Teratogenic effects of Silastic intrauterine devices in the rat with or without added medroxyprogesterone acetate*

Vol. 39, No.2, February 1983 Printed in U.8A.

FERTILITY AND STERILITY Copyright © 1983 The American Fertility Society

Teratogenic effects of Silastic intrauterine devices in the rat with or without added medroxyprogesterone acetate*

Susan M. Barlow, Ph.D.t Adrienne F. Knight Department of Pharmacology, Guy's Hospital Medical School, London, England

The teratogenicity of intrauterine devices (IUDs) made of silicone rubber (Silastic, Dow Corning Corporation, Midland, MI) with or without added medroxyprogesterone acetate (MPA) has been investigated in the rat. Small rod-shaped IUDs were inserted into the uterus, one between each embryo, on day 9 of pregnancy and left in place until the rats were killed just before term for examination of the fetuses. MPA exposure caused masculinization of the external genitalia of female fetuses and feminization of the external genitalia of male fetuses. There was no increase in other, nongenital malformations in MPA-exposed fetuses, compared with fetuses exposed to Silas tic alone, but both Silastic-exposed groups had significantly more malformations than untreated control rats. In a second experiment, a significant increase in malformations in fetuses exposed to Silastic alone, compared with untreated control. fetuses, was confirmed. The malformation rate in control rats that underwent sham operations was not significantly increased, compared with untreated control rats. Fertil Steril 39:224, 1983

Silicone rubber (Silastic, Dow Corning Corporation, Midland, MI) intrauterine devices (IUDs) and intravaginal rings (IVRs) incorporating steroids such as medroxyprogesterone acetate (MPA) have been used in human clinical trials to ascertain their suitability as contraceptives. I, 2 The failure rates ofIUDs and IVRs are not negligible; and, should the use of steroid-releasing devices become widespread, a substantial number of pregnancies would be carried to term with the device remaining in situ for part or the entire

Received January 20, 1982; revised and accepted September 8,1982. *Supported by grant 780-0350 from the Ford Foundation, New York, New York. tPresent address: Susan M. Barlow, Ph.D., Department of Environment, Room A347, Romney House, 43 Marsham Street, London SW1P 3PY, England.

224

Barlow and Knight Teratogenicity of IUDs in the rat

gestation. Under these circumstances, the embryo and fetus could be exposed to relatively high concentrations of chemicals released from the devices. To date, there is insufficient information on the outcome of such pregnancies in women for one to assess whether there might be any adverse effects on development. Few animal studies have been carried out on the potential teratogenicity of MPA,3 and none have investigated the effects of direct exposure of the embryo and fetus to MP A released from IUDs. The present experiments were carried out as part of a program to develop suitable methods for teratogenicity evaluation of medicated IUDs in the rat. 4 In the first experiment, the effects of MPA released from Silastic IUDs were investigated. However, it became apparent that Silastic itself might be teratogenic, and a second experiment was therefore carried out to investigate this possibility in more detail. Fertility and Sterility

r I MATERIALS AND METHODS

ANIMALS

Virgin female rats of the Wistar strain (purchased from Charles River, Manston, Kent, UK), 12 weeks old and weighing 200 to 250 gm on arrival, were used. For 3 weeks before mating and throughout the experiment they were kept in the animal house, under controlled lighting (12 hours light:12 hours dark) and maintained at 21 0 C to 240 C. Food and water were freely available. The females were housed in groups of three, and a proven male of the same strain was introduced into each cage. Vaginal smears were taken each morning and the day of finding spermatozoa in the smear was designated day 1 of pregnancy. Pregnant females were removed from the mating cages and rehoused in groups of three. They were weighed on days 1 and 21 of pregnancy. '

INSERTION OF IUDs

Silastic IUDs were made by adding two drops of a polymerizing catalyst, stannous octoate, to 10 ml of Silastic 382 Medical Grade Elastomer (Dow Coming Corporation). The mixture was extruded into plastic cannulas with an internal diameter of 1.5 mm and left to vulcanize overnight; it was then removed from the cannulas and cut into rods 3 mm in length. MPA IUDs were made as described above but with the addition of MPA (Sigma Chemical Company, Poole, Dorset, UK), 20% by weight, to the elastomer before vulcanization. Each 3-mm MPA IUD rod contained approximately 2 mg of steroid. On the morning of day 9 of pregnancy, rats were assigned randomly to treatment groups. Animals receiving IUDs were anesthetized with ether, and the IUDs were inserted into both uterine horns, one between each implantation site, with the use of the surgical technique described elsewhere. 4 To control for the stress of the operation, in the second experiment only, some animals underwent sham operations; incisions were made between each implantation site, but IUDs were not inserted. After recovery from the anesthetic, all animals were returned to their home cages and left undisturbed until killed on day 21 of pregnancy. One group of animals was left undisturbed throughout pregnancy to serve as untreated control animals. Vol. 39, No.2, February 1983

EXAMINATION OF THE FETUSES

On day 21 of pregnancy the rats were killed, and the uterus was exposed and opened. The number and position of live and full-term dead fetuses (no signs of maceration), late resorptions (maceration, death occurring at the fetal stage), and early resorptions (death occurring at the embryonic stage) were noted. In IUD-bearing animals the positions of the IUDs were noted; then they were removed and counted. The fetuses and placentas were weighed individually, and the fetuses were examined for any gross external abnormalities. The fetuses were alternately fixed in Bouin's fluid for examination of the soft tissues, viewed as I-mm slices under the dissecting microscope or in 70% alcohol for subsequent staining with alizarin red S for skeletal examination. Before further processing, fetuses placed in alcohol were sex-assigned by visual examination of the external anogenital distance. The anogenital distance was then measured with calipers, and the fetuses were opened so that we could check the true sex from the internal anatomy for comparison with the initial sex of assignment. Fetal malformations were classified as abnormalities or anomalies, the former being major malformations rarely seen in control fetuses, the latter being soft tissue or skeletal variants commonly present in a proportion of control fetuses. STATISTICAL ANALYSIS

All group means are given ± standard error of the mean (SEM). Differences between group means ± SEM were compared by Student's twotailed t-test. All other comparisons were examined by the two-tailed Fisher exact probability test. Fetal weight is expressed as the mean of the litter means for each treatment group. RESULTS EXPERIMENT 1

In the first experiment the teratogenicity of MPA IUDs was evaluated by comparison of the three groups of rats: (1) untreated control rats (n = 17); (2) rats carrying IUDs made of Silastic only (n = 16); and (3) rats carrying IUDs made of Silastic with added MPA (n = 16). There were no significant differences between groups in maternal body weight on day 1 of pregnancy. Maternal body weight on day 21 of pregnancy was Barlow and Knight Teratogenicity of IUDs in the rat

225

Table 1. Experiment l-Outcome of Pregnancy After Insertion of IUDs on Day 9 and Sacrifice on Day 21 Group

No. of

rats

No. of implantation sites

Fetuses Live

Resorptions Dead

Early

Late

Mean live litter size

Mean fetal weight gm

Untreated control Silastic IUD MPAIUD

17

243

232

o

9

2

13.6 ± 0.5

3.76 ± 0.06

16 16

224 226

192 205

o

1

23a 15

8a 6

12.0 ± 0.6 b 12.8 ± 0.6

3.64 ± 0.06 3.72 ± 0.08

ap < 0.001, compared with untreated control group. bp < 0.05, compared with untreated control group.

significantly reduced in Silastic IUD (371 ± 6 gm) and MPA IUD (374 ± 5 gm) groups, compared with untreated control rats (394 ± 6 gm) (P < 0.02). At sacrifice on day 21, all the inserted IUDs were recovered lying free, in the uterine lumen, between the amniotic membranes and the endometrial surface. The outcome of pregnancy is shown in Table 1. The incidence of early and late resorptions was increased in both IUD groups, but only significantly so in the Silastic IUD group, compared with untreated control rats. Similarly, the mean litter size was reduced in both IUD groups, significantly so in the Silastic IUD group. There was no effect on mean fetal weight. Placental weight was significantly reduced in the MPA IUD group to 422 ± 12 mg, compared with 503 ± 10 mg in the Silastic IUD group, or 514 ± 10 mg in untreated control rats (P < 0.00001). Anogenital distance was significantly shortened in male fetuses exposed to MPA and significantly lengthened in female fetuses exposed to MP A, compared with either the Silastic IUD group or the untreated control group (Table 2). Consequently, there were errors in sex assignment from external appearance in the MPA IUD group; 13 of 98 fetuses were found to have been incorrectly assigned when the internal reproductive organs were examined. No errors of assignment from external appearance were made with fetuses from the Silastic IUD group or the untreated control group. The appearance of the genital papilla was also abnormal in fetuses exposed to MPA. Fetuses from the untreated control group and the Silastic IUD group showed the normal circular groove at the tip of the papilla, which in male fetuses surrounds the urethral opening. In fetuses exposed to MPA, this groove was absent. In female fetuses of 21 days' gestation the genital papilla is normally hypospadiac, with an obvious cleft on its caudal surface, which does not close 226

Barlow and Knight Teratogenicity of IUDs in the rat

until 2 to 4 days after birth. This cleft was absent or indistinct in female fetuses exposed to MPA, compared with females in the Silastic IUD and untreated control groups. Male fetuses exposed to MPA had a distinct cleft on the caudal surface of the genital papilla, suggesting hypospadias, which is not normal in males at this stage of gestation. Clefts were not seen in males in the Silastic IUD and untreated control groups. There were no abnormalities in the structure or position of the internal reproductive organs when viewed under the dissecting microscope. The results of the examination of the fetuses for abnormalities and anomalies other than those of the genitalia described above are given in Table 3. The types of abnormality found are listed in Table 4. Types of anomaly found were edema, subcutaneous hemorrhage and a fluid-filled sac around the umbilicus (by gross external examination), tracheobronchomegaly and displacement of the heart to the left (by soft tissue examination), abnormally fused or split sternebrae, short or extra ribs, extra vertebrae, and split centra (by skeletal examination). The incidence of abnormalities was significantly increased in both Silastic IUD and MPA IUD groups, compared with Table 2. Experiment 1-Anogenital Distance in Male and Female Fetuses Anogenital distance Group Males

Females mm

Untreated control Silastic IUD MPAIUD

3.33 ± 0.03 (61)a 2.16 ± 0.03 (56) 3.28 ± 0.03 (50) 2.13 ± 0.03 (48) 2.76 ± 0.06 (5W 2.29 ± 0.03 (48)C

aFigures given in parentheses are number of fetuses by internal sex. bp < 0.0001, compared with Silastic IUD or untreated control groups. cp < 0.005, compared with Silastic IUD or untreated control groups.

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Table 3. Experiment 1-Examination of Fetuses for Abnormalities and Anomalies (Excluding Genital Abnormalities) Untreated control Gross external examination Fetuses examined Fetuses abnormal Fetuses anomalous Soft tissue examination Fetuses examined Fetuses abnormal Fetuses anomalous Skeletal examination Fetuses examined Fetuses abnormal Fetuses anomalous Total no. of abnormal fetusesd Percentage of abnormal fetuses

Silastic IUD

MPAIUD

232 1 0

192 9" 3

205

115 6 6

93 12 7

107 9 8

117 9 28

99

98

18c

17c

38

34

16

34

38 b

17.7

18.5

6.9

Q

16b

2

ap < 0.005, compared with untreated control group. bp < 0.0005, compared with untreated control group. cp < 0.025, compared with untreated control group. dFetuses with more than one abnormality only counted once.

untreated control rats. Five fetuses in theSilastic IUD group and four fetuses in the MPA IUD group had multiple abnormalities, but none did in the untreated control group. The differences in abnormality rates between Silastic IUD and MPA IUD groups were not significant.

increased in the Silastic IUD group, compared with untreated control rats, but there were no significant differences in mean live litter size (after exclusion of unilateral pregnancies) or mean fetal weight. The results of the examination of the fetuses for abnormalities and anomalies are given in Table 6. The types of abnormality found are listed in Table 7. Types of anomaly found were subcutaneous hemorrhage (by gross external examination), tracheobronchomegaly and displacement of the heart to the left (by soft tissue examination), abnorm,al fusion of sternebrae, short or extra ribs, and split centra (by skeletal examination). The overall incidence of abnormal fetuses was significantly increased in the Silastic IUD group, compared with untreated control rats. The group that underwent sham operations did not differ significantly from untreated control rats. One fetus in the Silastic IUD group had multiple abnormalities, but none of the rats that underwent sham operations and none of the untreated control rats had multiple abnormalities. DISCUSSION

The results of the first experiment show that prenatal exposure to MPA released from IUDs affects development of the external genitalia,. causing masculinization of female fetuses and Table 4. Experiment 1-Types of Abnormality (Excluding Genital Abnormalities)

EXPERIMENT 2

The results of the first experiment indicated that Silastic itself might be teratogenic. A second experiment was therefore carried out to inves~ tigate this further by comparison of three groups of rats: (1) untreated control rats (n = 10); (2) control rats that underwent a sham operation (n = to); and (3) rats carrying IUDs made of Silastic only (n = 11). There were no significant difrerences between groups in maternal body weight on day 1 of pregnancy. Maternal body weight on day 21 of pregnancy was significantly reduced in the Silastic IUD group (387 ± 7 gm), compared with untreated control rats (415 ± 8 gm) (P < 0.02). Body weight in· the group that underwent sham operations was intermediate (396 ± 8 gm) and did not differ significantly from Silastic IUD or untreated control groups. The outcome of pregnancy is shown in Table 5. The incidence of late resorptions was significantly Vol. 39, No.2, February 1983

Exencephaly and open eye Encephalocele Deformed skull and brain Hydrocephaly Microphthalmia Folded retina Cleft palate ± cleft lip Exomphalos Diaphragmatic hernia Hypoplastic kidney Hydronephrosis Partially descended testis Club foot Absent footpad Scoliosis Kinked tail Kinked ribs

Untreated control

Silastic IUD"

MPAIUD b

0

2

0

0 0

0 2

1 3

0 0 0 0

0 1 0 4

1 1 1 4

0 5 0 0 1

1 1 1 3 4

3 3 1 0 3

0 1 0 0 9

1 1 1 1 18

6 0 0 1 17

QFive fetuses had multiple abnormalities. bFour fetuses had multiple abnormalities. Barlow and Knight Teratogenicity of IUDs in the rat

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Table 5. Experiment 2-Outcome of Pregnancy After Insertion of IUDs on Day 9 and Sacrifice on Day 21 Group

No. of rats

No. of implantation sites

Fetuses Live

Resorptions Dead

Early

Mean live litter size

Late

Mean fetal weight gm

Untreated control Sham operation Silastic IUD

10

153

146

0

6

1

14.6 ± 0.7

3.79 ± 0.07

10

141

131

0

S

2

13.1 ± 0.7

3.63 ± O.OS

11

144

126

0

10

sa

11.5 ± LIb

3.64 ± 0.11

ap < 0.05, compared with untreated control group. btJ'wo pregnancies unilateral; mean live litter size excluding these two 12.3 ± 1.1 (not significantly different from untreated control group).

feminization of male fetuses. Many experimental workers have confirmed the virilizing effects on the female fetus of certain synthetic progestogens that exhibit androgenicity in other test systems, but the explanation for the virilizing activity of a nonandrogenic progestogen such as MP A 5-8 is less clear. The seemingly paradoxical capacity to cause both masculinization of females and feminization of males has been reported previously with subcutaneous administration of MPA in the rat8. 9 and the rabbit. 9 Other agents also cause these paradoxical effects, including ethisterone in the rabbit,10 cyproterone acetate in the guinea pig,9 and aminoglutethimide and analogs of dehydroepiandrosterone in the rat. 11 There is limited evidence that MPA may act in a similar way to the analogs of dehydroepiandrosterone, which inhibit 3~-hydroxysteroid dehydrogenase. 12 In man, a rare form of congenital adrenal hyperplasia due to a genetic deficiency in the 3~-enzyme results in short anogenital distance and severe hypospadias in male infants and virilization with clitoral hypertrophy in female infants.12 Prenatal exposure to MP A, used in the treatment of threatened abortion, has also resulted in a number of cases of feminization of male infants (hypospadias) and virilization of female infants (clitoral hypertrophy with or without labioscrotal fusion).12. 13 The question remains' whether the amount of MP A released from IUDs and IVRs might be sufficient to affect genital development in the human fetus in the event of accidental pregnancy with the device remaining in situ for some time between 6 and 24 weeks' gestation, when differentiation of the genitalia is taking place. Release rates for MPA from Silastic both in vitro and in vivo are initially high but level off after the first few days.14-16 In rats, Silastic rods of 5% MPA containing approximately 470 flg MPA, inserted 228

Barlow and Knight Teratogenicity of IUDs in the rat

into the uterus, released around'28 flg/day in the first week and 21 J..Lg/day in the second week. 16 In monkeys and baboons, Silastic rods with 10% MPA (1.5 mg) released 24 to 43 flg/day after the initial equilibration period. 16 In the present study, release rates were not determined, but it seems likely from the above studies that release rates of 50 to 80 flg/day might be achieved with the 20% MPA Silastic rods containing a total of 2 mg MPA used in the present experiments. Silastic IUDs and IVRs containing 50 to 400 mg MP A have been used successfully to prevent ovulation in human trials. 1. 2. 17 In women, release rates of 0.9 to 1.6 mg/day from IVRs containing a total of 100 or 200 mg MPA and 0.5 to 1.2 mg/day from IVRs containing 50 to 400 mg MPA have been recorded. 2, 17 The metabolic clearance rate for MPA is similar in rats and human beings. 18 In Table 6. Examination of Fetuses for Abnormalities and Anomalies (Experiment 2) Untreated control Gross external examination Fetuses examined Fetuses abnormal Fetuses anomalous Soft tissue examination Fetuses examined Fetuses abnormal Fetuses anomalous Skeletal examination Fetuses examined Fetuses abnormal Fetuses anomalous Total no. of abnormal fetuses Percentage of abnormal fetuses

Sham operation

Silastic IUD

146 1 2

131 0 0

126 5 2

73 0 1

64 0 5

65 3 2

73 1 14

67 4 19

61 4 24

2

4

11a

1.4

3.1

S.7

ap < 0.01, compared with untreated control group (one fetus with gross external and soft tissue abnormalities counted only once).

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Table 7. Types of Abnormality (Experiment 2)

Deformed skull and brain Hydrocephaly Cleft palate Exomphalos Hypoplastic kidney Hydronephrosis Polydactyly Kinked ribs

Untreated control

Sham operation

Silastic

0

0

3

0 0 0 0 0 1 1

0 0 0 0 0 0 4

1 1 2 1 1 0 4

luna

aOne fetus had multiple abnormalities.

the present experiments, mean litter size and therefore number of IUDs inserted averaged 12 per rat, and so it may be estimated that a total of approximately 0.5 to 1.0 mg MPA per day was released into the rat uterus, which is similar to the release rate in women. There are, of course, considerable differences in uterine volume and in embryonic and fetal weight between rats and women; so it is likely that the MPA exposure per gram of fetal tissue would be less in human pregnancy than was achieved in the present experiments. The genital abnormalities discussed above were not seen in fetuses exposed to Silastic alone. However, the overall rate for other malformations seen in fetuses exposed to Silastic containing MPA was similar to the rate seen in fetuses' exposed to Silastic alone, and both were increased, compared with the untreated control group. The range and types of malformation seen in the two Silastic IUD groups were also similar. This suggests that Silastic itself may be teratogenic, but MPA is not a general teratogen in the rat, confirming the observations of Andrew and Staples, 3 who administered MPA subcutaneously in doses of 30 to 3000 mg/kg/day on days 8 to 16 of pregnancy and found no significant effects on survival, weight, or development of the fetuses. The possibility that Silastic might be teratogenic was further investigated in the second experiment, which included control animals that underwent sham operations. As in the first experiment, there were significant increases in resorption and malformation rates in rats carrying Silastic IUDs, compared with untreated control rats; and the malformation rate in the Silastic IUD group was nearly three times higher than in the control group that underwent sham operations. While sham operations will control for some of the stress and trauma of the operation, they do not control for possible physical effects of VoL 39, No.2, February 1983

the presence of the devices in the uterus. However, in a previous study,4 we found that inert stainless steel devices of dimensions (2 mm x 1 mm) similar to those of the Silastic IUDs caused a significant increase in resorption but did not increase malformation rates when compared with the rates of animals that underwent sham operations. The occurrence of an increase in malformations in fetuses exposed to Silastic in the absence of any effect on fetal weight in both experiments also suggests a direct teratogenic effect, rather than abnormal development secondary to maternal toxicity. Silastic 382 Elastomer, used in the present experiments, is made from polydimethylsiloxane polymer and silica filler as a base: to which is added the vulcanizing catalyst, stannous octoate. Polydimethylsiloxanes administered subcutaneously or orally were found to have no fetotoxic or teratogenic effects in one study on rats and rabbits, although there was some evidence of increased intrauterine mortality in rats. 19 Alcohol is known to be released when cross-linking of the polymer occurs and may also be released after setting, with as much as 1% by weight being lost as alcohol. 20 The alcohol released from Silastic 382 is n-propanol,21 but the teratogenicity of this alcohol is unknown. The catalyst, stannous octoate, would almost certainly be leached out of the IUDs in the uterus, because biologic fluids extract organotin stabilizers from plastic materials,22 and preliminary migration studies indicate about 6 ,...,g tinlgm Silastic diffuses out in vitro.21 A number of organotin stabilizers are toxic to chick embryo cells in culture23 and to neonatal rat kidney cells in culture,24 causing necrosis in both cases. Two other organotin stabilizers, given orally by gavage to rats, have been shown to reduce fetal weight significantly and increase intrauterine mortality in a dose-related manner.25 However, the reproductive effects of stannous octoate, if any, are not known. Further studies are therefore needed to identify which component(s) of Silastic may be teratogenic. REFERENCES 1. Stryker JC: Recent clinical results with medroxyproges-

terone acetate (MPA) IUDs. In Intrauterine Devices, Edited by RG Wheeler, GW Duncan, JJ SpeideL London, Academic Press, 1974, p 211 2. Thiery M, Vandekerckhove D, Dhont M, Vermeulen A, Decoster JM: The medroxyprogesterone acetate intravaginal Silastic ring as a contraceptive device. Contraception 13:605, 1976 Barlow and Knight Teratogenicity of IUDs in the rat

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3. Andrew FD, Staples RE: Prenatal toxicity ofmedroxyprogesterone acetate in rabbits, rats and mice. Teratology 15:25,1977 4. Barlow SM, Knight AF, House I: Intrauterine exposure to copper IUDs and prenatal development in the rat. J Reprod Fertil 62:123, 1981 5. Foote WD, Foote WC, Foote LH: Influence of certain natural and synthetic steroids on genital development in guinea pigs. Fertil Steril 19:606, 1968 6. Cupceancu B, Neumann F: Sensitivity differences of different genital tract structures of female rat fetuses due to the effect of medroxyprogesterone acetate or norethisterone acetate. Endokrinologie 54:66, 1969 7. Kawashima K, Nakaura S, Nagao S, Tanaka S, Kuwamura T, Omori Y: Virilising activities of various steroids in female rat fetuses. Endocrinol Jpn 24:77, 1977 8. Revesz C, Chappell CI, Gaudry R: Masculinisation of female fetuses in the rat by progestational compounds. Endocrinology 66:140, 1960 9. Neumann F, GrafKJ, Elger W: Hormone-induced disturbances in sexual differentiation. Adv Biosci 13:71, 1974 10. Courrier R, Jost A: Intersexualite filetale provoquee par la pregneninolone au cours de la grossesse. CR Soc BioI (Paris) 136:395, 1942 11. Goldman AS: Abnormal organogenesis in the reproductive system. In Handbook of Teratology, Vol 2, Edited by JG Wilson, FC Fraser. London, Plenum Press, 1977, p 391 12. Goldman AS, Bongiovanni AM: Induced genital anomalies. Ann NY Acad Sci 142:755, 1967 13. Schardein JL: Congenital abnormalities and hormones during pregnancy: a clinical review. Teratology 22:251, 1980 14. Roseman TJ, Higuchi WI: Release of medroxyprogester-. one acetate from a silicone polymer. J Pharm Sci 59:353, 1970

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15. Pharriss BB, Hendrix JW: Absorption of steroids through Silastic in animals and humans: results of experiments in animals. Adv Planned Parenthood 5:146,1969 16. Doyle LL, Stryker JC, Clewe TH, Harrison RA: Selection of steroids for incorporation into Silastic intrauterine devices. J Steroid Biochem 6:885, 1975 17. Mishell DR: Absorption of steroids through Silastic in animals and humans: results of studies in humans. Adv Planned Parenthood 5:151, 1969 18. Bardin CW, Musto NA, Feil PD, Bullock LP: The in vivo metabolism of progestins. VI. Species differences in the metabolic clearance rate of medroxyprogesterone acetate and 6a-methylprogesterone. In Animal Models for Research on Contraception and Fertility, Edited by NJ Alexander. Hagerstown, Harper & Row, 1979, p 165 19. Kennedy GL, Keplinger ML, Calandra JC: Reproductive, teratologic and mutagenic studies with some polydimethylsiloxanes. J Toxicol Environ Health 1:909, 1976 20. Brown D: Factors affecting the dimensional stability of elastic impression materials. J Dent 1:265, 1973 21. Sarginson NJ: Unpublished data 22. Piver WT: Organotin compounds: industrial applications and biological investigations. Environ Health Perspect 4:61,1973 23. Guess WL, Haberman S: Toxicity profiles of vinyl and polyolefinic plastics and their additives. J Biomed Mater Res 2:313, 1968 24. Grasso P, Gaydon J, Hendy RJ: The safety testing of medical plastics. II. An assessment oflysosomal changes as an index of toxicity in cell cultures. Food Cosmet Toxicol 11:255, 1973 25. Nikonorow M, Mazur H, Piekacz H: Effect of orally administered plasticizers and polyvinyl chloride stabilizers in the rat. Toxicol Appl Pharmacol 26:253, 1973

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