Environmental Health Perspectives

Vol. 57, pp. 261-271, 1984

Comparative In halation Teratogenicity

of Four Glycol Ether Solvents and

an Amino Derivative in Rats*

by B. K. Nelson,t James V. Setzer,t W. Stephen Brightwell,t

Patricia R. Mathinos,* Maureen H. Kuczuk,*

Timothy E. Weavert and Phillip T. Goad§

Previous research demonstrated the inhalation teratogenicity of the solvent 2-ethoxyethanol in rats and

rabbits. As this is one of a class of widely used industrial solvents, we investigated the teratogenicity of five

structurally related compounds. Each chemical was vaporized and administered to approximately 15

pregnant rats in one to three concentrations for 7 hr/day on gestation days 7 to 15, and dams were sacrificed

on day 20. Fetuses were individually weighed, and two-thirds of them were fixed in Bouin's solution and

examined for soft-tissue anomalies. The other one-third were fixed in alcohol, stained with Alizarin Red

and examined for skeletal defects. Data were analyzed on a litter basis; three solvents were compared with a

pooled group (N = 34) of sham-exposed controls, and the remaining two were compared with a group of 15

controls. At concentrations which were apparently not maternally toxic, 2-methoxyethanol was highly

embryotoxic, producing complete resorptions at 200 ppm; increased resorptions, reduced fetal weights and

skeletal and cardiovascular defects occurred at both 100 and 50 ppm. 2-ethoxyethyl acetate at 600 ppm

induced complete resorption of litters; 390 ppm reduced fetal weights and induced skeletal and

cardiovascular defects, but only a single defect was observed at 130 ppm. 2-Butoxyethanol evidenced slight

maternal toxicity at 200 ppm but produced no increase in congenital defects at that concentration. Neither

2-(2-ethoxyethoxy)ethanol (100 ppm) nor 2-methylaminoethanol (150 ppm) was maternally toxic or

embryotoxic. In summary, shorter alkyl chained glycol ethers produced greater embryotoxicity than those

having longer chains, and the ester produced effects equivalent to the ether, both patterns predictable from

the biochemical literature.

Of the thousands of chemicals to which workers are

exposed, only a few have even minimal experimental

animal data from which to evaluate or predict

reproductive toxicity. One class of chemicals that has

demonstrated reproductive toxicity is a class of monoand dialkyl ethers of ethylene glycol and their

derivatives, collectively referred to as cellosolves or

glycol ethers. Approximately 700 million pounds (318

thousand metric tons) of glycol ethers were produced in

1977, with the ethylene glycol monoethers representing

about 78% of the total production (1). Ethoxyethanol,

the prototype of this class, has the largest production

volume, followed by butoxyethanol and methoxyethanol

(1). The glycol ethers are widely used in industry as

*This manuscript was not presented as part of the conference

proceedings.

'Division of Biomedical and Behavioral Science, NIOSH 4676

Columbia Parkway, Cincinnati, OH 45226.

tDivision of Pathologic Embryology, Children's Hospital Research

Foundation, Cincinnati, OH 45229.

§Intox Laboratories, Inc., 914 Barber Street, Little Rock, AR

72202.

solvents for nitrocellulose, lacquers, inks, dyes, enamels,

varnishes, varnish removers, and dry cleaning compounds. In addition, they are used in some commercial

products such as cosmetics and liquid household

cleaners. Produced and distributed by at least six major

American chemical companies, the glycol ethers have a

combined potential exposure population of around 1.5

million workers in the United States.

The earliest evidence of reproductive toxicity of this

chemical group was observed when skeletal defects

were reported in offspring of rats following 100

,ug/kg/day IP injections of ethoxyethanol throughout

gestation, although no defects were noted in mice or

rabbits (2). The same investigators administered

ethoxyethanol at 400 ,uL/kg/day for 4 weeks or 200

,uL/kg/day for 13 weeks to male rats and observed

testicular damage after both exposure regimens. Recently increased skeletal variations were observed following intubation of doses as low as 31.25 mg/kg/day

methoxyethanol on gestation days 7 to 14 in mice (3).

Only recently, however, have there been reports of

teratogenic effects produced by the glycol ethers after
NELSON ET AL.

administration using the common routes of occupational

exposure, inhalation and cutaneous absorption. Skeletal

and cardiovascular defects were observed in rats after

dermal exposure to 1.0 mL (0.25 mL, four times/day)

ethoxyethanol on gestation days 7 to 16, with nearly

half of the litters entirely resorbed at that level, and all

litters completely resorbed at twice that level (4).

Gestational exposure via inhalation to 600 to 750 ppm

ethoxyethanol produced complete embryomortality in

rats and rabbits, whereas 200 or 160 ppm ethoxyethanol

induced skeletal and cardiovascular defects in rats and

rabbits, respectively (5). Increased neonatal mortality

following inhalation exposure of pregnant rats to

ethoxyethanol at the current Federal occupational standard (200 ppm) and behavioral and neurochemical

alterations in the offspring of pregnant rats exposed to

one-half that concentration (viz. 100 ppm) have been

reported (6).

Dose-dependent increases in testicular atrophy have

been reported in mice following oral administration of

various glycol ethers at 250 to 4000 mg/kg/day, 5

days/week for 5 weeks (7). The lowest levels causing

significant deviations from controls were as follows:

methoxyethanol 250 mg/kg, methoxyethyl acetate 500

mg/kg; ethoxyethanol 1000 mg/kg, ethoxyethyl acetate

1000 mg/kg; butoxyethanol and phenoxyethanol produced mortality in all rats at 2000 mg/kg but had no

effect on testicular weight at 1000 mg/kg. The most

important implication of these results is the indication

that the chemicals having shorter attached alkyl groups

produced the most damage and that reproductive toxicity decreases with increasing chain length. Esterification

of the compounds (i.e, the acetate forms) resulted in

less testicular atrophy when the dose was calculated on

a weight per body weight (mg/kg) basis (at least with

methoxyethanol), but produced effects equivalent to

those of the alcohol form when calculated on a molar

basis (thus implying that the acetate was removed prior

to the toxic action).

The present research evaluated the teratogenic effects of structurally related glycol ethers in an attempt

to determine the structure activity relationships for

teratogenicity. Table 1 presents the glycol ethers evaluated for teratogenicity in the present research, along

with the same data for ethoxyethanol. As seen from the

table, a chemical having a shorter alkyl chain was

selected (methyl rather than ethyl), as was one with a

longer chain (butyl), and were the diethylene glycol

ethyl ether and the ethyl acetate. The latter was chosen

rather than the ether in order to verify that the acetate

would produce effects equivalent to the ether, as would

be predicted from the ubiquitous nature of esterases

within the body and as shown for the male reproductive

effects cited above. Finally, the amino analog of

methoxyethanol was tested to determine the structural

specificity required to produce teratogenic effects.

Materials and Methods

Subjects

Virgin female and male Sprague-Dawley rats specified

to be free of mycoplasma and Sendai virus and of

internal and external parasites (Charles River Breeding

Laboratories, Wilmington, MA) were acclimated to a

12-hr light-dark cycle (lights on at 6 am) and to a

temperature of 24 + 2°C for 2 weeks. The humidity, not

controlled, typically was in the range of 40 + 20%.

Purina Lab Chow and tap water were available ad

libitum except when pregnant animals were in exposure chambers. Bedding consisted of cleaned, heattreated sawdust from a local supplier (Absorb-Dri,

Tasty Foods, Cincinnati, OH).

Apparatus and Procedures

Males weighing over 300 g were placed individually

into a cage with three females weighing 200 to 300 g.

Table 1. General information on the glycol ethers included in the present research.

Rat toxicity data

Common name Skeleton Oral, g/kg Inhalation, ppm/hr OSHA Standard NIOSH estimated

and formula formula LD50 LCL (TWA), ppm exposure population

2-Methoxyethanol

CH3OC2H4OH C-O-C-C-OH 2.46 2000/4H 25 97,285

2-Ethoxyethanola C-C-O-C-C-OH 3.0 4000/4H 200 359,790

C2H5OC2H4OH

2-Ethoxyethyl acetate 0

C2H5OC2H402C2H3 C-C-O-C-C-O-C-C 5.1 3000/4H 100 231,970

2-Butoxyethanol

C4HqOC2H4OH C-C-C-C-O-C-C-OH 1.48 500/4H 50 1,279,707

2-(2-Ethoxyethoxy)-

ethanol (carbitol) C-C-O-C-C-O-C-C-OH 6.5 None None 20,000

C2H5OC2H4OC2H4OH

2-Methylaminoethanol

CH3NHC2H4OH C-N-C-C-OH 2.34 None None Unknown

aInvestigated in previous research.

262
GLYCOL ETHER TERATOGENICITY

Vaginal smears were taken each morning, and the

presence of sperm marked day zero of gestation. These

females were placed alone in 38 x 33 x 17-cm polycarbonate cages with filter tops. Feed and water intake and

maternal weight were recorded weekly (i.e., on days 7,

14, and 21); any other signs of maternal toxicity were

noted daily.

The glycol ethers (technical grade, 98 to 99.5% pure;

Eastman Kodak Co., Rochester, NY) were heated in a

flask set in hemispherical mantles. Vaporization was

achieved by bubbling air through each solvent which

was heated to approximately 40°C (for the higher

concentrations). This vapor was then mixed with filtered

room air to obtain the desired concentration and introduced into 0.5 m3 exposure chambers (Charles Spengler

and Associates, Cincinnati, OH). The concentration was

continually monitored by a Miran I infrared spectrophotometer (Foxboro Analytical, South Norwalk, CT) with

the concentration recorded hourly. Typically, two or

three charcoal tube samples (silica gel tubes for 2-

methylaminoethanol) were taken weekly from the chamber air for independent verification of the concentration

by gas chromatograph. Two exceptions are noted below

where more were taken (9). Throughout this paper

concentrations are expressed in parts of solvent per

million parts of air; hence values for the different

chemicals are comparable on a molar equivalent basis.

Air flow through the chambers provided approximately

four air changes per minute. Exposures were conducted

sequentially in one or two chambers, with a third

chamber for sham exposure of control subjects.

Pregnant females were transported from the animal

quarters to the inhalation chambers in their home cages

with filter tops (Hazleton Systems, Aberdeen, MD).

They were placed individually in 13 x 25 x 189-cm

stainless steel wire mesh cages within exposure

chambers. Control animals were placed in similar chambers for the same hours as the exposed animals; a

pooled group of controls (N = 34) served as the

comparison group for the first three chemicals

examined. Another group of 15 controls served as the

comparison group for the last two chemicals examined,

as these groups were exposed at a later time

(approximately 6 months later) than the first three.

Exposures, as outlined above, were conducted 7 hr/day,

and the animals were left in the chamber for at least one

additional hour blow-off time after vapor generation

terminated. They were then returned in their individual

housing cages to the animal quarters, where water

bottles were replaced. Exposures were conducted on

gestation days 7-15. On day 20, the females were

individually weighed and euthanized by chloroform

asphyxiation. The entire uterus was removed and numbers of resorption sites (classified as early, middle or

late) and live fetuses wvere determined. Fetuses were

serially removed, blotted of excess fluids, weighed,

examined for external malformations and external sex

determined. One third of the fetuses were randomly

selected and placed in 95% ethanol, and the remaining

fetuses were placed in Bouin's solution. After being in

the Bouin's solution for at least 1 week, these fetuses

were examined for visceral abnormalities using Wilson's

razor blade sectioning technique (10). The viscera were

examined with the aid of a dissecting microscope. A

representative sample of sections with malformations

was identified by dam number and saved in 70% alcohol.

Fetuses were examined for skeletal defects by using a

modified Staples technique (11). They were fixed in 95%

alcohol, eviscerated and macerated in 2% KOH/Alizarin

Red S solution. The fetuses were further macerated and

cleared in the appropriate solutions of 2% KOH/glycerin

(60:40, 40:60, 20:80) and stored in 100% glycerin. A

crystal of thymol was added to each storage vial to

retard fungal growth. Storage vials were individuallv

identified by dam number.

Statistical Analysis

Numbers of implants and proportions of resorptions

were independently analyzed by using a Kruskal-Wallis

Table 2. Concentrations of vapors generated in inhalation study.

Vapors generated, ppm

Days of By infrared By gas chromatography

exposure (X ± SD) (X ± SD)

Methoxyethanol 11 199.2 ± 0.98 b

29 101.4 ± 2.2 99.3 ± 16.3

19 50.6 ± 1.0 47.4 ± 5.9

Butoxyethanol 11 194.4 ± 6.1 b

20 153.3 ± 6.8 176.8 ± 8.3c

Ethoxyethyl acetate 12 601.0 ± 3.47 594.9 ± 109.9

21 a 387.2 ± 8.7

12 a 128.9 ± 1.a

2-(2-Ethoxyethoxy) ethanol 29 a 102.2 ± 15.6

2-Methylaminoethanol 28 a 150.0 ± 15.2

aDue to equipment problems, suitable data for concentrations from the infrared analyzer were not available for these chemicals.

bGas chromatograph samples were not taken in these exposures. cThis charcoal tube result is approximately 15% higher than that from the infrared analyzer (IR); since the IR provided an essentially continuous daily record of exposures, we have used the figures (usually rounded to the nearer 10 ppm) from the IR throughout the text for all

chemicals where suitable data from the IR were available. Where IR data were not available, the charcoal tube results were used within the text.

263
NELSON ET AL.

test corrected for ties, with subsequent multiple comparisons to determine where the differences occurred

(12). Analysis of pup weights involved a mixed model

analysis of covariance (with the number of live pups in

the litter as the covariate) using maximum likelihood

estimation (13,14). The model was mixed, since there

was both within-litter and between-litter variation.

Subsequently, pairwise comparisons between the pooled

control group and each treatment group were

performed. Incidence of total defects and of total

variants were compared using a Kruskal-Wallis test

with multiple comparisons (12) with the litter as the

experimental unit and the level of significance at

p< 0.05.

Results

The concentrations within the exposure chambers as

measured by the infrared analyzer were relatively close

to those obtained from gas chromatography (Table 2).

Control

No visceral or skeletal malformations were observed

in fetuses from control dams. The types of visceral and

skeletal variants observed in fetuses from the control

treated dams were similar to those variants observed in

chemically exposed groups.

Methoxyethanol (ME)

Concentrations of ME (Table 2) were easily generated

and were rapidly cleared from the exposure chambers.

There were no overt signs of toxicity in the pregnant

rats in any of the groups. The only weight decrements

in the maternal animals were likely due to increased

resorptions. As shown in Table 3, all litters at the

highest concentration (200 ppm) were entirely resorbed.

At 100 ppm, about half of the litters (18 of 34) were

entirely resorbed, and all litters had some resorptions;

the mean weight of the live fetuses was reduced by

approximately one-third from the control value. At 50

ppm there was approximately a 3-fold increase in

resorptions and fetal weights were reduced by 20%

compared to the control values.

Visceral malformations (Table 4) were found in fetuses from both the 50 ppm and 100 ppm ME-treated

dams, but only the incidence of those from the 100 ppm

group was significantly different from the controls (p S

0.025). Heart abnormalities were the predominant malformations observed in both the 100 ppm and 50 ppm

ME groups. Malformations of the retina, eye, umbilicus

and lungs of ME-exposed fetuses were also observed

but at a frequency lower than the aforementioned.

Wavy, fused, and absent ribs, plus extra vertebrae

were observed in the 100 and 50 ppm ME treatment

groups (Table 5). Additionally, tail malformations were

observed in the 100 ppm group. Statistical analyses of

these pooled data indicate significant differences from

controls for both the 50 ppm (p < 0.025) and 100 ppm

(p < 0.05) treatment groups. Fetuses in the 50 ppm

group demonstrated more rib malformations than fetuses in the 100 ppm group. The failure of the incidence

of malformations to increase with dose may be the

result of increased embryo lethality observed in the

high dose group. These data indicate that inhalation of

50 or 100 ppm ME is teratogenic in the rat.

Table 3. Effects of maternal exposure to glycol ethers on pregnancy outcome.

2-Methoxyethanol

Number pregnant/number mated

Implants/dam (X)

Litters with resorptions, %

Resorptions/litter (X)

Controls 50

ppm

34/41 14/17

12.7 12.5

14 9

(42) (64)

0.64 1.6a

100

ppm

34/38

12.5

34

(100)

9.8a

200

ppm

8 /11

12.6

8

(100)

12.6a

2-Butoxyethanol

150 200

ppm ppm

16/19 15/18

12.9 15.1a

4 9

(25) (60)

0.23 1.2

2-Ethoxyethyl acetate

130 390 600

ppm ppm ppm

15/15 15/20 9/9

14.5a 12.9 14.7a

7 9 9

(47) (56) (100)

0.49 2.3a 14.7a

Resorptions

Early

Middle

Late

Tbtal

(% of implants)

Tbtal live fetuses

Live fetuses/litter (X)

(% of implants)

Live fetal weights (X)

Female

Male

Standard error of meanb

21 15

1 2

1 2

23 19

(5) (11)

192

77

61

330

(77)

407 156 96

12.2 11.1 2.8

(95) (89) (23)

3.46 2.84a 2.29a

3.64 2.91a 2.49a

0.07 0.07 0.28

101

(100)

4 16 8 20

0 1 0 7

0 1 0 8

4 18 8 35

(2) (8) (4) (17)

0 206 203 211

0 12.6 13.9 14.0

(98) (92) (96)

175

10.8

(83)

78

19

6

103

(100)

0

0

3.28a 3.49 3.31a 2.74a

3.55a 3.66 3.46a 2.85a

0.10 0.10 0.10 0.10

aSignificantly different from controls at p < 0.05 (only data on implants, X resorptions, and fetal weights were analyzed).

bIn no case was there a significant difference between sexes on this variable; hence, only the larger is presented.

264
GLYCOL ETHER TERATOGENICITY

Table 4. Visceral malformations.

ME BU EEAC

50 100 150 200 130 390

Controls ppm ppm ppm ppm ppm ppm

Number of litters 34 14 11 16 15 15 15

Number of fetuses examined 270 103 65 135 135 142 116

Cardiac malformation, no. (%)

IV septal defect 0 4(4) 13(20) 0 0 0 6(5)

Ringed aortic arch 0 1(1) 4(6) 0 0 1(1) 2(2)

Rt.-sided aortic arch 0 1(1) 1(2) 0 0 0 0

Rt.-sided ductus arteriosus 0 0 0 0 0 0 1(1)

Pulmonary valve stenosis 0 0 1(2) 0 0 0 0

TImcus arteriosus communus 0 0 1(2) 0 0 0 0

Levocardia 0 0 1(2) 0 0 0 0

Pulmonary malformations

Hypoplastic lungs 0 0 2(3) 0 0 0 0

Optical malformation

Deformed retina 0 1(1) 0 0 0 0 0

Reduced eye size 0 0 1(2) 0 0 0 0

Miscellaneous

Umbilical hernia 0 1(1) 0 0 0 0 2(2)

Tbtal fetuses normala 191 55 27 104 99 95 55

Ibtal fetuses malformed 0 8 19 0 0 1 7

Tbtal litters affected 0 6 10 0 0 1 3

Statistical significance NSb p < 0.025 NS NS NS p < 0.01

aNormal defined as having no visceral variations or malformations.

bNS = not significant at p = 0.05.

Table 5. Skeletal malformations.

ME BU EEAC

50 100 150 200 130 390

Control ppm ppm ppm ppm ppm ppm

Number of litters 34 14 9 16 15 15 15

Number of fetuses examined 137 53 31 68 71 69 59

Vertebra malformations, no. (%)

Extra 0 0 2(6) 0 0 0 0

Ribs

Rudimentary 13tha 0 0 0 1(1) 0 0 0

Wavy 0 15(28) 5(16) 0 0 0 2(4)

Fused 0 2(4) 0 0 0 0 1(2)

Absent 0 1(2) 0 0 0 0 0

Tail

Shortened 0 0 4(6) 0 0 0 0

Absent 0 0 1(2) 0 0 0 0

Total fetuses normalb 137 2 1 37 52 30 2

Tbtal fetuses malformed 0 16 10 0 0 0 4

Total litters affected 0 8 4 0 0 0 3

Statistical significance p < 0.025 p < 0.05 NSC NS NS p < 0.01

aConsidered a minor malformation.

bNormal defined as having no skeletal malformations or deviations.

CNS = not significant at p = 0.05.

265
NELSON ET AL.

Table 6. Visceral variations.

ME BU EEAC

50 100 150 200 130 390

Control ppm ppm ppm ppm ppm ppm

Number of litters 34 14 11 16 15 15 15

Number of fetuses examined 270 103 65 135 135 142 116

Cranial variations, no. (%)

Enlarged lateral ventricles 5(2) 21(20) 24(37) 0 5(4) 7(5) 22(19)

Cardiac

Enlarged atria 0 0 1(2) 0 0 0 0

Urogenital

Hydronephrosis 4(2) 1(1) 2(3) 2(1) 3(2) 6(4) 6(5)

Bilateral 1 0 1 2 1 1 5

Unilateral 3 1 1 0 2 5 1

Dilated renal pelvis 39(14) 23(22) 2(3) 13(10) 19(14) 25(18) 14(12)

Bilateral 13 16 2 7 9 13 7

Unilateral 26 7 0 6 10 12 7

Low set kidney 6(2) 0 0 0 1(1) 2(1) 8(7)

Hypoplastic kidney 0 0 0 0 0 0 1(1)

Hydroureter 52(19) 8(8) 3(5) 17(13) 12(9) 34(24) 19(16)

Bilateral 21 5 1 9 3 25 11

Unilateral 31 3 2 8 2 9 8

Misplaced testes 20(7) 11(11) 5(8) 10(7) 12(9) 5(4) 10(9)

Bilateral 5 1 1 0 1 1 2

Unilateral 15 10 4 10 11 4 8

Tbtal fetuses normal 191 55 27 104 99 95 55

Total fetuses with variations 79 44 31 31 36 47 58

TItal litters affected 26 13 10 12 13 14 15

Statistical significance p < 0.01 p < 0.01 NSa NS NS p < 0.01

aNS = not significant at p = 0.05.

Visceral variants observed in fetuses from both

ME-treated groups were similar to those variants

observed in control fetuses (Thble 6); however, their

frequency in exposed groups was significantly increased

above that for the control group (p < 0.01). Skeletal

variants were significantly more numerous in fetuses

from both the 50 (p < 0.01) and 100 ppm (p < 0.025)

ME-exposed dams than were variants in fetuses from

control dams (Table 7). These data indicate that

inhalation of ME did cause growth retardation in the

rat.

Butoxyethanol (BU)

Concentrations of BU were more difficult to generate

in that the solvent required heat to generate even the

low concentrations. Clearance time from the chambers

was also longer than for ME. This was the only one of

the chemicals which produced apparent maternal toxicity [ranging from hematuria, as has been reported in

the literature (15), to death at concentrations from

250 to 500 ppm. Based on the maternal toxicity,

In a dose finding study conducted at the outset of the project,

nonpregnant rats (250-300 g) were exposed to 250, 350 or 450 to 500

ppm BU (no charcoal tube verification). Three of four rats exposed to

450 to 500 ppm of BU for 6.5 hr died within 36 hr after termination of

exposure levels of 150 and 200 ppm were chosen for

the teratology study.

Pregnant rats exposed to 200 ppm BU showed some

hematuria, but only on the first day of exposure. With

that exception, the dams showed no adverse effects.

There was a significant increase in the mean number of

implants in the 200 ppm BU group, but since no

treatment began until day 7, this increase was not

treatment-related. There was also no apparent adverse

effect upon the pups, as the number of resorptions, fetal

weights, and incidence of malformations did not differ

from controls (Tables 3-7). (There was a statistically

significant decrease in fetal weights in the 150 ppm BU

groups, but since the differences were slight and there

were no effects at the higher level, these differences

exposure, and two of three nonpregnant rats died within 18 hr after

exposure to approximately 350 ppm BU for 7 hr. Of three nonpregnant rats exposed for 7 hr to 250 ppm BU, one died within 18 hr after

exposure, and a second died after day 2 of exposure. With each of

these concentrations, the exposed rats began showing various (legrees of hematuria 4 to 6 hr after onset of exposure, as has been

reported elsewhere (15). From each of these concentrations, one

rat survived, and these three animals were placed together in a

41 x 32 x 18-cm steel cage. Approximately 1 week after exposure,

the distal portion of the tail (about one-half the tail length) became

necrotic and fell off or was chewed off. Observations of a small number

of singly housed rats exposed cutaneously to butoxyethanol woul(d

suggest that each rat chewed its own tail off.

266
GLYCOL ETHER TERATOGENICITY

Thble 7. Skeletal variations.

ME BU EEAC

50 100 150 200 130 390

Control ppm ppm ppm ppm ppm ppm

Number of litters

Number of fetuses examined

Skull variations, no. (%)

Incomplete ossification

Frontal

Parietal

Interparietal

Supraoccipital

Maxilla

34

137

14

53

1(1) 16(30)

3(2) 16(30)

3(2) 15(28)

2(2) 17(32)

0 0

9 16 15

31 68 71

4(13) 0

15(48) 0

12(39) 1(1)

18(58) 0

5(16) 0

1(1)

1(1)

0

0

0

Vertebra

Absent

Sacral/caudal

Incomplete ossification

Thoracic

Lumbar

Sacral/caudal

Centra

Absent

Thoracic

Lumbar

Sacral/caudal

Incomplete ossification

Thoracic

Lumbar

Sacral/caudal

Abnormal

Thoracic

Lumbar

Sacral/caudal

0

0

0

20(15)

0

0

0

0

0

0

6(4)

0

0

35(66)

0

1(2)

21(40)

14(26)

0

23(43)

0

0

1(2)

40(75)

16(30)

3(6)

3(10) 4(6)

0 0

0 0

12(39) 9(13)

16(52) 0

1(3) 0

5(16) 0

0 0

0 0

0 0

21(68) 15(22)

16(52) 0

3(10) 0

Sternebrae

4-incomplete ossification

Abnormal

2(2)

1(1)

Ribs

Rudimentary 14th

Pelvic girdle

Pubis

Absent (not ossified)

Incomplete ossification

Illium

Absent (not ossified)

Incomplete ossification

Ischium

Absent (not ossified)

Incomplete ossification

5(4)

1(1)

0

0

0

0

2(2)

Other

Metacarpals (3 ossified)

Total fetuses normal

Tbtal fetuses with variations

Total litters affected

0

100

37

19

Statistical significance

"NS = not significant at p = 0.05.

b130 ppm vs. 390 ppm, p < 0.05.

17(32)

11(21)

4(8)

9(17)

3(6)

0

0

1(2)

11(21)

19(36)

2

50

13

23(74) 3(4)

5(16) 1(1)

14(45) 3(4)

6(19) 0

5(16) 0

12(39) 0

5(16) 0

9(29) 0

6(19) 0

12(39) 0

1 37

30 31

9 10

p < 0.01 p < 0.025 NSa

0 1(1) 12(21)

0 0 5(9)

9(13) 10(14) 36(63)

0 1(1) 2(4)

0 0 0

0 0 0

0 1(1) 7(12)

0 0 0

0 1(1) 3(5)

O 1(1) 11(19)

52 30 2

19 39 56

8 14 15

NS p < 005b p < 0.01

15

69

15

59

1(1)

1(1)

1(1)

1(1)

0

12(21)

12(21)

10(17)

11(19)

0

0

0

0

7(10)

0

0

0

0

0

0

7(10)

1(1)

0

0

0

0

22(32)

1(1)

0

0

0

0

5(7)

11(16)

0

0

2(4)

0

0

26(46)

17(30)

0

1(2)

0

0

8(14)

32(56)

17(30)

0

267
NELSON ET AL.

Table 8. Effects of maternal exposure to 2-(2-ethoxyethoxy)ethanol (2-EEE) or 2-methylaminoethanol (2-MAE) on pregnancy outcome.

Controls 2-EEE 2-MAE

Number pregnant/number mated 15/16 20/21 18/24

Implants/dam (X) 13.1 13.4 11.9

Litters with resorptions (%) 10(67) 12(60) 10(56)

Resorptions/litter (X) 1.3 0.9 0.7

Tbtal live fetuses 174 250 212

Live fetuses/litter (X) 11.6 12.5 11.8

(% of implants)

Live fetal weights (FX)

Female 3.26 3.19 3.59

Male 3.33 3.25 3.69

Standard deviation 0.30 0.25 0.31

Number (%) of litters 5(33) 6(30) 7(39)

with abnormal fetusesa

aLitter with at least one fetus having either skeletal or visceral anomalies; detailed in Tables 9 and 10.

Table 9. Summary of visceral observations after exposure to 2-(2-ethoxyethoxy)ethanol or 2-methylaminoethanol.

Number of litters (fetuses)/percenta

Control 2-EEE 2-MAE

Number of litters 15 19 18

Number of fetuses examined 116 166 143

Malformations

Cardiovascular:

Double aortic arch 0 1(1)/.53 0

Missing innominate 0 1(1)/.53 0

Brain:

Hydrocephalus (mild) 3(6)/8 1(4)/4 3(5)/4

Variations

Testicular: (number males examined) 62 76 74

Undescended testes 0 0 1(l)/i

Position variation 10(22)/39 13(17)/22 9(13)/17

Urinary:

Dilated renal pelvis 3(4)/3 7(13)/7 5(5)/3

Dilated ureter 3(6)/5 9(19)/11 6(18)/11

Hydronephrosis 2(2)/2 3(4)/3 0

Hydroureter 3(3)/2 4(7)/4 5(6)/4

Ectopic kidney 1(1)/i 2(2)/1 0

Other:

Abdominal hemorrhage 1(l)/i 1(l)/i 0

Liver hemorrhage 0 1(1)/i 0

Number of fetuses with variations 30 43 27

Percent normalb,c 89 ± 26 91 ± 21 92 ± 15

Number of fetuses 107 153 131

Percent abnormalb 11 ± 26 9 ± 21 8 ± 15

Number of fetuses 9 13 12

aMean of litter mean percent.

bMean of litter mean percent ± SD.

cIncludes fetuses with variations.

were likely not of biological significance.) After exposure to 150 ppm BU, rats occasionally showed slight

hematuria on the first day of exposure, but no other

adverse effects were noted in the dams or pups; one

fetus with cephalothoracomphalopagus was observed,

but this was likely a chance occurrence and not

treatment-related.

2-Ethoxyethyl Acetate (EEAC)

Concentrations of EEAC were relatively easy to

generate and cleared rapidly from the chamber. Maternal animals showed no overt toxicity; a weight reduction

was observed at higher concentrations, but this was

likely due to increased resorptions. As shown in Table

3, all offspring from dams exposed to 600 ppm EEAC

were resorbed. At 390 ppm there was a significant

increase in resorptions and fetal weights were reduced

by 21%, and at 130 ppm there were slight but significant

decreases in fetal weights.

Visceral malformations of the heart and umbilicus

were observed in fetuses from the 390 ppm EEACtreated dams (Table 4). Only one fetus from the 130 ppm

group had a heart defect. Statistical analysis of the data

268
GLYCOL ETHER TERATOGENICITY

T!able 10. Summary of skeletal observations after exposure to 2-(2-ethoxyethoxy)ethanol or 2-methylaminoethanol.

Number of litters (fetuses)/percenta

Control 2-EEE 2-MAE

Number of litters 14 19 18

Number of fetuses examined 58 84 69

Malformations

Missing ribs 0 1(1)/1 0

Decreased thoracic vertebrae 0 1(1)/1 0

Variations

Ribs:

Rudimentary 14th 1(1)/2 5(7)/8 1(2)/4

Rudimentary lumbar 0 0 1(1)/1

Skull:

Incomplete ossification

Supraoccipital 0 1(1)/1 1(1)/1

Interparietal 1(1)/2 2(2)/3 1(2)/2

Parietal 1(1)/2 3(3)/4 0

Frontal 0 2(2)/3 0

Sternebrae:

Decreased number ( < 4) 1(1)/2 1(1)/1 0

Split 0 0 1(1)/1

Misaligned 1(1)/2 6(6)/7 5(5)/7

Centra

Thoracic

Missing, half-sized 0 4(4)/5 0

Misshapen 1(1)/2 3(3)/3 5(5)/8

Appendages

Decreased metatarsals ( < 4) 0 3(3)/4 0

Decreased metacarpels ( < 3) 0 1(1)/1 0

Pelvic girdle

Pubis, missing, pinpoint 0 1(1)/1 2(2)/3

Ischium, missing, reduced size 0 1(1)/1 1(1)/1

Number of fetuses with variations 4 23 16

Number of fetuses with no variations 14(54)/93 19(61)/73 18(53)/77

or malformations

Percent normalb,e 100 ±0 99 ± 6 100 ± 0

Number of fetuses 58 83 69

Percent abnormal 0 1(6) 0

Number of fetuses 0 1 0

'Mean of ltter mean percent. bMean of litter mean percent ± SD.

'Includes fetuses with variations.

indicated significant differences between EEAC treated

and control groups (p < 0.01); furthermore, since heart

defects rarely occur spontaneously, and none were

observed in control fetuses, these data indicate that

inhalation of either 130 ppm of 390 ppm EEAC is

teratogenic in the rat.

Malformations of the ribs were observed in three

fetuses from 390 ppm EEAC-exposed dams (Table 5); no

skeletal malformations were observed in fetuses from

the 130 ppm EEAC group. Statistical analysis of these

data indicated significant differences from the controls

(p < 0.01); however, due to the low frequency of

occurrence of these malformations, treatment-induced

cause cannot be positively concluded.

Visceral variants observed in fetuses from EEACtreated dams were similar to those variants observed in

control fetuses (Table 6); however, the frequency of

variants in the exposed groups was significantly

different from the control-treated group Significant

statistical differences in skeleltal variants (Table 7) were

observed between control and 130 ppm (p < 0.05) and

390 ppm (p < 0.01) and also between 130 ppm and 390

ppm treatment groups (p < 0.01). (p < 0.05). These

data indicate that inhalation of EEAC at either dose

level during the period of organogenesis can cause

growth retardation in the rat.

2-(2-Ethoxyethoxy)ethanol

(Carbitol, 2-EEE)

Due to the low vapor pressure of 2-EEE, higher

concentrations could not be tested due to probable

formation of an aerosol (this was observed during

chamber calibration). No toxicity was observed in

maternal animals or in the offspring (Tables 8-10) after

exposure to 100 ppm 2-EEE (mean concentration from

65 charcoal tubes or 2 to 3 per day = 102 ppm).

Consequently, 2-EEE is likely not a teratogenic hazard

after inhalation exposure. Nor is it of teratogenic risk

when given in comparable molar equivalents to a

cutaneously teratogenic (4) level of ethoxyethanol

(Nelson, unpublished observations).

269
NELSON ET AL.

2-Methylaminoethanol (2-MAE)

Low vapor pressure also prevented our generating

high concentrations of 2-MAE. At 150 ppm 2-MAE

(mean concentration from 28 silica gel tubes, one per

day, analyzed in duplicate = 150.0 ppm), no maternal or

fetal toxicity was observed (Tables 8-10).

Discussion

In this study of the inhalation teratogenic effects of

four glycol ethers and an amino derivative, methoxyethanol was the most embryotoxic; ethoxyethyl acetate

was less potent; but butoxyethanol, 2-(2-ethoxyethoxy)-

ethanol, and 2-methylaminoethanol were not embryotoxic at the concentrations tested. Congenital malformations in mice have been reported after the mice were

intubated with various levels of ME on gestation days

7-14 (3). Comparing the frequency of resorptions in

that study with the frequency of resorptions we

observed, it appears that our level of 100 ppm ME

would roughly correspond to their dose of 250 mg/kg. At

one half that level (50 ppm and 125 mg/kg) in both

studies, there was little increase in resorptions, but

malformations were observed. Since the study in mice

(3) found malformations at levels lower than this (even

at their lowest dose of 31.25 mg/kg), it is possible that

malformations could be induced by concentrations lower

than our low level of 50 ppm.

The results from ethoxyethyl acetate were as

expected. Based upon the ubiquitous nature of the

esterases within the body, and upon work with testicular atrophy (7), we had predicted at the outset of the

study that, calculated on a molar basis, EEAC would

have precisely the same effects as ethoxyethanol (EE).

Consequently, 600 ppm EEAC was expected to, and

did, induce complete embryomortality as 600 ppm EE

had done (5,16). At 390 ppm EEAC, resorptions were

only slightly increased, though fetal weights were

depressed and malformations were observed. At 130

ppm EEAC, fetal weights were still depressed and

there was one defective fetus, indicating that this level

is near the teratogenic threshold but should still be

considered teratogenic in the rat. Thus, there is a

relatively steep dose-effect curve with EEAC, and the

same can be said of 2-ethoxyethanol and ME.

Butoxyethanol appears to be rather unique in this

series, in that it produces maternal toxicity at levels

below those which produce embryotoxicity. Its toxicity

and hemolytic effects have been described (15), with a

recent study verifying and extending the previous

report (17). The same investigators also indicated (15)

that the likely metabolic product is butoxyacetic acid,

and that the majority of the metabolite is excreted

within 24 hr. That the excretion is fairly rapid is also

suggested from our results in that the hematuria was

observed only on the first day of exposure. As is

apparently true with most of the glycol ethers, BU is

readily absorbed through the skin (18). Our incidental

observation of necrosis in the tails of rats exposed to

BU was not noted in the literature. Whether the

necrosis was due to damage in the extremity of the

peripheral nervous system (in the tail) or to the lack of

oxygen and nutrients provided by the blood of the

region is unknown, though the known effects of BU on

the blood suggest the latter as a strong possibility.

Theoretically, one of the biotransformation products

of 2-EEE would be 2-ethoxyethanol (or its biotransformation products). Thus it may be that high levels of

2-EEE would be teratogenic, though this remains as

only speculation since we could not generate higher

levels of 2-EEE as a vapor. Considering all of these

results in conjunction with the previously reported

teratogenic effects of 2-ethoxyethanol, it appears that

longer chain glycol ethers are less embryotoxic than the

short-chain glycol ethers; this would be expected due to

the relatively lower lipid solubilities of the longer chain

compounds (19). Further, based on our results with

2-ethoxyethyl acetate, it is likely that the acetates of

other glycol ethers would have similar reproductive

toxicity to their corresponding ethers.

Finally, the lack of teratogenic response of 2-methylaminoethanol was interesting and from a mechanistic or

theoretical point of view, would merit follow up using a

different route of exposure. At first glance, one might

expect that its biotransformation would be similar to

that of 2-ME. However, our results of no maternal or

fetal toxicity at 150 ppm 2-MAE suggest that this may

not be the case; since the amine is likely more lipidsoluble and less water-soluble than the methoxy portion,

the absorption and excretion of the 2-MAE is likely

quite different from that of 2-ME. Thus it would be of

interest to see if a higher dose of 2-MAE would be

teratogenic, though a route other than inhalation would

be required, since the vapor concentration we used was

near the saturation point.' This lack of teratogenicity

at three times the concentration of a teratogenic level

of its structurally similar glycol ether, points to a

relatively strict structural requirement to produce

teratogenic effects.

The present study does not elucidate the mechanism

of action by which the solvents induce embryotoxicity.

Nor did we determine biotransformation pathways,

though it is likely that each glycol ether undergoes

* One purpose of this study was to select another glycol ether for

inclusion in a behavioral teratology study. As exposure to 200 ppm

2-ethoxyethanol on gestation days 14-20 was previously reported to

cause neonatal deaths (6), small groups of animals were also exposed

to the chemicals in the present study on gestation days 14-20.

Maternal animals reared their young (litters culled to four female and

four male pups on the day of birth) to weaning, and the offspring were

weighed weekly for 5 weeks. The results indicated that 100 ppm 2-ME

(N = 6) delayed parturition by approximately 36 hr and no pups

survived for 1 week; 50 ppm 2-HE (N = 5) produced little, if any, delay

in parturition, and all pups survived. Exposure to 600 ppm EEAC (N

= 3) delaved parturition by around 24 hr and produced 75% mortality

in the offspring; 390 ppm (N = 3) delayed parturition by about 12 hr,

but all offspring survived; neither 130 ppm EEAC (N = 4) nor 150

ppm BU (N = 4) produced any adverse effects in the offspring.

270
GLYCOL ETHER TERATOGENICITY 271

biotransformation via alcohol and aldehyde dehydrogenase, yielding its corresponding acetaldehyde and

subsequently excreted primarily as the acetic acid

derivative. We observed that embryotoxicity decreases

as alkyl chain length increases, similar to observations

with testicular atrophy (7). We found that short-chain

glycol ethers and their corresponding esters are

embryotoxic and teratogenic in rats. Since previous

research found similar effects induced by methoxyethanol in mice via intubation (3) and ethoxyethanol in

rabbits (5) via inhalation, and in rats via cutaneous

exposure (4), teratogenic effects have been induced in

three lower species of animals by common routes of

human exposure. Such effects call for careful examination of human populations exposed to this class of

solvents, particularly since these effects in animals

were observed at concentrations which are at or below

current permissible exposure limits.

NOTE: Since the time this paper was prepared,

there has been a proliferation of research on the glycol

ethers; we refer readers to the other paper in this issue

for more recent research.

We thank Dr. Bill Scott and the staff at Children's Hospital

Research Foundation for their assistance, Dr. Kent Anger for his

support and review of the manuscript, Mr. Randy Smith for his help

with the statistical analyses, and Mrs. Nadine Dickerson for her

uncomplaining typing and correcting of the manuscript.

Mention of company names or products does not constitute endorsement by the National Institute for Occupational Safety and Health.

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