A study of the prenatal and postnatal toxicity of a sulfhydryl resin in rats

TOXICOLOGY

AND APPLIED

A Study

PHARMACOLOGY,

(1974)

27,621-628

of the Prenatal Sulfhydryl

and Postnatal Resin in Rats

Toxicity

of a

B. A. SCHWETZ,H. C. SPENCERAND P. J. GEHRING Chemical

Biology

Research,

The Dow

Received

May

II,

Chemical 1973;

Company,

accepted

JuIy

Midland,

Michigan

48640

31,1973

A study of the Prenatal and Postnatal Toxicity of a Sulfhydryl Resin in Rats. SCHWETZ, B. A., SPENCER, H. C. AND GEHRING, P. J. (1974). Toxicol. Appl. Pharmacol., 27, 621-628. This study evaluates the effects of a sulfhydryl resin on embryonal, fetal and neonatal development in rats, Doses of 150 or 1500 mg/kg/day were administered on days 3 through 15 of gestation in a prenatal toxicity study and on day 16 of gestation through day 14 of lactation in a perinatal toxicity study. In the prenatal study, fetuses were delivered by cesareansection on day 21 of gestation and were examined grossly, measured and weighed. Following routine preparations, the soft tissues and skeletons were examined. Evidence of retarded fetal development (delayed ossification of skull bones and sternebrae) was observed at the high dose level. Teratogenic effects were not observed at either dose level. Perinatal administration of the sulfhydryl resin had no effect on parturition or neonatal growth and survival. A finding at necropsy of the 3-week-old neonates of resin-treated dams, dilated urinary bladder, was not associated with any functional problems or microscopic morphologic abnormality of urinary tract structures. In both the prenatal and perinatal studies, 150 mg of sulfhydryl resin/kg/day had no effect on the developing embryo, fetus or neonate. Prenatal exposure to methylmercury causes brain damage to the conceptus (Lofroth, 1969) even among pregnant females showing little or no evidence of toxicity. A treatment which effectively lowered the plasma concentration of mercury might prevent such brain damage in the developing embryo and fetus. In animals, the neonate is also highly susceptible to the toxic effect of methylmercury. Spyker (1972) has recently reported on a postnatal study among methylmercury-treated mice. Toxicity, including neuropathologica1 changes, was observed in the offspring of methylmercury-treated females during later development, despite apparent normality at birth. Offspring of treated mothers fostered by control mothers had a better survival rate than offspring of treated mothers nursed by other treated mothers. Offspring of control mothers fostered by treated mothers showed signs of toxicity which were probably related to the exctietion of mercury in the milk. These results suggest that an effective drug would be beneficial in methylmercury poisoning if administered during lactation as well as during pregnancy. Ion-exchange resins have recently been suggested as a potential treatment for methylmercury poisoning in man (Clarkson, 1971). Recent studies by Norseth and 621 Copyright 0 1974 by Academic Press, Inc.

All rig& of reproduction Printed in Great Britain

in any form reserved.

622

SCHWETZ,

SPENCER

AND

GEHRING

Clarkson (1971) have demonstrated that the excretion of methylmercury takes place primarily via the feces and that methylmercury undergoes extensive enterohepatic circulation. These results suggest that if some agent, such as an ion-exchange resin, could be used to trap the secreted methylmercury within the intestinal lumen, the fecal excretion would be greatly enhanced. In subsequent studies, Clarkson er al. (1971) fed groups of mice a diet containing either methylmercury alone or methylmercury plus a sulfhydryl-containing ion-exchange resin (1 “/o by weight of the diet of the same resin used in the presently reported study). The mice receiving the resin in the diet excreted mercury approximately 2.5 times as rapidly as the mice receiving mercury without resin. Clarkson has also found the sulfhydryl resin to significantly reduce the plasma concentration of mercury in children exposed to methylmercury. The resin was administered orally at dose levels of approximately 35-150 mg/kg/day (personal communication). The purpose of this study was to determine the effect of the sulfhydryl resin on the developing embryo and fetus by administration of the resin to rats during pregnancy and to determine the effect of the resin on developing neonates by administration of the resin to lactating rats. METHODS

Test material. The test substance’ was a modified synthetic organic ion-exchange resin containing sulfhydryl (-CH,SH) groups attached to a macroporous styrene-divinylbenzene copolymer such as described by Kun, K. A., U.S. Patent 3,278,487 (1966). Animals. Pregnant adult, Sprague-Dawley (Spartan) female rats weighing approximately 250 g were used. The day following mating was considered day 0 of gestation. Animals were housed individually in wire-bottom cages and maintained on Purina Laboratory Chow and water ad libitum. Experimental design. In the prenatal toxicity study, groups of rats were treated with 150 or 1500 mg sulfhydryl resin/kg/day on days 3 through 15 of gestation. In the perinatal toxicity study, groups of rats were treated with 150 or 1500 mg sulfhydryl resin/ kg/day from day 16 of pregnancy through day 14 of lactation. In both studies, the resin was given as a water suspension by oral gavage. The dose of the suspension was 6 ml/kg. Control rats received 6 ml/kg of water orally. Dosages were adjusted daily for changes in body weight. Maternal observations. In the prenatal toxicity study, animals were observed daily throughout the gestation period for indications of toxicity from the test material. Maternal body weight was recorded on days 3, 9 and 16 of gestation. In addition, maternal body weight and the weight of the maternal liver and adrenals were recorded at the time of cesarean section on day 21 of gestation. The average daily food consumption was measured over the intervals of days 3-7, 8-11, 12-15 and 16-20 of gestation. In the perinatal toxicity study, animals and their litters were observed daily during treatment for evidence of toxicity from the test material. The maternal body weight was recorded on days 16 and 21 of gestation and on days 7,14 and 21 of lactation. Fetal examination. All pregnant females of the prenatal study were sacrificed by carbon dioxide on day 21 of gestation. After exteriorizing the uterine horns through a midline incision in the abdominal wall, the number and position of live, dead and 1 The test material (sulfhydryl resin 7864-43 and 46, ground) for thisstudywassupplied byHamish Small, Physical Research Laboratory, The Dow Chemical Company, Midland, Michigan 48640.

PERINATAL

TOXICITY

OF A SULFHYDRYL

623

RESIN

resorbed fetuses were noted. Corpora lutea were counted grossly. Umbilical cords were clamped and severed distally. The weight and sex of the fetuses were recorded and then they were examined for external anomalies. The crown-rump length of the fetuses was measured with a Vernier caliper. Each litter was divided into 2 sub-groups for preservation and subsequent examination. One subgroup, preserved in Bouin’s solution, was examined by the method of Wilson (1965) for evidence of soft tissue anomalies. The second subgroup, preserved in alcohol, was cleared and stained with Alizarin red-S (Dawson, 1926) for examination of the skeleton for evidence of abnormalities. Postnatal obseruation. Dams from the perinatal toxicity study and their pups were observed daily after parturition for changes in behaviour and appearance. The observations recorded include the date of parturition, number of live and dead newborn, number of live pups at days I, 7, 14 and 21, litter weights at days I, 7 and 14, individual weanling weights at day 21, sex of each weanling at day 2 I, and any abnormalities noted at 21 days of age. The neonate survival indices were calculated at birth and days I, 7, 14 and 21 of age. Statistical evaluation. Statistical evaluation of the frequency of anomalies and resorptions among the fetal populations and among litters as well as the postnatal indices were made by the Fisher Exact Probability test (Siegel, 1956). Analyses of maternal, fetal and neonatal body weights and body measurements and food consumption values were made by an analysis of variance. Group means were compared to controls using Dunnett’s test (Steel and Torrie, 1960). In all cases, the level of significance wasp < 0.05. RESULTS

Prenatal Toxicity Study. The maternal food consumption and body weight gain during gestation were not affected by the administration of 150 or 1500 mg sulfhydryl resin/kg/day on days 3 TABLE EFFECT

OF A SULFHYDRYL

RESIN

ON MATERNAL RATS DURING

I FOOD CONSUMPTION GESTATION

AND

BODY

WEIGHT

OF

Sulfhydryl resin” Statistic

Control (water)

Number of dams 21 Food consumption (g/rat/day, mean f SD) Days 3-7 22 * 2 8-l I 12-15 I 6-20

Body weight (g, mean + SD) Day 3 9 15 21

23 f 2 26 k 2 27 + 2 258 278 309 399

5 13 k 15 + 17 -t- 24

150 mg/kg/day

1500 mg/kg/day

16 21 23 26 28 253 276 307 393

* 1 f 2 f 2 + 2 f rt f *

16 13 13 30

21 22 23 25 28

+ + + +

2 3 3 2

257 281 310 397

k & * k

12 15 22 42

aAdministered orally on days 3 through 15 of gestation. No values were significantly different from control by an analysisof varianceand Dunnett’s test,p < 0.05. 21

624

SCHWETZ,

SPENCER

AND

GEHRING

TABLE 2 EFFECT

OF A SULFHYDRYL

RESIN

ON THE WEIGHT PREGNANT

OF THE RATS

Number of animals 21 Liver weight Absolute (g, mean k SD) 14.81 + 1.29 Relative 37.1 -c 2.4 (mg liver/g body wt, mean k SD) Adrenal weight (mg, mean * SD) a Administered b Significantly

orally greater

AND

ADRENAL

GLANDS

OF

Sulfhydryl resin” 150 mg/kg/day 1500 mg/kg/day

Control (water)”

Statistic

LIVER

16

21

15.59 If: 1.42 39.8 k 4.3b

15.37 * 2.11 38.9 f 4.4

67 k 9

68 f 8

61 f 10

on days 3 through 15 of gestation. than control by Dunnett’s test, p < 0.05.

through 15 of gestation (Table 1). No signs of maternal toxicity were observed among dams treated with either dose level of sulfhydryl resin during gestation or at the time of cesarean section and necropsy on day 21 of gestation. The absolute liver weight and adrenal weight were not affected by administration of either dose level of sulfhydryl resin (Table 2). The relative liver weight (mg liver/g TABLE 3 EFFECT

OF A SULFHYDRYL

RESIN

ON MEASURES

RELATED

TO PREGNANCY

Sulfhydryl resin” Control (water)

Statistic Number of litters Implantation sites,/litterb Corpora lutea/damb Living fetuses/litter Resorptions/implantation sites (%) Litters with resorptions (%I Litters totally resorbed ResorptionsJitters with resorptions Sex ratio, M : F Fetal body weight (g)” Fetal crown-rump length (mm) ’ Administered b Mean f SD.

orally

greater

than

3 through

SD. control

1500 mg/kg/day

21 11 f2 14i2 11+2 4 (9/235)

16 10 + 4 14+2 10+4 4 (7/165)

21 I1 &4 14*3 10f 5 I5 (36/238)d

29 (6/21)

31 (5/16)

52 (11/21)

o/21 1.5 (9/6)

O/16 1.4 (7/5)

l/21 3.3 (36/l 1)

103 : 123 5.74 + 0.27 43.5 f 0.9

on days

’ Mean of litter meansk d Significantly

150 mg/kg/day

78 180 5.74 f 0.27 43.6 + 0.8

15 of gestation.

by Fisher

Exact

Probability

test, p < 0.05.

90:112 5.71 _+0.50 43.5 f 1.5

625

PERINATALTOXICITYOFASULFHYDRYLRESIN TABLE

4

EFFECTOFASULFHYDRYLRESINONTHEINCIDENCEOFRATFETALANOMALIES

Sulfhydryl resin’ Control (water)”

Anomalies

(%)

Gross

Omphalocele Acaudia Runts (d-3 Soft

SD)

1 (2/226)

Pb L’ P L P L

2 (4/226) 14 (3/21)

P L P L

2 9 3 19

(2/l 12) (2/21) (4/l 12) (4/21)

8 28 3 19 2 9 0 0

(9/l 14) (6/21) (4/l 14) (4/21) (2/l 14) (2/21) (O/l 14)

9 (2/21) 0 (O/226)

0 (O/21)

150 mg/kg/day

(%I 1 (l/158) 6 U/16) 0 0 5 25

(O/158) (O/l 6) (S/l 58) (4/l 6)

1500 mg/kg/day

(%I 0 (O/202)

0 @/m 0.5 5 7 30

(I /202) (l/20) (1 4/202)d (6120)

tissue

Dilated renal pelvis Dilated ureters

1 (l/79)

6 (l/16) 1 (l/79) 6 (l/16)

0 (O/101) 0 wm 1 (l/101) 5 (l/20)

Skeletal

Delayed ossification of the skull Lumbar ribs or spurs Bipartite vertebral centra De!ayed ossification of sternebrae

4 (3/79) 12 (2/16) 1 (l/79)

6 U/16) 5 (4/79) 25 (4/16) 4 (3/79)d

17 42 2 5 3 16 5 21

W1) 6 U/16) _” Administered orally on days3 through 15 of gestation. b Population ‘A incidence(number affected fetuses/numberfetusesexamined). c Litter % incidence(number affected litters/number litters examined). d Significantlydifferent from control by the Fisher Exact Probability test, p < 0.05.

(17jloly (8/19)d (2/101) (l/19) (3/101) (3/19) (5jlOl)” (4/19)d

body weight) was slightly increased among rats receiving 150 mg sulfhydryl resin/kg/day but was not statistically significantly increased among rats receivir,g the higher dose level. Administration of either dose level of sulfhydryl resin had no effect on the number of corpora lutea per dam or the number of implantation sites or live fetuses per litter (Table 3). There was a statistically significant increase in the incidence of fetal resorptions at the high dose level. This effect was observed among the fetal population but not among litters. The average number of resorptions among litters with one or more resorptions was more than twice the number in control litters. Thus, litters were not randomly affected. Treatment had no effect on the sex ratio of the offspring or on the fetal body weight or crown-rump length (Table 3). There were no gross or soft tissue anomalies which occurred at an incidence which was significantly increased by treatment with the sulfhydryl resin. There was a statistically significant increase in the number of runts (animals with a body weight less than the control mean minus 3 SD) among the fetal population of dams receiving 1500 mg/kg/day, but this effect was not evenly distributed among litters (Table 4).

626

SCHWETZ,

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GEHRING

TABLE 5 EFFECT OF PERINATAL ADMINISTRATION OF A SULFHYDRYL RESIN TO PREGNANT AND LACTATING RATS

Sulfhydryl resin” Statistic Number of pregnant females Gestation survival indexb 24-Hour survival index” ‘I-Day survival indexd 1CDay survival indexe 21-Day survival indexf

Control (water) 18 99 99 98 98 98

150 mg/kg/dw 23 94 99 97 96 96

Neonatal body weight (g, mean of litter means + SD) Day 1 6.80 f 0.59 7.07 f 0.63 Day 7 14.16 k 1.55 14.10 * 2.49 Day 14 27.29 & 3.77 27.00 + 3.18 Day 21, male 43.05 rf: 7.54 43.22 f 6.56 Day 21, female 40.54 + 7.08 41.18 + 5.80 Sex Ratio on day 21, M : F 110:94 98 : 123 Litter incidence of neonates with dilated urinary bladder-g at necropsy (day 21)

0% (O/18)

25 % (5/20)

1500 w/kg/day 17 94 99 95 95 95 7.09 + 0.64 14.72 f 1.40 28.06 + 3.28 44.83 k 5.02 43.61 + 6.04 67 174 54 % (7/13)h

a Administered orally from day 16 of gestation through day 14 of lactation. b The c The d The e The J-The

‘A of newborn pups that were alive. % of liveborn pups that survived 24 hr. ‘A of liveborn pups that survived 7 days. % of liveborn pups that survived 14 days. ‘A of liveborn pups that survived 21 days. gLitters with 1 or more neonateswith a bladder more than twice the normal sizefor a full bladder. h Significantly higher than control value, Fisher Exact Probability test,p < 0.05.

Two skeletal anomalies-delayed ossification of the skull and delayed ossification of sternebrae-were found at a statistically significantly increased incidence among the fetal population and litters of dams treated with 1500 mg/kg/day of the sulfhydryl resin. No skeletal anomalies were found at a significantly increased incidence among the fetal population and litters of dams treated with 150 mg/kg/day of the resin.

Perinatal Toxicity Study In the perinatal toxicity study, administration of a sulfhydryl resin to pregnant and lactating rats had no effect on the survival indices at birth or on days 1, 7, 14 and 21 (Table 5). The neonatal body weight at days 1, 7, 14 and 21 after birth was unaffected by administration of the sulfhydryl resin to the lactating dams. There were no signs of toxicity in the dams or the neonates during the entire observation period. Dilation of the urinary bladder was noted at necropsy among litters of dams treated with either dose of resin. Among litters of dams treated with 1500 mg/kg/day, the incidence was significantly greater than among the control litters. No signs of toxicity were observed among the dams at necropsy.

PERINATAL

TOXICITY

OF A SULFHYDRYL

RESIN

627

DISCUSSION An ion-exchange resin which effectively reduces the blood concentration of mercury in man and animals exposed to methylmercury has been found to have no significant effect on the developing embryo, fetus and neonate when administered to pregnant and lactating rats. Administration during gestation and/or lactation caused no evidence of toxicity in maternal animals. In a separate study, groups of 5 male and female rats were given a single oral dose of 10 or 15 g/kg. There were no deaths or signs of toxicity that were related to treatment with the resin. These results indicate that single or repeated oral administration of high doses of this sulfhydryl resin to rats causes little or no untoward effects. Prenatal or perinatal administration of 150 mg/kg to rats had no effect on the developing embryo, fetus and neonate. At 1500 mg/kg/day during organogenesis, there was evidence of retarded fetal development--delayed ossification of the skull and sternebrae. In previous studies in this laboratory, retarded fetal development of this nature was of no consequence to neonatal growth, development and survival (Schwetz et al., 1971). At necropsy at the end of the perinatal study, a statistically significant number of 3-week-old offspring of resin-treated mothers were found with urinary bladders 2-3 times the size of a normally full bladder. There was no evidence of urinary obstruction in these animals; no other part of the urinary tract was involved; no other smooth muscle structures were affected elsewhere in the body; microscopic examination of hematoxylin and eosin-stained sections of bladder and urethra from fetuses and 21-dayold neonates revealed no morphological differences between treated and control groups. Thus, although some of the bladders were dilated, there was no morphological or functional evidence of a deleterious effect of treatment with the resin. In conclusion, this study provides evidence that administration of sulfhydryl resin to pregnant and lactating rats had no significant deleterious effects on the developing embryo, fetus or neonate. Treatment with 1500 mg/kg/day on days 3 through 15 of gestation was associated with retarded fetal development, but no terata were observed. This same dose administered from day 16 of gestation through day 14 of lactation had no effect on parturition or neonatal growth and survival. The one finding in the 3-weekold neonates at necropsy, dilated urinary bladders, was not associated with any functional problem or microscopic morphological abnormality of urinary tract structures. In both the prenatal and perinatal studies, 150 mg sulfhydryl resin/kg/day had no effect on the developing embryo, fetus or neonate. ACKNOWLEDGMENT The authors are grateful to Mrs. P. A. Keeler, Dr. R. J. Kociba, Mrs. R. W. Lisowe and Dr. L. W. Rampy for their assistancein all aspectsof this study. REFERENCES CLARKSON, T. W. (1971). Epidemiological and experimental aspects of lead and mercury

contamination of food. Food Cosmet. Toxicol. 9,229-243. CLARKSON, T. W., SMALL, H. AND NORSETH, T. (1971). The effect of a thiol containing resin

on the gastrointestinal absorption and fecal excretion of methylmercury compounds in experimental animals. Fed. Proc., Fed. Amer. Sot. Exp. Biol. 30,543 (Abstract).

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GEHRING

A. B. (1926). A note on the staining of the skeleton of cleared specimenswith Alizarin Red-S. Stain Technol. 1, 123. LOFROTH, G. (1969). A review of health hazards and side effects associated with emission of mercury compounds in natural systems. Ecological Res. Commun. Bull., No. 4, Swedish Natural ScienceResearch Council, Stockholm. NORSETH, T. AND CLARKSON, T. W. (1971). Intestinal transport of 203Hg-labeled methylmercury chloride. Role of biotransformation in rats. Arch. Environ. Health. 22, 568-577. SCHWETZ, B. A., SPARSCHU, G. L. AND GEHRING, P. J. (1971). The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) and estersof 2,4-D on rat embryonal, foetal and neonatal growth and development. Food Cosmet. Toxicol. 9, 801-817. SIEGEL, S. (1956). Nonparametric Statistics for the Behavioral Sciences, pp. 96104. McGrawHill, New York, New York. SPYKER, J. M. (1972). Subtleconsequences of methylmercury exposure. Teratology 5,267. STEEL, R. G. D. AND TORRIE, H. H. (1960). Principles andprocedures of Statistics, pp. 101-l 15, 194-205. McGraw-Hill, New York, New York. WILSON, J. G. (1965). Methods for administering agents and detecting malformations in experimental animals. In: Teratology Principles and Techniques (J. G. Wilson and T. Warkany, eds.), pp. 262-277. Univ. of Chicago Press,Chicago, Illinois. DAWSON,