Embryotoxic effects of ethylene glycol monomethyl ether in mice

Embryotoxic effects of ethylene glycol monomethyl ether in mice

Toxicology, 20 (1981) 335--343 © Elsevier/North-Holland Scientific Publishers Ltd. EMBRYOTOXIC EFFECTS OF ETHYLENE GLYCOL MONOMETHYL ETHER IN MICE K...

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Toxicology, 20 (1981) 335--343 © Elsevier/North-Holland Scientific Publishers Ltd.

EMBRYOTOXIC EFFECTS OF ETHYLENE GLYCOL MONOMETHYL ETHER IN MICE

K A S U K E N A G A N O * , EIKI N A K A Y A M A , HISAO OOBAYASHI, T S U T O M U YA_MADA, HIDEMI ADACHI, T O M O S H I NISHIZAWA, H A R U M I O Z A W A , M O M O K O NAKAICHI, H I R O K A Z U O K U D A , K Y O K O M I N A M I and K A Z U N O R I Y A M A Z A K I Occupational Health Service Center, 35-1, 5-chome, Shiba Minato-ku, Tokyo, 108 (Japan)

(Received October 22nd, 1979) (RevisionreceivedFebruary 15th, 1981) (Accepted February 18th, 1981)

SUMMARY

An embryotoxicity study on ethylene glycol monomethyl ether (EGM) was carried out in ICR mice. They were given EGM daily at 6 dose levels (31.25, 62.5, 125, 250, 500 or 1000 mg/kg body wt) by gastric intubation on days 7 through 14 of gestation. On day 18 of gestation all fetuses were examined. Marked and dose-related embryotoxic effects were observed. Skeletal and gross anomalies, reduced fetal weight and fetal death were all observed at lower dosages of EGM, while marked leucopenia of the dams occurred at the highest dose.

INTRODUCTION

Being miscible with water and many organic solvents, EGM is widely used as an industrial solvent for resins, lacquers, dyes and inks. Some cases of human EGM poisoning reported to date indicate that the main pathological findings are disorders of the central nervous system, changes of blood picture and kidney damage [ 1--4]. Wiley et al. described toxic effects of EGM on the testis [5], and Werner et al. on the blood of laboratory animals [6,7].We also reported toxic effects of ethylene glycol monoalkyl ethers including EGM on testis and leucocytes of adult mice [8]. These effects of EGM resemble those of cytotoxic alkylating chemicals such as ethyleneimine and certain epoxy compounds which interfere with cell proliferation. According to Hemsworth et al. administration of these compounds to pregnant animals caused embryotropic effects [ 9]. *To whom correspondence and reprint requests should be addressed.

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One report on ethylene glycol monoalkyl ethers by Stenger, in which he gave ethylene glycol monoethyl ether to pregnant rodents orally, describes an increased occurrence of fetal skeletal anomalies [10]. The present report describes the embryotoxicity and teratogenicity of EGM. METHODS

Female JCL-ICR mice (Clea Japan, Inc., Tokyo, Japan) weighing 30--34 g were housed overnight with males of the same stock, and the day on which a vaginal plug was observed was designated d a y 0 of gestation. The pregnant mice were kept in shoe-box cages containing wood chips in a room under controlled environmental conditions (temperature 23 -+ 0.5°C, r.h. 60-+ 5%, 12-h light-dark sequences). They were fed a commercial laboratory diet for reproduction use, OA-2 (Clea Japan, Inc., Tokyo, Japan) and tapwater was provided ad libitum. They were randomly assigned to experimental and control groups, the number of individuals in each being 21--24. On days 7---14 of gestation, EGM (Wako Chemical Industries, Ltd. Osaka, Japan) at various doses (31.25, 62.5, 125, 250, 500 or 1000 mg/kg body wt) dissolved in deionized water, was given daily by gastric intubation. The group administered vehicle only served as the control. The general condition and symptomatology of the dams was checked daffy throughout the experiment and maternal body weight was noted at 2--4-day intervals. On day 18 of gestation mice were sacrificed by cervical dislocation, their reproductive status was observed and blood taken for leucocyte counting. The number and position of live, dead and resorbed fetuses were noted; those living were weighed and examined for external malformations. From each dose group living fetuses with no external malformations or with only abnormal fingers were eviscerated, preserved in alcohol, subsequently cleared and stained with Alizarin red S for skeletal examination. Fetuses with abnormal fingers were stained with Alcian blue and Alizarin red S [ 11]. The number of post-lumbar vertebrae, proximal and middle phalanges was determined to evaluate the degree of ossification. The Wflcoxon rank sum test [12] was used to compare the incidence of fetal death and gross and skeletal abnormalities among litters. Litter size, maternal and fetal body weight, maternal leucocyte counts and ossification of fetuses were analyzed by the Student t-test. Differences were considered significant if P was < 0.05. RESULTS (1) Effects on the dams There were no maternal deaths, but the maternal body weight gain was lower in 250, 500 and 1000 mg/kg groups than in the control (Fig. 1). The significant decrease in the leucocyte count was observed only in the 1000 mg/kg group (Table I).

336

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50

45 ~O

=:

40

w

=- 35 3O

====¢"25 ~

0

T

0

(1

(4

1"8

DAYS OF GESTATION Fig. 1. Body weight changes of pregnant mice treated with EGM. (@) Control group; (m) 31.25 mg/kg group, (o) 62.5 mg/kg group; (A) 125 mg/kg group; (A) 250 mg/kg group; (o) 500 mg/kg group; (o) 1OOOmgikg group.

(2) Effects on the fetuses (A) Observations made at the time o f caesarean section (Table I). There was an increase in the incidence of dead fetuses compared with controls in the 250, 500 and 1000 mg/kg groups. All litters given 1000 mg/kg were dead, and only 1 fetus lived in the 500 mg/kg group. Reduction of fetal weight was significant at 125 and 250 mg/kg doses. (B) Incidence o f gross and skeletal anomalies (Table II). The incidence of gross and skeletal anomalies are indicated in Table II. In 500 and 1000 mg/kg groups, skeletal observations were n o t made, since all fetuses were dead or had gross anomalies. (a) Gross anomalies. The incidence of gross anomalies observed by external examination of fetuses was significantly greater than in controls for the 250 mg/kg group. A total of 57 abnormal fetuses were f o u n d among 130 live fetuses in the 250 mg/kg group. Of these 24 had exencephaly, 3 umbilical hernia, 29 abnormal finger(s), and 1 had both exencephaly and abnormal finger. Only 1 living fetus in the 500 mg/kg group had exencephaly and abnormal fingers. 337



21 12.7 ± 2.7 239 (90.3 -+ 7.7) 6 (2.1 ± 3.8) 21 (6.8 ± 5.2) 11.4 ± 1.4 1384 ± 84 0 3636 ± 1114

22 13.0 ± 2.2 270 (94.0 + 7.4) 7 (2.3 ± 4.5) 10 (4.1 ± 6.8) 12.3 ± 2.4 1367 -+ 76 0 3267 ± 1647

31.25

304 (90.8 ± 8.0) 5 (1.6 ± 3.8) 25 (7.O ± 6.1) 12.7 ± 2.0 1321 ± 106 0 3075 ± 1781

24 13.5 ± 1.4

62.5

269 (88.6 ± 14.0) 7 (2.6 ± 4.2) 25 (8.8 ± 13.3) 11.2 ± 2.6 1185 ± 193 c 0 2190 ± 736

24 12.5 ± 2.0

125

aMean ~ S.D. b M e a n o f litters + S.D. CSignificantly d i f f e r e n t f r o m c o n t r o l by the S t u d e n t t-test, P < 0.01. dSignificantly d i f f e r e n t f r o m c o n t r o l by t h e Wilcoxon rank s u m test, P < 0.01.

Resorbed (%)b Live f e t u s e s / l i t t e ra Fetal b o d y weight (rag) b No. o f maternal d e a t h L e u c o c y t e c o u n t o f dams a

(%)b

No. o f litters Implantation sites/dama No. o f fetuses Live (%)b Dead

0

Dosage ( m g / k g / d a y )

130 (46.5 ± 29.0) d 153 (50.0 ± 28.7) d 10 (3.2 -+ 4.3) 5.7 ± 3.5 c 864 ± 171 c 0 3100 ± 1384

23 12.7 ± 2.1

250

1 (0.3 ± 1.5) d 283 (97.7 ± 4.4) d 6 (2.0 ± 4.2) 0.04 ± 0.2 c 485 0 2789 ± 692

24 12.1 ± 2.2

500

0 1763 ± 607 c

278 (96.9 ± 5.2) d 10 (3.2 ± 5.2) 0

0

24 12.0 ± 2.5

1000

R E S U L T S OF O B S E R V A T I O N M A D E AT THE TIME O F C A E S A R E A N S E C T I O N O F MICE G I V E N E T H Y L E N E G L Y C O L M O N O M E T H Y L E T H E R BY G A S T R I C I N T U B A T I O N ON DAYS 7--14 O F G E S T A T I O N

TABLE I

T A B L E II I N C I D E N C E O F G R O S S A N D S K E L E T A L A N O M A L I E S O F MICE G I V E N E T H Y L E N E GLYCOL MONOMETHYL ETHER BY GASTRIC INTUBATION ON DAYS 7--14 OF GESTATION Dosage ( m g / k g / d a y ) 0 Gross a n o m a l i e s N u m b e r o f fetuses e x a m i n e d (litters) Anomalies, type and incidence Total (%)c Exencephaly Umbilical hernia A b n o r m a l fingers

31.25

62.5

125

250

270 (22)

239 (21)

304 (24)

269 (24)

130 (23)

1 (0.4) 1 0 0

1 (0.4) 1 0 0

1 (0.5) 0 1 0

5 (3.1) 5 0 0

49 (36.5) b 25 a 3 30 b

174 (15)

229 (18)

178 (17)

77 (16)

142 (82.1) b 7 76 74 a 1 27 40

207 (90.9) b 23 131 108 b 11 61 b 66

178 (100) b 55 b 170 b 101 b 63 b 50 b 59

77 (100) b 44 b 64 b 68 b 39 b 29 4

2 (1.0) 0 0 0 0 0 0 0 0 2 0 0 0 0

21 (9.1) a 1 2 0 1 0 0 0 0 19 a 0 0 0 0

85 (43.7) b 48 a 0 0 8 2 28 b 41 b 5 25 b 0 0 0 0

77 (100) b 71 b 0 5 32 b 13 b 63 b 60 b 55 b 17 b 24 b 9 17 b 3

Skeletal anomalies N u m b e r o f fetuses e x a m i n e d 173 (litters) (15) Variations, type and incidence Total 99 (%)c (57.7) R i b s , cervical 1 Ribs, l u m b a r 63 Cervical v e r t e b r a e , b i f u r c a t e d or split 46 Lumbar vertebrae, supernumerary 0 Sternebrae, asymmetrical 14 Sternebrae, supernumerary 22 Malformations, type and incidence Total 1 (%)c (0.5) Ribs, fused 0 Ribs, wavy 0 Ribs, agenesis 0 Cervical v e r t e b r a e , fused 0 Cervical v e r t e b r a e , agenesis o f 0 T h o r a c i c v e r t e b r a e , fused 0 L u m b a r v e r t e b r a e , fused 0 P o s t - l u m b a r v e r t e b r a e , fused 0 S p i n a bifida o c c u l t a 1 A b n o r m a l fingers 0 Syndactyly 0 Oligodactyly 0 Polydactyly 0

a S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l b y t h e W i l c o x o n r a n k s u m test, P ~ 0.05. b S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l b y t h e W i l c o x o n r a n k s u m test, P ~ 0.01. CMean o f litters.

339

llb,m, ~ gbQ G

ko4 h

4

5

6

Fig. 2. Fused ribs. EGM, p.o. 250 mg/kg on days 7--14 of gestation. Alizarin red S strain. Fig. 3. Fused lumbar. EGM, p.o. 250 mg/kg on days 7--14 of gestation. Alizarin red S stain. Fig. 4. Right fore paw, control fetus. Aleian blue and Alizarin red S stain. Fig. 5. Syndactyly. Right fore paw, EGM, p.o. 250 mg/kg on days 7--14 of gestation. Alcian blue and Alizarin red S stain. Fig. 6. Oligodactyly, Right fore paw, EGM, p.o. 250 mg/kg on days 7--14 of gestation. Alcian blue and Alizarin red S stain.

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(b) Skeletal variations. In the experimental groups the incidence of skeletal variations such as cervical ribs, lumbar ribs, bifurcated or split cervical, supernumerary lumbar, asymmetry of sternebrae, or supernumerary sternebrae, showed a tendency to a dose-dependent increase. The skeletal variations observed at the lowest dose, 31.25 mg/kg, were difurcated and split cervicals. (c) Skeletal malformations. The incidence of skeletal malformation was significantly higher in 62.5, 125 and 250 mg]kg groups than in the control. All 77 fetuses examined from the 250 mg/kg group had skeletal malformations, including fusion and/or agenesis of ribs (Fig. 2), fusion and/or agenesis of vertebrae (Fig. 3), spina bifida occulta, syndactyly {Fig. 5), oligodactyly (Fig. 6) and polydactyly. In the 125 mg/kg group 91 of 178 fetuses had skeletal malformations including fused ribs, fusion and/or agenesis of vertebrae and spina bifida occulta. Twenty fetuses having skeletal malformations were found in 229 examined fetuses from dams given 62.5 mg]kg. Of these, 16 had spina bifida occulta, 2 spina bifida occulta and wavy ribs, 1 spina bifida occulta and fused cervical vertebrae and 1 had fused fibs. A low frequency of skeletal malformation was observed in the fetuses from the 31.25 mg/kg and control groups. Two of 174 fetuses from the 31.25 mg/kg and 1 of 173 fetuses from the control group had spina bifida occulta. (C) Ossification of fetuses (Table III) The ossification of fetuses was retarded in all treated groups. The average number at proximal and middle phalanges of fore and hind limbs showed a tendancy to a dose-dependent decrease throughout all observations. The T A B L E HI R E S U L T S O F O S S I F I C A T I O N O F MICE G I V E N E T H Y L E N E G L Y C O L M O N O M E T H Y L E T H E R BY G A S T R I C I N T U B A T I O N ON DAYS 7 - - 1 4 O F G E S T A T I O N

Dosage (mg/kg/day) 0 Number of fetuses 173 examined (litters) (15) Number of post14.1 ± 1.2 lumbar vertebraea Number of proximal and m i d d l e phalanges a F o r e limbs 7.4 + 0.5 Hind limbs 7.6 ± 1.2

31.25

62.5

125

250

174

229

178

77

(15) 14.3 ± 0.9

(18) 13.5 ± 1.5

(17) 12.4 + 2.1 b

(16) 8.7 -+ 2.7 c

5.5 -+ 1.1 c 4.8 ± 1.4 c

3.2 ± 2.2 c 2.2 ± 2.5 c

6.5 -+ 0.5 c 6.1 ± 0.9 c

5.8 • 1.0 c 5.0 -* 1.2 c

aMean o f litters + S . D . b S i g n i f i c a n t l y d i f f e r e n t f r o m c o n t r o l b y t h e S t u d e n t t-test, P < 0.05. CSignificantly d i f f e r e n t f r o m c o n t r o l b y t h e S t u d e n t t-test, P < 0.01.

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significant decrease in the number of post-lumbar centers was found at doses of 62.5 mg/kg and more. DISCUSSION

Many chemicals can induce embryotoxic effects during organogenesis. Our experiments show that EGM is such a compound when administered orally to pregnant mice at doses greater than 31.25 mg/kg body wt. The severity of response was dependent on the dose of EGM and in almost all parameters, the dose-response relationship was expressed by a sigmoid curve. The various malformations or anomalies which occurred were dose dependent as described in Table IV. Fetuses have been proved to be more susceptible to many chemicals than the dams. EGM apparently is such a substance, inducing skeletal anomalies of fetuses at 31.25 mg/kg, but leucopenia of the dams at 1000 mg/kg. In 1971 Stenger et al. reported the embryotoxic properties of EGE given orally to rat [10]. Although it is difficult to compare data from different species, it is likely that ethylene glycol monoalkyl ethers of lower molecular weight have teratogenic properties. Within the limits of the above data, it is concluded that EGM produces teratogenic effects in mice although it exerts only mild maternal toxicities. TABLE IV THE LEAST DOSE AT WHICH OBSERVED ITEMS SIGNIFICANTLY D I F F E R E N T BETWEEN TEST GROUPS CONTROL BY THE WILCOXON RANK SUM TEST OR THE STUDENT t-TEST Dose

Observed items

31.25 mg/kg

Skeletal variations as a whole. Number of proximal and middle phalanges. Cervical vertebrae, bifurcated and split.

62.5 mg/kg

Asymmetrical sternebrae. Skeletal malformations as a whole. Spina bifida occulta.

125 mg/kg

Fused and wavy ribs and their agenesis. Fused lumbar vertebrae. Fused thoracic vertebrae. Fetal body weight loss. Number of post-lumbar vertebrae. Cervical and lumbar ribs. Supernumerary lumbar vertebrae.

250 mg/kg

Fetal mortality. Gross anomalies as a whole. Fused post-lumbar vertebrae. Fused cervical vertebrae and their agenesis. Abnormal fingers. Live fetal number/litter.

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ACKNOWLEDGEMENT This w o r k was r e p o r t e d , in p a r t , a t t h e 5 1 s t A n n u a l M e e t i n g o f J a p a n A s s o c i a t i o n o f I n d u s t r i a l H e a l t h , J u n e 1st, 1 9 7 8 , M a t s u m o t o , J a p a n . T h e a u t h o r s are p a r t i c u l a r l y i n d e b t e d t o Dr. J. K u b o t a a n d Dr. H. M a t s u f u j i f o r h e l p in t h e p r e p a r a t i o n o f this p a p e r a n d m a n y u s e f u l suggestions f o r its improvement. REFERENCES 1 F.A. Patty, Industrial Hygiene and Toxicology, Interscience Publishers, New York, 1963, p. 1537. 2 E. Browning, Toxicology and Metabolism of Industrial Solvents, Elsevier, Amsterdam, 1965, p. 601. 3 M.R. Zvon, Am. Ind. Hyg. Assoc. J., 24 (1963) 36. 4 G. Ohi and D.H. Wegman, J. Occup. Med., 20 (1978) 675. 5 F.H. Wiley, W.C. Hueper, D.S. Bergen and F.R. Blood, J. Ind. Hyg. Toxicol., 20 (1938) 269. 6 H.W. Werner, C.Z. Nawrocki, J.L. Mitchell, J.W. Miller and W.F. yon Oettingen, J. Ind. Hyg. Toxicol., 25 (1943) 374. 7 H.W. Werner, J.L. Mitchell, J.W. Miller and W.F. yon Oettingen, J. Ind. Hyg. Toxicol., 25 (1943) 409. 8 K. Nagano; E. Nakayama, M. Koyano et al., Jpn. J. Ind. Health, 21 (1979) 29 (in Japanese). 9 B.N. Hemsworth and H. Jackson, Embryopathies induced by cytotoxic substances, in J.M. Robson (Ed.), Embryopathic Activity of Drugs, J. & A. Churchill LTD, Gloucester Place, 1965, p. 116. 10 E.G. yon Stenger, L. Aeppli, D. Miiller et al., Arzneim.-Forsch., 21 (1971) 880. 11 M. Inouye, Congenital Anomalies, 16 (1976) 171. 12 F. Wilcoxon, S. Katti and R.A. Wilcox, Critical Values and Probability Levels for the Wilcoxon Rank Sum Test and the Wilcoxon Signed Rank Test, American Cyanamid Company, Pearl River, 1963.

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