tDiffers significantly from water controls (p < 0.001) by two-tail Fisher-Irwin test of litters affected.

72
DEVELOPMENTAL TOXICITY OF CUTANEOUSLY APPLIED GLYCOLS 73

Table 4. Skeletal malformations and variations: number of litters (fetuses) affected.

Treatment group

EGEE EGEEA EGBEa diEGEE Water

(0.25 mL) (0.35 mL) (0.12 mL) (0.35 mL) (control)

Tbtal examined 11(36) 5(11) 9(45) 13(67) 17(90)

Malformations

Ribs: total 2(2) 2(2) 1(1) 2(4) 1(1)

Missing ribs 2(2)b 0 0 2(3) 1(1)

Fused, malformed 1(1)b 2(2) 0 1(2) 0

Cervical 0 0 1(1) 0 0

Vertebrae: total 4(5) 0 0 2(4) 0

Fused arches 2(2)b 0 0 1(1) 0

Extra lumbar 2(2) 0 0 1(3) 0

Reduced lumbar 2(3)b 0 0 0 0

Reduced thoracic 1(1)b 0 0 1(1) 0

Absent arch 0 0 0 1(1) 0

Scoliosis 0 1(1) 0 0 0

Missing tympanic ring 0 0 0 1(1) 0

Total malformations 4(5) 2(2) 1(1) 3(5) 1(1)

Variations

Ribs: total 6(13)* 5(6). 3(4) 5(12) 1(1)

Extra thoracic 2(6) 0 0 0 0

Extra lumbar 5(7)b 5(6) 3(4) 5(12) 1(1)

Vertebrae: total 10(32)t 5(11) 1(1) 3(6) 0

Reduced sacral (< 6) 2(2)b 0 0 0 0

Missing lumbo-sacral arch 2(2) 1(1) 1(1) 0 0

Increased thoracic 1(3) 0 0 0 0

Centra: misshaped 7(16)b 5(10) 0 2(3) 0

Centra: absent, incomplete 7(23) 5(11) 0 2(5) 0

Sternebrae: total 11(32) 4(9) 6(13) 7(18) 13(28)

Misaligned 6(8) 0 0 1(3) 2(2)

Split 0 1(1) 0 0 0

Decreased number (< 6) 9(29) 4(9) 6(13) 7(17) 13(27)

Reduced ossification: total 5(14) 5(11) 3(3) 3(3) 1(1)

Skull 2(8) 5(11) 3(3) 2(2) 0

Pelvic girdle 4(8)b 4(6) 0 0 0

Decreased metatarsals (< 4) 2(3)b 4(7) 0 1(1) 0

Decreased metacarpals (< 3) 2(5) 2(5) 1(1) 0 1(1)

Tbtal variations 11(34) 5(11) 7(16) 9(22) 13(29)

aReplicate 2 only. All other groups are combined data for replicates 1 and 2.

bIndicates two fetuses with acaudia and imperforate anus.

*Differs significantly from water controls (p < 0.01) by two-tail Fisher-Irwin exact test of litters affected.

tDiffers significantly from water controls (p < 0.001) by two-tail Fisher-Irwin exact test of litters affected.

We wish to thank Peggy L. Johnson, Garth E. Peterson, Ronald L.

Schuler and Terry W Thrner for their assistance with treatments and

necropsies. We are also grateful to Sandra L. Clark for her help in

preparing the manuscript.

Mention of a company name or product does not constitute

endorsement by the National Institute for Occupational Safety and

Health.

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