The developmental toxicity of ethylene glycol in rats and mice

TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

The Developmental

81,

113-127 (1985)

Toxicity of Ethylene Glycol in Rats and Mice’

CATHERINE J. PRICE,* CAROLEA. KIMMEL,~-*~ ROCHELLEW.TYL,*'~ ANDMELISSAC.MARR* *Chemistry 12194,

and Life Sciences, Center for Life Sciences and Toxicology, Research Triangle Institute, P. 0. Box Research Triangle Park, North Carolina 27709-2194, and, tPerinata1 and Postnatal Evaluation Branch, Division of Teratogenesis Research, National Center for Toxicological Research/National Toxicology Program, Jefferson, Arkansas 72079

Received

September

20, 1984; accepted

May

24,

The Developmental Toxicity of Ethylene Glycol in Rats and Mice.

1985

C. J., KIMMEL, C. A., 81, 113-127. Timedpregnant CD rats and CD-I mice were dosed by gavage with ethylene glycol (EC) in distilled water on gestational days (gd) 6 through I5 (0, 1250, 2500, or 5000 mg kg-’ day-’ for rats; and 0, 750, 1500, or 3000 mg kg-’ day-’ for mice). Females were observed daily during treatment, but no maternal deaths or distinctive clinical signs were noted. Dose-related decreases in maternal weight gain during treatment were significant at all doses in rats and at the mid and high doses in mice. Gravid uterine weight was reduced in both species at the mid and high doses, and corrected maternal gestational weight gain showed a significant decreasing trend. At termination (gd 20, rats; gd 17, mice), the status of uterine implantation sites was recorded, and live fetuses were weighed and examined for external, visceral, and skeletal malformations. Dose-related increases in postimplantation loss per litter were observed in both species with the high dose significantly above controls only in rats. Fetal body weight per litter was significantly reduced at the mid and high doses in rats and at all doses in mice. The percentage of malformed live fetusesper litter and/or the percentage of litters with malformed fetuses was significantly elevated in all EC dose groups and 295% of litters were affected at the high dose for each species. A wide variety of malformations were observed; the most common in both species were craniofacial and neural tube closure defects and axial skeletal dysplasia. EC produced severe developmental toxicity in two rodent species at doses that apparently failed to produce any serious maternal effects. o 1985 TYL,

Academic

R.

W.,

AND

MARR,

M.

C. (1985).

Toxicol.

Appl.

PRICE,

Pharmacol.

Press, Inc.

Ethylene glycol (EG) is a high-volume industrial chemical with diverse applications and an estimated annual production of 1.9 X lo6 tons (Miller, 1979). EG is used as an antifreeze, an industrial humectant, a solvent for borates ’ Presented at the 24th Annual Meeting of the Teratology Society, Boca Raton, Florida, June 3-7, 1984 [Teratology 29, 52A, 19841. ’ To whom correspondence should be addressed, at Reproductive Effects Assessment Group, RD 689, U.S. Environmental Protection Agency, 401 M Street, S.W., Washington, DC 20460. ’ Present address: Bushy Run Research Center, R.D. 4, Mellon Road. Export, PA 15632.

in electrolytic condensers, a solvent in the paint and plastics industries, in the formulation of various types of inks, as a softening agent for cellophane, and as a stabilizer for soybean foam used to extinguish oil and gasoline fires. It is also employed in the synthesis of a variety of chemical products (Windholz, 1976) and has been used as a cryoprotectant in the laboratory (Kasai et al., 1982). EG may represent little hazard to human health in normal industrial handling, except possibly when used as an aerosol or at elevated temperatures. EG at ambient temperatures has a low vapor pressure and is not very irritating I13

0041~008X/85 $3.00 Copyri%t 0 1985 by Academic Press, Inc. All rights of reproduction in any form reserved.

114

PRICE ET AL.

to the eyes or skin (Rowe, 1963). However, structural developmental effects observed by accidental or intentional ingestion of anti- Lamb et al. (1985) and the reduced growth freeze products (approximately 95% EG) is and viability of offspring reported by Hazelden toxic and may result in death. The acute tox- (1983). icity of EG in laboratory species is similar to METHODS4 that in man, including CNS effects (ataxia, convulsions, coma), metabolic acidosis, renal Animals and animal husbandry. The experimental antoxicity, and death. Chronic exposure to EG imals were CD rats [(COBS) CrkCD (SD)BR] and CD-l results most notably in renal pathology (Balazs mice [(COBS) CrlCD-1 (ICR)BR].5 Female rats weighed et al., 1982; FDA, 1978a; Lyon et al., 1966) 200 to 275 g and female mice weighed 20 to 35 g on gd day of sperm or vaginal plug detection). and does not appear to increase tumor inci- 0 (i.e., Animals were individually identified by ear tags during dence in laboratory animals (Morris et al., their 7-day quarantine. During the study, animals were 1942; Glaser et al., 1977). housed on Ab-Sorb-Dri cage l&err in solid-bottom polyThe potential reproductive toxicity of EG propylene or polycarbonate cages (8 X 19 X 10 $ in.) with has been evaluated recently in several labo- stainless-steel wire lids’ and molded filter tops.’ Feed’ and deionized/filtered water were available ad libitum ratories. Exposure of pregnant CD- 1 mice on throughout the study. Animal rooms were equipped with gestational days (gd) 8 through 15 to EG ( 11.09 automatic light cycles (lights on 7:00 AM to 7:00 PM). Relg kg-’ day-‘, po) was toxic to the dams (5/50 ative humidity and temperature were maintained at 55 = 10% mortality) and adversely affected the + 10% and 20 f 1“C, respectively, for the rat study, and growth and viability of the offspring in the 47 k 10% and 20 -C 1“C respectively, for the mouse study. perinatal period (Hazelden, 1983). EG was also Air in each animal room was exchanged 12 to 14 times per hr. evaluated at lower concentrations (i.e., 0.25, Individual female rats in estrus or proestrus were placed 0.50, or 1.OO%, or approximately 0.4, 0.8, or overnight in the home cage of singly housed males of the 1.6 g kg-’ day-’ in the drinking water) in a same stock. On the following morning, vaginal smears were examined for the presence of sperm. Female mice continuous breeding ( 14-week cohabitation) were “primed” prior to breeding (Whitten, 1956) by placing protocol in CD-l mice (Lamb et al., 1985). a single male mouse in a small wire mesh cage inside the EG at 1.OO% slightly decreased the fertility of home cage of 10 females. Forty-eight hours later. each the exposed parental generation and produced female was placed overnight in the cage of one male for adverse effects on the growth, skeletal devel- mating, and then examined the next morning for the presopment (especially the cranium, ribs, and ver- ence of a vaginal plug. Sperm-positive female rats were singly housed and plug-positive mice were group housed tebral column), and fertility of the Fi gener- (maximum of four per cage). ation. Maronpot et al. ( 1983) recently reported Two replicates of the teratology evaluation were conno evidence of embryotoxicity nor any in- ducted for each species. Approximately equal numbers of crease in the incidence of major malformasperm- or plug-positive females were assigned to each of tions when EG was administered to Fischer four dose groups in each replicate (minimum of 10 per 344 rats in the feed at doses approximating 0, 40, 200, or 1000 mg kg-’ day-’ on gd 6 4 All studies were conducted in accordance with the through 15. In that study, a statistically sig- Food and Drug Administration Good Laboratory Practice nificant increase in the incidence of poorly os- Regulations for Nonclinical Laboratory Studies (FDA, sified and unossified vertebral centra was 1978b). Copies of the final study reports (Price et al.. 1984a,b; PB85 105385/GAR and PB85 104594/GAR) are observed in fetuses of high-dose dams in available from the National Technical Information Service. the absence of growth retardation or other Springfield. VA 22 16 1. embryotoxicity. Since the doses used in that 5 Charles River Breeding Laboratories, Inc., Kingston, NY. study failed to produce any signs of maternal 6 Laboratory Products, Garfield, NJ. toxicity, further testing was warranted to ex’ Laboratory Products, Rochelle Park, NJ. amine the potential teratogenicity and other ’ Ancare Corporation, Ma&asset, NY. developmental effects of EG. Thus, the present 9 Purina Certified Rodent Chow (5002). Ralston Purina studies were designed to further elucidate the Co., St. Louis, MO.

DEVELOPMENTAL

TOXICITY

dose group per replicate) for a total of at least 20 confirmedpregnant females per dose group. Breeding dates were on two consecutive days within replicates, but at least 2 weeks apart between replicates. Females of each species were assigned to dose groups by the method of stratified randomization so that body weight on gd 0 was not significantly different across dose groups within individual replicates or for each completed study (Tables 1 and 4). Test chemical and treatment. Ethylene glyc01’~ (CAS No. 107-21-l) of greater than 99% purity” was dissolved in distilled water. Dosing solutions were verified to be within + 10% of the theoretical concentration by gas chromatography both prior to administration and following completion of dosing. Each dosing solution was coded so that treatment and examination of animals were performed without knowledge of the dose concentrations. Animals were dosed daily by gavage with EG solutions or distilled water (vehicle) between 8:OOAM and 10:00 AM on gd 6 through 15. The actual volume administered (10 ml/kg) was based on body weight taken daily during the dosing period. The doses selected were 0, 1250, 2500, or 5000 mg kg-’ day-’ for rats and 0,750, 1500, or 3000 mg kg-’ day-’ for mice based on a preliminary toxicity study performed using timed-pregnant rats and mice (6 to 10 per dose group) treated with 0, 2500, 5000, 10,000, or 11,090 (undiluted) mg kg-’ day-’ EG on gd 6 through 15 (Price et al., 1984a,b). Observations. Rats were weighed on gd 0, 3,6 through 15, 18. and 20; mice were weighed on gd 0.6 through 15, and 17. Dams were observed during treatment for clinical signs of toxicity. Water consumption was measured in rats on gd 3.6,9, 12, 15. 18, and 20. On gd 20 all mated rats were anesthetized with carbon dioxide, and both rats (gd 20) and mice (gd 17) were killed by cervical dislocation. Maternal liver weight, kidney weights (rats only), and gravid uterine weight were measured. Uterine contents (i.e., number of implantation sites, resorptions, dead fetuses, and live fetuses) were evaluated, and live fetuses were dissected from the uterus and anesthetized by placing on ice (Lumb and Jones, 1973; Blair, 197 1). Each live fetus was weighed and examined for external morphological abnormalities, then the viscera were examined by a fresh tissue dissection technique (Staples, 1974). Half of the fetuses were decapitated prior to dissection and the heads were fixed in Bouin’s solution for free-hand sectioning and examination (Wilson, 1965). All fetal carcasses

OF ETHYLENE

GLYCOL

115

were prepared with Ahz.arin Red S stain and examined for skeletal malformations, as modified from Pehzer and Schardein ( 1966) and Crary (1962). Statistical analyses. Analyses of data were carried out by the General Linear Model (GLM) procedure in the SAS software library (SAS Institute, 1982a,b). Prior to analysis, an arcsine-square root transformation was performed on all litter-derived percentage data (Snedecor and Cochran, 1967). Dose-response relationships for selected measures were evaluated with a Test for Linear Trend. Analysis of variance (ANOVA) was used to determine whether significant dose effects, replicate effects,or dose X replicate interactions had occurred. When ANOVA revealed significant differencesamong groups then Williams’ Multiple Comparison Test (Williams, 197 1, 1972) and Dunnett’s Test (Dunnett, 1955, 1964) were used to compare EG-treated groups to the vehicle control group (alpha level = 0.05). Nominal scale measures were analyzed by a Test for Linear Trend on Proportions and a Chi-Square Test for Independence among treatment groups (Siegel, 1956). When Chi-Square revealed significant (p < 0.05) differences among groups, then a one-tailed Fisher Exact Probability Test (alpha level = 0.05) was used for pairwise comparisons between each EG-treated group and the vehicle control group.

RESULTS

Rats. No maternal deaths or distinctive clinical signs were associated with EG treatment, except for piloerection which was seen in all EG-treated groups but not in controls. Maternal body weight at termination (gd 20) weight gain during treatment, weight gain during gestation, corrected weight gain (i.e., weight gain during gestation minus gravid uterine weight), and gravid uterine weight each showed a significant decreasing trend as dose increased (Table 1). Weight gain during treatment was significantly reduced in all EGtreated groups relative to controls, and body weight on gd 20, weight gain during gestation, and gravid uterine weight were reduced in the mid and high dose groups. No individual EGtreated group was significantly reduced below controls for corrected maternal weight gain. lo Obtained for Research Triangle Institute from Maternal liver weight (g), but not relative liver Southern Research Institute, Birmingham, AL. Manufacutred by Ashland Chemical Company, Ashland, KY (Lot weight (% body weight), was decreased signifNo. A02 1180). icantly at the high dose. Relative kidney weight ” Purity determinations were conducted by Midwest (% body weight), but not absolute kidney Research Institute (NC1 Contract No. NO l-CP-956 l5), and involved titration, thin-layer and gas chromatography, and weight (g), was increased in the mid- and highinfrared, uv/visible, and NMR spectroscopy. See Price et dose groups, possibly due to the associated real. (1984a,b) for details. duction in maternal body weight on gd 20.

’ Includes all dams pregnant at termination; X f SEM. b Weight gain during gestation minus gravid uterine weight. t p < 0.05, Test for Linear Trend. tt p < 0.001, Test for Linear Trend. * p < 0.05 versus Controls. ** p < 0.01 versus Controls. NSNot significant, p > 0.05.

Maternal body weight (gd 0) (g)a Maternal body weight (gd 20) (g)” Maternal weight gain (treatment, gd 6-l 5) (g)” Maternal weight gain (gestation, gd O-20) (g)” Maternal weight gain (corrected) (g)“*’ Gravid uterine weight (g)” Maternal liver weight (g)” Relative maternal liver weight (W body wt.)” Maternal kidney weight (g)” Relative maternal kidney weight (% body weight)“ Maternal water consumption (g/kg/day) on gd 6- 15 a Maternal water consumption (g/kg/day) on gd 15-20”

Total(%) No. treated pregnant at termination

Subjects (Dams)

I

NS tt tt tt t tt tt NS NS j-t tt tt + 4.3

+ 4.8

130.3

148.2

1.920 + 0.030 0.517 f 0.012

k 0.06

154.1

0.531 128.4

k4.1

f 0.007 + 3.0

1.880 f 0.037

f 0.05

4.23

3.76 4.15 1.73** 3.90 3.60 1.79

15.01 f 0.28

-e -c f f f k

4.23

f 3.31 f 1.63

56.41 73.04

237.18 354.21 34.81 117.09 49.40 67.69

28 $0.0,

1250

15.47 + 0.26

+ 3.41 k 3.94 f 1.96

236.56 366.06 42.03

129.50 f 3.06

28 (100.0) 28

0

6 THROUGH 15

237.43 345.69 29.45 108.26 50.27 58.00 14.95 4.33 1.977 0.573 154.4 178.8

+ k f f + k + k + f + +

3.57 5.86** 1.38** 4.06** 2.57 2.97** 0.27 0.07 0.032 0.008** 4.0** 4.5**

29 $0.0)

2500

Ethylene glycol (mg kg-’ day-‘, po)

MATERNALTOXICITYINCDRATSEXPOSEDTOETHYLENEGLYCOLONGESTAT~ONALDAYS

TABLE

f + iz + + + f k

3.73 6.64** 1.93** 6.35** 4.57 3.75** 0.35* 0.08 0.615 165.0 172.1

+ 0.021** + 3.8** k 5.8**

1.979 & 0.059

234.82 324.99 20.68 90.17 43.56 46.61 13.70 4.22

27 $5.4)

5000

DEVELOPMENTAL

TOXICITY

Maternal water consumption (g kg-’ day-‘) during treatment (gd 6 to 15) and after treatment (gd 15 to 20) was increased in dose-related manner, with significantly more water consumed by mid and high dose dams (Table 1).

OF ETHYLENE

117

GLYCOL

Treatment-related adverse effects on pregnancy outcome (Table 2) included a significant increase above controls in the percentage postimplantation loss (resorptions plus dead fetuses) per litter in the high-dose group, as well as a trend toward an increased percentage

TABLE 2 DEVELOPMENTAL

TOXICITY IN CD RATS FOLLOWING MATERNAL EXPOSURE TO ETHYLENE GLYCOL ON GESTATIONAL DAYS 6 THROUGH 15

Ethylene glycol (mg kg-’ day-‘, po)

All litters” No. implantation sites per litterb Percentage postimplantation loss per litterbac Percentage litters with postimplantation loss at one or more sites Live litters’ No. live fetuses per litterb Percentage males per litterb Average fetal body weight (g) per litterb Percentage live fetuses malformed per litter’ Percentage litters with one or more malformed live fetuses

0

1250

2500

5000

28

28

29

27

NS 14.21 f 0.26

13.64 f 0.33

12.72 f 0.62

13.44 + 0.56

tt 4.70 + 1.23

6.35 f 1.85

6.27 + 1.35

21.34 f 5.24*

t 39.3

50.0

55.2

28 tt 13.54 f 0.28

28 12.75 + 0.38

29 11.90 + 0.60*

26 11.04 + 0.79*

NS 43.87 rt 3.55

47.62 f 2.51

46.06 f 2.86

46.63 + 3.88

2.9 16 2 0.056**

74.1 d

tt 3.404 + 0.052

3.312 + 0.058

tt 1.37 k 0.97

6.65 + 2.04

25.11 i 4.84**

73.53 t 6.42**

tt 7.14

39.29**

68.97***

96.15***

’ Includes all dams pregnant at termination; litter size = no. implantation sites per dam. b Reported as X f SE. ’ Postimplantation loss includes all resorptions and dead fetuses. ’ One dam had all resorptions. ’ Includes only dams with live fetuses; litter size = no. live fetuses per dam. t p < 0.01, Test for Linear Trend. tt p < 0.00 1, Test for Linear Trend. * p < 0.05 versus Controls. ** p < 0.01 versus Controls. *** p < 0.001 versus Controls. NSNot significant, p > 0.05.

2.388 + 0.089**

118

PRICE TABLE

ET

AL. 3

CD RATS FOLLOWINGMATERNAL EXPOSURETOETHYLENEGLYCOLON GESTATIONAL DAYS 6 THROUGH 15: LISTING BY DEFECT TYPE”

MORPHOL~GICDEFECTSOBSERVEDIN

Ethylene glycol (mg kg& day-‘, po) 1250

0

All malformations Malformed fetuses/total examined b Litters with malformed/total examined’ External malformations No. fetuses with one or more defects No. litters with malformed fetuses

NA 51319

2128

ttt

NAO tt 0

2500

5000

211357

861345

1971281

1l/28**

20/29***

25/26-

0

5

65

0

4

15***

I

36 33

Cleft palate Cleft lip Anophthalmia (bilateral, right, or left) Micrognathia Meningoencephalocele Exencephaly Meningocele Gastroschisis Umbilical hernia Visceral malformations No. fetuses with one or more defects No. litters with malformed fetuses

1

21 2 18 9 3

5

11 4

NAO

9

3

to

6*

2

Anomalies of the great vesselsd Hydroureter (bilateral, left. or right) Hydronephrosis (bilateral or right) Diaphragmatic hernia Testes l/2 normal size Ectopic testes Displaced kidney Renal agenesis (left) Missing liver lobe Missing small intestines Missing thymus Skeletal malformations No. fetuses with one or more defects No. litters with malformed fetuses Ribs (short, missing, branched, and/or fused)

1

8** 9

2 I

15

1

2

3 2

1

12

NA5

2

tit 5

83

6

19***

8

50

193 24*** 141

DEVELOPMENTAL

TOXICITY

OF ETHYLENE

119

GLYCOL

TABLE 3-Continued Ethylene glycol (mg kg-’ day-‘, po) 0 Arch (enlarged, small, fused, and/or missing) Centra (misaligned, unilateral ossification, off center, fused, and/or missing) Stemebrae (fused or scrambled)

1250

2500

5000

18

73

4

57

179

2

7

4

’ A single fetus may be represented more than once in listing individual defects;statistical analysis was not performed for the incidence of individual malformations. * Only live fetuses were examined for malformations. ’ Includes only litters with live fetuses. d Anomalies of the great vesselsincluded two left carotid arteries, retrotracheal and/or retroesophageal aorta, missing right subclavian artery, reversed aorta, pulmonary artery stenosis, and left common carotid artery arising from the innominate artery. t p < 0.05, Test for Linear Trend. tt p < 0.0 1, Test for Linear Trend. ttt p < 0.00 1, Test for Linear Trend. * p < 0.05 versus Controls. ** p i 0.01 versus Controls. *** p < 0.001 versus Controls, NA Not analyzed statistically.

of litters with postimplantation loss at one or more sites. The live litter size was reduced at the mid and high doses, but the reduction at the mid dose may have been related to the lower number of implantation sites per litter at that dose relative to controls. The fetal sex ratio (percentage of male fetuses per litter) was not affected by EC treatment. In the mid- and high-dose groups, there was a significant decrease in average fetal body weight, and a significant increase in the percentage of malformed fetuses per litter. At the low dose ( 1250 mg kg-’ day-‘), the percentage of fetuses malformed per litter was elevated but the value was not statistically significant relative to controls. However, the percentage of litters with one or more malformed fetuses was significantly increased at all doses tested. A variety of malformation types were seen (Table 3 and Fig. 1). When malformations were analyzed

by general category (Table 3), fetuses with external malformations (craniofacial, neural tube closure, and abdominal wall defects) occurred in a significantly higher number of litters in the high-dose group relative to controls. Fetuses with visceral malformations occurred in a higher number of litters in all groups, but only the low- and high-dose groups were significantly higher than controls. Visceral malformations involved several organ systems including the major vessels, kidneys, liver, thymus, diaphragm, testes, and gastrointestinal tract. Fetuses with skeletal malformations occurred in a significantly higher number of litters in the mid- and high-dose groups. Severe dysmorphogenesis of the axial skeleton was characterized by malformed or fused ribs, vertebral arches, and centra (Table 3). Mice. The maternal effects of EG were essentially the same in mice (Table 4) as in rats,

120

PRICE ET AL.

FIG. I. Rat fetusesexhibiting cleft lip and palate (left) or axial skeletal dysmorphogenesis (right) following maternal exposure to 5000 mg kg-’ day-’ EC on Days 6 through 15 of gestation.

except that weight gain during treatment was significantly reduced in the mid- and high-dose groups relative to controls but not at the low dose. Maternal liver weight (g), but not relative liver weight (% body weight), was decreased significantly in both the mid- and high-dose groups. A significant reduction in the number of implantation sites per litter (Table 5) was seen only in the mid-dose group versus controls; since this reduction was not dose-related, it was assumed unlikely to be related to EG treatment. Treatment-related adverse effects on pregnancy outcome included significant increasing trends for the percentage postimplantation loss (resorptions plus dead fetuses) per litter and for the percentage of litters with postimplantation loss at one or more sites. A

significant treatment-related decrease in live litter size was seen in the high dose group. The percentage of male fetuses per litter was not affected by EG treatment. A significant treatment-related decrease occurred for average fetal body weight per litter, and all dose groups were below vehicle controls. In all EG-treated groups, the percentage of malformed fetuses per litter and the percentage of litters with one or more malformed fetuses were significantly increased above controls (Table 5). As in the rat, craniofacial anomalies, neural tube closure defects, and axial skeletal defects were observed with the highest frequency. When malformations were analyzed by general category (Table 6) fetuses with external or visceral malformations occurred in a significantly higher number of litters in the high-dose group

DEVELOPMENTAL

TOXICITY

OF

TABLE

ETHYLENE

121

GLYCOL

4

MATERNAL TOXICITY IN CD- 1 MICE EXPOSED TO ETHYLENE GLYCOL ON GESTATIONAL DAYS 6 THROUGH 15 Ethylene glycol (mg kg-’ day-‘, po) 0 Subjects (Dams) Total treated No. (%) pregnant at termination Maternal body weight (gd 0) (g)” Maternal body weight (gd 17) (g)” Maternal weight gain (treatment, gd 6-15)(g)"

Maternal weight gain (gestation) (g)” Maternal weight gain (corrected) (g)“sb Gravid uterine weight (g)” Maternal liver weight (g)” Relative maternal liver weight (% body weight)“

1500

750

27 25 (92.6)

26 24 (92.3)

NS 29.80 f 0.46 tt 52.87 f 0.89

29.10 f 0.57 50.49 + 0.80

29.46 47.30

11.58 ? 0.39 21.38 f 0.72 5.92 kO.49

8.54 + 0.84* 17.84 f l.OO* 5.67 + 0.31

16.45 + 0.61*

t 0.62

15.46 r 0.55

12.17 + 0.93*

11.58 f 0.59;

f 0.05

2.63 f 0.04

2.49 f 0.06;

2.47 f 0.06*

5.22 k 0.06

5.27 + 0.08

5.35 f 0.10

tf

12.40 f 0.49 f 0.87 f 0.49

tt 23.07 t 5.94 tt 17.13 tt 2.72

NS 5.15 + 0.07

25 23 (92.0)

3000

f 0.43 k 1.08*

26 23 (88.5) 29.66 + 0.46 46.12 f 0.81* 8.42 + 0.54* 4.87 + 0.32

’ Includes all dams pregnant at termination; X + SE. b Weight gain during gestation minus gravid uterine weight. 7 p < 0.05, Test for Linear Trend. ti p < 0.001, Test for Linear Trend. * p < 0.01 versus controls. NSNot significant, p > 0.05.

relative to controls. External malformations were similar to those observed in rats, except that no abdominal wall defects were seen in mice. In mice, two fetuses in one litter in the high-dose group each exhibited a mid-facial cleft (Fig. 2), which is a particularly unusual defect for this species. Visceral malformations were restricted to a smaller number of organ systems than in the rat, but involved major malformations of the cardiovascular system, kidneys, and lungs. Fetuses with skeletal malformations occurred in a higher number of litters in all EG-treated groups relative to controls. The types of skeletal defects seen in mice were very similar to those observed in rats, involving fusion or malformation of the ribs, vertebral arches, and centra.

DISCUSSION

In a previous reproductive toxicity evaluation (Lamb et al., 1983, several neonatal CD1 mouse pups exhibited cleft lip following continuous exposure to the parents to 1.00% EG in the drinking water (N 1A g kg-i day-r), thus indicating a possible teratogenic effect of EG following in utero exposure. In the same study, some of the F, progeny displayed modified craniofacial characterisitics as adults, including short snout and wide-set eyes, which had not been apparent in the neonatal period. Skeletal evaluation of four adult control and four treated F, progeny of each sex also revealed defects in treated animals which af-

122

PRICE

ET

TABLE DEVELOPMENTAL

TOXICITY

AL. 5

IN CD-l MICE FOLLOWING MATERNAL ON GESTATIONAL DAYS 6 THROUGH

EXPOSURE 15

TO ETHYLENE

GLYCOL

Ethylene glycol (mg kg-’ day-‘, po)

All litters’ No. implantation sites per litter Ir Percentage postimplantation loss per litte+ Percentage litters with postimplantation loss at one or more sites Live litters’ No. live fetuses per litter6 Percentage males per litterb Average fetal body weight (g) per litter6 Percentage live fetuses malformed per litter& Percentage litters with one or more malformed live fetuses

0

750

1500

3000

25

24

23

23

13.32 + 0.44

12.92 k 0.50

II.57

f 0.70*

id 10.92 f 2.25

10.49 k 2.39

19.35 + 5.37

t 68.0

62.5

25 tt 11.88 + 0.49 NS 44.05 + 2.57

24 11.50 + 0.49 48.69 k 3.54

22 10.41 * 0.74 42.28 k 4.33’

0.882 f 0.017**

0.787 + 0.024**

ttt

10.00 -+ 1.96**

37.77

zk 0.25

t 4.00

66.67***

20.49

+ 6.30’;

8 1.82***

+ 3.58

91.3

13.9

ttt 0.974 + 0.013 0.25

12.30 + 0.43

23 9.83 ? 0.56; 44.06 + 3.72 0.7 12 k 0.022** 56.54

f 6.80**

95.65***

’ Includes all dams pregnant at termination; litter size = number of implantation sites per dam. b Reported as Z+ SE. ’ Postimplantation loss includes all resorptions and dead fetuses. d Significant dose effect by ANOVA (p < 0.05) no significant pairwise comparisons. ‘Includes only dams with live fetuses; litter size = number of live fetuses per dam. ‘One litter had no males. t p < 0.05, Test for Linear Trend. tt p < 0.0 1, Test for Linear Trend. ttt p c 0.001, Test for Linear Trend. * p < 0.05 versus Controls. ** p cc 0.01 versus Controls. *** p c: 0.001 versus Controls. NS Not significant, p > 0.05.

fected not only the skull, but also the ribs, vertebrae, and stemebrae of both sexes. The studies reported here confirm and extend the findings of Lamb et al. (1985) by demonstrating that exposure to EG only during organogenesis results in a significantly increased incidence of major fetal malformations in rats at doses of EG 3 1250 mg kg-’ day-‘, and in mice at doses of EG 3750 mg kg-’ day-‘. The types of malformations observed in the present studies included many

of those seen in the study of Lamb et al. (e.g., cleft lip, fused ribs, abnormally shaped vertebrae, and abnormally shaped stemebrae). However, the shortened frontal, nasal, and parietal bones observed in adult F1 mice following continuous pre- and postnatal exposure (Lamb et al., 1985) were not observed in fetal rats or mice following exposure restricted to the period of organogenesis. These results suggest that EG continues to exert an effect upon cartilage development and/or skeletal ossifi-

DEVELOPMENTAL

TOXICITY

cation patterns when exposure extends into the postnatal period. We are currently following the postnatal development of animals exposed to EG only during organogenesis for more detailed observation of skeletal and other types of defects. Systematic evaluation of fetal anatomical defects in the present study revealed a wider variety of anomalies than reported by Lamb et al. ( 1985). For example, neural tube closure defects were seen in both fetal rats and mice, but were not seen in neonatal mice in the previous study possibly because of early maternal cannibalism of nonviable and/or moribund pups. Visceral examination (performed only in the present studies) indicated that in utero exposure to EG was also capable of affecting the development of the major blood vessels, kidneys, liver, thymus, diaphragm, testes, and gastrointestinal tract in rats, as well as the cardiovascular system, kidneys, and lungs in mice. In the present studies, the mouse appeared to be somewhat more sensitive to EG than the rat, both in terms of maternal toxicity and fetal effects. For example, a 30 to 3 1% reduction in maternal weight gain during treatment was observed at 1500 mg kg-’ day-’ in mice and at 2500 mg kg-’ day-’ in rats. In mice, a dose of 1500 mg kg-’ day-’ was associated with reduced maternal weight gain throughout gestation, as well as during treatment, but in rats a dose of 1250 mg kg-’ day-’ only reduced maternal weight gain during treatment (17% below the control group). In both species there was a significant increase in the percentage of malformed fetuses per litter or in the percentage of litters with malformed fetuses even at the lowest dose tested (750 mg kg-’ day-’ for mice; 1250 mg kg-’ day-’ for rats). Although a no-observed-effect level (NOEL) for fetal effects was not established in either species, there was a clear dose-response relationship, and comparison with the data of Maronpot et al. (1983) would suggest that the 1250 mg kg-’ day-’ dose in rats may be near the NOEL, since an estimated dose of 1000 mg

OF

ETHYLENE

GLYCOL

123

kg-’ day-’ in the feed in that study had no effect on Fischer 344 rats. The marked fetal effects at 750 mg kg-’ day-’ in mice in the present study suggest that the NOEL may be much lower in that species, but further testing is necessary in both species to clearly establish these values. Under the conditions of the present study, the lowest doses of EG which caused adverse fetal effects produced only mild signs of maternal toxicity (i.e., a slight decrease in body weight gain during treatment) in the rat and no obvious maternal effects in the mouse. Corrected maternal body weight gain was not significantly decreased in any dose group (although there was a significant dose-related trend and an obvious reduction at the highest dose in both species), suggesting that most of the effects on body weight gain throughout gestation were due to fetal effects, as reflected in the reduction in gravid uterine weight in the mid and high dose groups in both species. The reduction in maternal liver weight (but not relative liver weight) observed in rats at the high dose, and in mice at the mid and high doses probably reflects the general adverse effect of EG upon pregnancy status rather than a hepatotoxic effect, since the liver normally increases in weight during a successful pregnancy (unpublished data). In rats, maternal water consumption was significantly increased in the mid and high dose groups, suggesting the possibility of renal toxicity in those animals. However, no gross changes in the appearance of the maternal kidneys were observed, and kidney weight did not differ among groups. Increased relative kidney weight in the mid- and high-dose groups appeared to be due to the associated reduction in maternal body weight on gd 20. Maternal water consumption was not measured in mice, and other functional or histopathological indices of maternal renal toxicity were not specifically investigated for either species in this study. Further investigation of the embryotoxic response to EG in laboratory rodents appears to be warranted, to determine whether EG it-

124

PRICE ET AL. TABLE 6

MORPHOLOGIC DE!=ECXSOBSERVED IN CD-l ON GESTATIONAL

MICE FOLLOWING MATERNAL EXPOSURE TO ETHYLENE DAYS 6 THROUGH 15: LISTING BY DEFECT TYPE’

GLYCOL

Ethylene glycol (mg kg-’ day-‘, po) 750

1500

3000

NA I/297

261276

89/229d

I 29/226d

t 1125

16/24**

18/22**

22/23**

0

All malformations Malformed fetuses/total examined b Litters with malformed/total examinedC External malformations No. fetuses with one or more defectsb No. litters with malformed fetuses

NAO NSO

Exencephaly Meningoencephalocele Cleft palate Cleft lip Facial cleft Visceral malformations No. fetuses with one or more defects No. litters with malformed fetuses

16 8* 6

NAO to

0 0

Aortic stenosis Hydronephrosis (bilateral or m-4 Hydroureter (right) Retrotracheal and/or retroesophageal pulmonary artery One papillary muscle right ventricle Extra vesselbetween right subclavian and carotid arteries Missing lung lobe Skeletal malformations No. fetuses with one or more defects No. litters with malformed fetuses Rib (short, missing, branched, and/or fused) Arch (enlarged, fused, small, and/or missing) Centra (misaligned, off center, unilateral ossification, and/or fused) Stemebrae (fused or scrambled)

9 4 2

I

2 2

9 7* 2

1

2 2 2

NA 1 tt 1

24

84

15**

17**

126 22**

I

13

75

113

5

22

55

2 8

17 1

49 5

a A single fetus may be represented more than once in listing individual defects; statistical analysis was not performed for the incidence of individual malformations. b Only live fetuses were examined for malformations.

DEVELOPMENTAL

TOXICITY

FIG. 2. Mouse fetus exhibiting a mid-facial cleft after maternal exposure to 3000 mg kg-’ day-’ EG on Days 6 through I5 of gestation.

self is the proximal toxicant, and also to examine possible relationships between EG and the developmental toxicity of structurally related compounds. EG is rapidly distributed throughout the body following intravenous or inhalation exposure (Marshall and Cheng, 1983; Marshall, 1982) and has been reported to cross the placental barrier (Bissonnette et al., 1979; Thornburg and Faber, 1977), thus raising the possibility of a direct action upon fetal tissues. The possible role of EG metabolites (e.g., glycoaldehyde, glycolic acid,

’ Includes only litters with live fetuses. ‘One fetal skeleton was not examined. t p < 0.05, Test for Linear Trend. $t p < 0.0 I, Test for Linear Trend. * p -c 0.01 versus Controls. ** p < 0.001 versus Controls. NANot analyzed statistically. NsNot significant, p > 0.05.

OF ETHYLENE

GLYCOL

125

glyoxylic acid, and oxalic acid) (Marshall, 1982; Balazs et al., 1982; Parry and Wallach, 1974; Marshall and Cheng, 1983) in mediating maternal and fetal toxicity also deserves additional attention. EG can be encountered as both an in vivo and in vitro degradation product of ethylene oxide (EO), a gaseous sterilant and fumigant pesticide. EO has been shown to have some potential for teratogenicity in mice but not in rabbits (see review by Kimmel et al., 1984), and only after high-dose intravenous administration (LaBorde and Kimmel, 1980). Notably, EG is also the parent compound for the ethylene glycol ether solvents which have been identified as priority chemicals for evaluation (NIOSH, 1983) because developmental and reproductive toxicity have been reported for several members of this structural class (Hardin et al., 1981; Nagano et al., 1981; Nelson et al., 1982; Schuler et al., 1984). The possible role of EG in the toxicity of either EO or the glycol ethers has not been evaluated. Gavage administration of EG (21250 mg kg-’ day-‘, rats; a750 mg kg-’ day-‘, mice) during organogenesis produced severe doserelated developmental toxicity including an increase in fetal malformation incidence with more than 95% of the litters in each species being affected at the highest dose (5000 mg kg-’ day-‘, rats; 3000 mg kg-’ day-‘, mice). The lack of apparently serious maternal effects at the lowest dose which produced malformations in both species, as well as the severity and frequency of fetal defects at higher doses, suggest that EG may carry a selective risk to the embryo and should be considered a potential developmental hazard in situations where major EG exposure is likely to occur.

126

PRICE ET AL.

ACKNOWLEDGMENTS The present studies were conducted at Research Triangle Institute (RTI), Research Triangle Park, North Carolina, under a contract from the National Toxicology Program and the National Center for Toxicological Research, NTP/ NCTR Contract 222-80-2031(C). The authors expresstheir appreciation to the following RTI personnel who contributed to the completion of this investigation: Ms. Frieda S. Gerling, Ms. Lynne S. King, Ms. Vi&i I. Wilson, Mr. Oliver L. Bullock, Mr. Fred D. Cole, Ms. Ellen B. Hahn, Dr. Brian M. Sadler, Mr. Philip V. Piserchia, Dr. W. Robert Handy, Ms. Doris J. Smith, and Ms. Julia C. Albert.

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