TOXICOLOGY

AND

APPLIED

PHARMACOLOGY

33,21-26

(1975)

Toxicity

of Sodium Pentafluorostannite, Anticariogenic Agent. I. Comparison of the Acute Toxicity Pentafluorostannite, Sodium Fluoride, and in Mice and/or Rats’>2

A New of Sodium Stannous Chloride

DAVID L. CONINE,~ MOONAHM YuM,~ ROBERT C. MARTZ, GEORGE K. JOSEPH C. MUHLER,~ AND ROBERT B. FORNEY Department

of Toxicology,

Receiaed

Indiana Unicersity School Indiana 46202

September

17,1974;

accepted

qf Medicine,

January

STOOKEY,'

Indianapolis,

15, 1975

Toxicity of Sodium Pentafluorostannite,A New Anticariogenic Agent. I. Comparison of the Acute Toxicity of Sodium Pentafluorostannite, Sodium Fluoride, and StannousChloride in Mice and/or Rats. CONINE. D. L., YUM, M., MARTZ, R. C., STOOKEY, G. K., MUHLER, J. C. AND FORNEY, R. B. (1975). Toxicol. Appl. Pharmacol. 33, 21-26. The toxicity of sodiumpentafluorostannite(NaSn,F,) in mice and rats hasbeeninvestigated. LD50 valuesin mice were 18.9, 80.9, and 592.9mg/kg in fed mice dosediv, ip, and po, respectively.Signsof toxicity in miceincludedataxia, muscle weakness,central nervous system depression,and occasionally tremors. LDSO values in female rats were 12.9, 65.0. and 218.7mg/kg in fed rats dosediv and ip and in fastedrats dosedpo, respectively.LDSO valuesin malerats were 12.9,75.4,573.1,and 223.1mg/kg in fed rats dosed iv, ip, and po and in fastedrats dosedpo, respectively.Signs,accompanying toxic dosesof NaSn,F,, sodium fluoride (NaF), or stannouschloride (SnCl,.2H,O) in the rat weresimilarand includedataxia, muscleweakness, and CNS depression.Sodium pentafluorostannitewas found to be more toxic than either NaF, basedon F, or SnCl,.2H,O, basedon Sn. Rats given either Sn compound developedpathologic changesin the kidneys grossly visible 4 days following administration. The data suggestthat both F and Sn play a role in the acutetoxicity of NaSnZFsin rats. The anticariogenic properties of the fluoride ion (F) are well established. Certain tinfluoride compounds, such as stannous fluoride, have proven to be especially useful in the prevention of caries.The preparation and properties of a new tin-fluoride compound, sodium pentafluorostannite (NaSn,F,), were reported by Muhler et al. (1967). It also was found to possessanticariogenic properties (Stookey et al., 1969). 1Supportedin part by PHSGM 1089. * Presented in part at the EleventhAnnual Meetingof the Societyof Toxicologyin Williamsburg, Virginia, March3-9,1972;at the TwelfthAnnual Meetingof theSocietyof Toxicologyin New York, March 18-22,1973; andat the Fall Meetingsof the AmericanSocietyfor Pharmacology andExperimentalTherapeutics, EastLansing,Michigan,August 19-23,1973. 3 Presentaddress:Abbott Laboratories,North Chicago,Illinois 60064. ’ Departmentof Pathology,IndianaUniversitySchoolof Medicine,Indianapolis,Indiana, ’ Oral HealthResearch Institute, Indianapolis,Indiana. 6 PreventiveDentistryResearch Institute,Fort Wayne,Indiana. Copyright 0 1975 by Academic Press. Inc. 21 All rights of reproduction Printed in Great Britain

in any form reserved.

22

CONINE

ET AL.

Muhler et al. (1967) reported the acute toxicity of NaSnzFS in mice. Their data suggested that based on F, NaSnzF, was less toxic than sodium fluoride (NaF). The present studies were performed to investigate and compare the acute toxicity of NaSn,F,, NaF, and stannous chloride (SnCI,*2H,O) in mice and/or rats. METHODS

Formulation and Dosing Solutions of NaSn,F5,7 NaF,* or SnCl,.2H,0,g in redistilled water were prepared just prior to use. The concentration of these solutions was adjusted to deliver volumes of 0.2-0.5 ml/20 g body wt in mice and 0.5-1.5 ml/100 g body wt in rats. Oral doses were administered by gavage. Statistics Least squares regression techniques were used to provide an initial estimate of the log-dose mortality relationship for subsequent evaluation by the methods of Litchfield and Wilcoxon (1949). Comparisons between NaSnzF, and NaF were made on F content while comparisons between NaSn,F, and SnCl,*2H,O were made on tin (Sn) content. Animals Male, albino, Swiss micelo weighing between 17 and 24 g were treated with NaSn,F, as indicated in Table 1. The mice were housed in the toxicology laboratory area and maintained on Purina laboratory mouse chow and tap water ad libitum throughout the ICday experimental period. Male and female, albino, Wistar ratsll weighing between 91 and 152 g were treated with NaSn,F,, NaF, or SnCI,*2H,O as indicated in Table 1. The rats were housed in pairs, according to sex, in the animal care facility and maintained on Purina laboratory rat chow and tap water ad lib. throughout the 1Cday observation period. Those animals used for determination of the LD50 in the fasted state did not receive food for 16-18 hr prior to drug administration. RESULTS

Toxicity of Sodium PentaJIuorostannite in Mice The LD50 values for NaSn,F, in mice are shown in Table 1. Almost all deaths in animals treated iv or po occurred within the first 24 hr. Most of the deaths in mice injected ip occurred within the first 72 hr. The data suggests that the route of administration affects the toxicity of NaSn,F, in mice. Signs of toxicity were primarily ataxia and depression which appeared soon after treatment. Fore- and hind-leg weakness were evident in those animals that died after being treated orally. More pronounced signs of toxicity were often observed in mice treated ip which died 48 hr or more posttreatment. These included loss of righting reflex, cyanosis, and tremors. 7 Ozark-Mahoning Company, Tulsa, Oklahoma. * Matheson, Coleman, and Bell Manufacturing Chemists, Norwood, g J. T. Baker Chemical Company, Phillipsburg, New Jersey. lo Cox Laboratory Supply Company, Indianapolis, Indiana. I1 Harlan Industries Incorporated, Cumberland, Indiana.

Ohio.

400.0 520.0 676.0 878.8

65.0 71.6 78.6 86.5 96.2

15.0 18.0 21.6 25.9

Dose (mg/kg)

l/10 3/10 7/10 9/10

ND“

7/10 7/10

2/10 ;\;l

lo/10

l/10 $;I

D/T0

LDSO b-@/kg)

mice

592.9 (486.0-723.3)

80.9~ (74.2-88.2)

18.9 (16.7-21.4)h

Male

(1 Mortality fraction (dead/total). b95% Confidence limits. ‘48-Hour LD50 data given. p No data.

Route

NaSn2Fs

7/10 s/10

170.4 210.0 233. I 258.7 287.2

ND

lO/lO

3/10 ;\I;

5110

31;;

D/T

57.0 71.2 89.1 111.2

16.2 14.8

7.6 8.3 12.2 13.4

Dose Om/W

NaSnzFs

65.0 (54.6-77.4)

(11.??4.8)

LD50 (mdkg)

rats

RATS

430.0 526.0 560.0 600.0 631.2

:::i

149.1 170.0 193.8 220.9 251.8

38.5 47.0 57.3 70.0 85.3

Ii::

1;:: 12.2 13.4

1

;‘12 5110 s/10

016

6/16 8/16 9110 IO/10

(530.6-618.9)

573.1

(20%%47.6)

(60.8-93.5)

$l; 6j10 6/10

$

75.4

12.9 (11.6-14.3)

LD50 @m/kg)

rats

l/10

8/10

o/10 3/10 3/10 ~~too

Male

310.0 430.0 580.0

230.0

l%l 150.0 175.0 200.0

72.0 56.0 90.0

45.0

35.0 50.0 55.0 60.0 70.0

Dose b-a/kg)

NaF

OR

SnClz.2HZ0

l/6 4/6 3/6

O/6

O/IO O/9 l/IO 3110 6/10

7/10 2/10 9/10 lO/lO

zjts IO/IO

o/10 l/l0

471.7 (336.9-660.4)

202.1 (168.4-242.5)

(44.3-61.7) 52.3

53.3 (47.6-59.7)

Male rats - -__-..~.LD50 D/T (m/W

TREATED WITH NaSn2Fs.NaF,

NaSnzFs ___~. Dose (mg/kd D/T

MICEAND/OR

Female

%-HOUR LDSO DATAIN

TABLE

$;WO:l 6000.0

1500.0

2400:0 4800.0

600.0 f3&;.;

400.0 500.0

6755.00 200.0 1oo:o

28.0 24.0 32.0 64.0

14.0 16.0

rats

2274.6 (1613.2-3207.2)

258.4 (179.4-372.1)

29.3 (24.2-35.3)

LDSO @%/kg)

Male

o/10 3190.1 I / 10 (2044.9-4976.6) 5!10 9/10

6/10 lO/lO

O/l0 ;/to”

lijl5 9/10

:f:x $I;

8/10 2/18 8/10 lo/IO

O/IO O/18

D/T

SnC12.2H20 Dose (mg/kg)

.-

24

CONINE

ET AL.

Toxicity qf Sodium Pentajluorostannite in Rats The LD50 values for NaSn,FS in rats are shown in Table 1. Almost all of the deaths occurred within the first 24 hr. Delayed toxicity was observed in male rats dosed ip. The ICday LD50 was 35.5 mg/kg compared with the 24-hr LD50 of 75.4 mg/kg. As observed in the mice, the route of administration of NaSn,F, to rats also affected its toxicity. The signs accompanying toxic doses of NaSn,FS were similar for all routes of administration. Depression and ataxia became evident soon after treatment. Animals near death displayed cyanosis, progressive muscle weakness, and did not respond to external stimuli. A yellow-brown colored, mucoid diarrhea was observed when NaSn,F, was given po to fasted animals. Animals dosed ip which died several days following treatment showed a significant weight loss and death occurred during severe CNS depression. Toxicity of Sodium Fluoride in Rats The LD50 values of NaF in male rats are shown in Table 1. Most of the deaths occurred within the first 24 hr. No difference in toxicity was seen between the iv or ip administration of NaF; however, between the other routes differences were observed. Of particular interest may be the difference in the toxicity of NaF to fasted and fed rats. Rats given NaF exhibited similar signs of toxicity regardless of the route of administration. Initially, ataxia and depression were evident. The signs progressed to muscle weakness, loss of righting reflex, tremors, cyanosis, and slow deep breathing as the animal approached death. As in the rats dosed orally with NaSn,F,, those dosed with NaF had a yellow-brown colored, mucoid diarrhea after treatment. Toxicity of Stannous Chloride in Rats The LD50 values of SnCl, . 2Hz0 in male rats are shown in Table 1. Delayed toxicity was seen in rats treated iv or ip. The 14-day LD50 of SnCI,*2H,O when administered iv or ip was 15.8 or 83.6 mg/kg, respectively. The route of administration of SnCl, .2H,O greatly affects its toxicity. Signs of toxicity observed in rats administered SnC1,*2H,O were similar to those already described in rats administered NaSn,F, or NaF. Ataxia, muscle weakness, and depression appeared soon after treatment. Animals dying 3 or more days after dosage often displayed tremors, weight loss, loss of righting reflex, and pronounced muscle weakness prior to death. Terminal convulsions were observed in a group injected with 64 mg/kg SnCl,.2H,O iv. Comparison of the Toxicity of Sodium Pentafluorostannite, Stannous Chloride, and Sodium Fluoride The mortality curves for male rats treated with NaF or SnCl, * 2H,O were parallel to those of male rats treated with NaSn,FS; therefore, estimations of the relative potency of NaSn,F5 compared with SnCl, . 2H20 or NaF based on Sn or F content, respectively, were made. Table 2 shows the results of these calculations. Sodium pentafluorostannite tended to be more toxic than either SnCl,*2H,O or NaF. When compared with NaF, this difference was most dramatic when the compounds were administered iv. Tin-Induced Renal Pathologic Changes Concomitant to these toxicity studies, it was observed that NaSn,F, or SnC1,*2H,O administered iv, ip, or orally to rats produced pathologic changes in the kidney which

ACUTE

TOXICITY

OF

25

NaSn,F,

appeared to be related to the Sn in each compound. The morphologic changes, grossly visible in animals sacrificed on the fourth day after drug administration, were identical. The kidneys were often swollen and the outer surface was mottled, resembling that of a “speckled” egg. These discolorations extended into the cortex. The medullary region of these kidneys was consistently hyperemic. Microscopically, tubular necrosis and tubular regeneration were observed. These changes have been investigated by M. Yum et al. (unpublished observations). The alterations in renal function which parallel these changes have been investigated by D. L. Conine et al. (unpublished observations). TABLE COMPARISON

Compound

OF THE TOXICITY

OF

Route

NaSn2FS NaF NaSn2FS NaF NaSn,F, NaF NaSnlFS NaF NaSn,F, SnC12.2H20 NaSn,F, SnCI, . 2H20 NaSnzF, SnC12. 2H20

iv

PO fasted

NaSn2FS SnC12.2H0

&

ip

faf:d E iv ip

2

NaSn,F,, NaF,

AND SnCI,.?H,O

24-hr LD50 (w/kg F)

24-hr LDSO G-w!W

12.9 53.3 75.4 52.3 223.1 202.1 573.1 471.7 12.9 29.3 75.4 258.4 223.1 2274.6 573.1 3190.1

3.4 24.1 20.2 23.7 59.6 91.4 153.2 213.4 -

-

IN MALE

24-hr LD50 (m/kg W

RATS

Difference in potency” Significant

-

Not significant Significant Not significant

8.6 15.5 50.4 135.9 148.8 1196.5 382.8 1678.1

Significant Significant Significant Significant

fl Significant = p < 0.05; not significant = p > 0.05.

DISCUSSION The signs of toxicity in mice or rats given NaSn,F, were similar and included mainly ataxia, muscle weakness, and CNS depression. Delayed deaths were usually accompanied by severe muscle weakness, loss of weight, and sometimes tremors. These signs are similar to those of F toxicity observed in either mice or rats given NaF (Muehlberger. 1930; Leone et al., 1956; Taylor et al., 1961; Lu et al., 1965). They are also similar to those observed following the administration of salts of inorganic Sn (Stoner et al., 1955). The mucoid diarrhea observed in rats dosed orally with NaSn,F, appears to be related to a local effect on F on the gastrointestinal tract, since it was also observed in animals given NaF. The 24-hr mortality curves of rats dosed with NaSn,F, are parallel to those of rats given either NaF or SnCl, .2H20. It might be expected that NaF and NaSn,F, curves

26

CONINE

ET AL.

would be parallel, since both are F-containing compounds. The mortality curves in rats given NaSn,F5 in the present studies are also parallel to those of animals given NaF in other studies (Leone et al., 1956; Taylor et al., 1961). The NaSn,F, mortality curves were also parallel to those from animals given SnCl,*2H,O. Hence, it is difficult to judge the toxicity of NaSn,F, in terms of F activity alone, but instead it is suggested that both F and Sn might be active. The fact that NaSn,F, is toxic to the kidney is not surprising, but that this toxicity is identical to that produced by SnCl,.2H,O was unexpected. The renal toxicity of F in rats is well established(Muehlberger, 1930; Gottlieb and Grant, 1931-1932; Taylor et al., 1961). The renal toxicity of Sn is lessclearly described(Salant and Rieger, 19131914; Stoner et al., 1955; Haddon, 1958). The renal toxicity observed in the present study appears to be connected with the Sn and is produced when either Sn compound is given. Apparently, when Sn is in the circulation, it contributes to the toxicity of NaSn,F,. In summary, the present data suggeststhat both the F and Sn in NaSn,F, contribute to the toxicity of the compound. The contribution of the Sn may account for the difference in the toxicity between NaSn,F, and NaF. ACKNOWLEDGMENTS The authors thank the Ozark-Mahoning Company for generouslysupplying the NaSn,F, usedin thesestudiesand alsothank Mrs. Alice B. Richards,Mr. John Preston,Mr. Gary Ray, and Miss Mary Ann Jacobsfor their technical assistance. REFERENCES S. B. (1931-1932).Diuretic action of sodiumfluoride. Proc. Sot. Exp. Biol. Med. 29, 1293-1294. HADDON, W., JR. (1958). Lower nephron necrosisof a heavy metal type produced in rats given an inorganic tin preparation intraperitoneally. Annual Report of the Division of Laboratories and Research,New York State Department of Health, p. 41. LEONE, N. C., GREEVER, E. F., AND MORAN, N. C. (1956).Acute and subacutetoxicity studies of sodiumfluoride in animals.Pub. Health Report, Wash. 71,459-467. LITCHFIELD, J. T., JR. AND WILCOXON, F. (1949). A simplifiedmethod of evaluating doseeffect experiments.J. Pharmacol. Exp. Ther., 96,99-113. Lu, F. C., GREWAL, R. S.,RICE, W. B. AND GRAHAM, R. C. B. (1965).Acute toxicity of sodium fluoride for rhesusmonkeys and other laboratory animals.Acta Pharmacol. Toxicol. 22, GOTTLIEB,

L. AND

GRANT,

99-106. MUEHLBERGER,

C. W. (1930). Toxicity studiesof fluorine insecticides.J. Pharmacol.

Exp.

Ther. 39,246-248.

J. C., STOOKEY, G. K. AND BECK, C. W. (1967).The preparation and propertiesof sodiumpentafluorostannite.J. Dent. Res. 46, 380-383. SALANT, W. AND RIEGER, J. B. (1913-1914).Further observationson the toxicity of tin. Proc. Sot. Exp. Biol. Med. 11, 128-129. STONER, H. B., BARNES, J. M. AND DUFF, J. I. (1955).Studieson the toxicity of alkyl tin compounds.Brit. J. Pharmacol. 10, 16-25. STOOKEY, G. K., HENNON, D. K. AND MUHLER, J. C. (1969). Skeletal retention and anticariogenic efficacy of fluoride when administeredin the presenceof a prenatal vitaminmineral supplement.J. Dent. Res. 48, 12241230. TAYLOR, J. M., SCOTI, J. K. AND MAYNARD, E. A. (1961).Toxic effect of fluoride on the rat kidney. 1. Acute injury from singlelarge doses.Toxicol. Appl. Pharmacol. 3, 278-289. MUHLER,