Acta pharmacol. et toxicol. 1975, 37, 425-428.
From the Department of Oral Histopathology, Faculty of Odontology, University of Lund, Malmo, Sweden
Acute Sodium Fluoride Toxicity in Rats in Relation to Age and Sex BY HPkan Mlrnstad (Received April 29, 1975; Accepted May 5, 1975)
Abstract: The 24 hour LD50 dose for intraperitoneally injected sodium fluoride was determined in developing and young adult rats of both sexes. The developing animals tolerated two and a half times as much fluoride as the young adults when expressed as mg F-/kg of body weight. There were no significant differences between the two sexes. The 24 hour LD50 values were: 4 day old females 45 (39-52), males 40 (34-48);20 day old females 47 (39-57), males 51 (47-55); 60 day old females 30 (21-43),males 28 (17-42); 90 day old females 21 (17-26), males 20 (17-24) mg F-Ag of body weight.
Key-words: Age - fluoride
- toxicity.
The beneficial effects of fluoride in the prophylaxis of dental caries are well established as well as its adverse effects on bone and teeth following chronic overdosage. In experimental studies it is sometimes valuable to study the tissue response to a single high dose of fluoride. In such experiments, it is important to take into account whether the effects on the tissues are caused by a local effect of the fluoride per se or secondarily to a general intoxication. For this reason it is of importance to know the LD50 values for the experimental animals. The 24 hour LD50 dose of intraperitoneally injected sodium fluoride in adult rats has been estimated at 15-25 mg F / k g of body weight (STOKINGER 1949; SHOURIE et al. 1950; MAYNARD et al. 1951a & b; DYBING & LOE 1956; TAYLOR et al. 1961). MAYNARD et al. (1951a) considered young rats more resistant than older rats, and males more resistant than females, but did not present any LD50 values for developing rats. The purpose of this study was to obtain LD50 values of developing and young adult rats of both sexes.
HAKAN MURNSTAD
426
Materials and methods Developing and young adult rats (Sprague-Dawley, randomly bred) of both sexes were used in the following age groups: 4, 20, 60 and 90 days. The body weights are as shown in table 1. Aqueous solutions of sodium fluoride were injected intraperitoneally using a constant volume of 0.01 mug of body weight by means of a microsyringe. The solutions were freshly prepared from analytical grade sodium fluoride and contained 0.15, 0.225, 0.30, 0.45, 0.525, 0.60 and 0.75 % F- respectively. The administered amount of F- was thus 15, 22.5, 30, 45, 52.5, 60 and 75 mg F-/kg of body weight. All injections were given in the morning. Groups of at least 10 animals of each age and sex were tested at four or five dose levels. After the injection the animals were observed for two hours. All mortalities up to 24 hours were counted and considered as being caused by fluoride toxicity. The 24 hour LD50 values and the 95 % confidence limits were calculated according & WILCOXON (1949). to LITCHFIELD
Results The LD50 values, including the 95 % confidence limits, expressed as mg F/kg of body weight for the different age groups and sexes are given in table 1 and fig. 1. The tolerated amount of injected fluoride decreased with increasing age of the animals. During the first three weeks of life the animals survived two and a half times as much fluoride as the young adults when expressed as mg F/kg of body weight. There were no significant differences between the two sexes. Symptoms of intoxication appeared five to ten minutes after the injec-
Table 1. LD50 values, including the 95 % confidence limits, for intraperitoneally injected sodium fluoride into developing and young adult rats. Age in days
Sex
Weight in gram
LD50 mg M k g
4
9 8
10.5 f 1.5 11.5 k 1.9
45 (39-52) 40 (34-48)
20
9
42 f 10 48 f 10
47 (39-57) 51 (47-55)
6
30 (21-43) 28 (17-42)
90
9 6
232 f 21 342 f 38
21 (17-26) 20 (17-24)
AGE AND SEX RELATION IN FLUORIDE TOXICITY
427
4 70
-
M
Y
'IM
Ei
F:
50-{L
...I
0
Qo 30
-
1
' ; I
10
*
e
tions. Doses around the LD50 values caused decreased motor activity, ataxia and occasionally marked tetanic contractions. The normally pink colour of uncovered skin turned cyanotic. A reddish brown exudate was evacuated from the eyes. In the majority of the cases death occurred in a comatous state half an hour after injection. Only a few animals died during the remainder of the 24 hour period. Gross autopsy revealed intestinal congestion and in some cases a moderate ascites.
Discussion
This study confirms the statement by MAYNARD et al. (1951a&b) that developing rats have a higher tolerance to injected fluoride than adult rats, but does not support the suggestions of a lower susceptibility for males than for females. The LD50 values obtained for the young adult rats are et al. (1950), consistent with those presented by STOKINGER(1949), SHOURIE MAYNARD et al. (1951a & b), DYBING & LOE (1956) and TAYLOR et al. (1961). The higher tolerance levels for the younger animals might be explained by a rapid transfer of fluoride from the blood and soft tissues into miner-
428
HAKAN MORNSTAD
alizing tissues, where fluoride is strongly bound to the forming apatite crystals (CARLSON et al. 1960; STOOKEY et al. 1963; ERICSSON 1966). Other possible explanations for this tolerance may be the lower absolute amount of injected fluoride in relation to the functions of excreting organs, mainly the kidneys, or the fact that some enzymatic functions are not fully developed in the younger age groups. Fluoride has been reported to interact with enzyme function, calcium homeostasis, circulation and, finally, with specific organ functions (SIMONS 1965). The mechanism of death caused by high doses of fluoride is still unknown, but a general inhibition of cellular respiration has most often been held responsible (TAYLOR et al. 1961).
REFERENCES Carlson, C. H., L. Singer & W. D. Armstrong: Radiofluoride distribution of normal and nephrectomized rats. Proc. SOC. Exp. Biol. Med. 1960, 103, 418420. Dybing, 0.& L. V. Loe: Fluoride poisoning and cholinesterases in rats. Acta pharmacol. et toxicol. 1956, 12, 364-368. Ericsson, Y.: Blood fluoride clearence in rats differing in age or previous fluoride exposure. Acta Odont. Scand. 1966, 24, 393-404. Litchfield, J. T.& F. Wilcoxon: A simplified method of evaluating dose-effect experiments. J. Pharmacol. 1949,96, 99-113. Maynard, E. A., W. L. Downs & M. F. LeSher: Univ. Rochester At. Energy Proj. Quart. Tech. Rept. 1951a, UR-164, 73-77. Maynard, E. A., W. L. Downs & M. F. LeSher: Univ. Rochester At. Energy Proj. Quart. Tech. Rept. 1951b, UR-200. Shourie, K. L., J. W. Hein & H. C. Hodge: Preliminary studies of the caries inhibiting potential and acute toxicity of sodium monofluorophosphate. J . Dent. Res. 1950, 29, 529-533. Simons, J. H.: Fluorine chemistry. Academic Press, New York and London, 1965. Stokinger, H. E.: Toxicity following inhalation of fluorine and hydrogen fluoride. In: Pharmacology and toxicity of uranium compounds. Eds.: C. Voegtlin & H. C. Hodge. McGraw-Hill, New York, 1949. chapt. 17. Stookey, G. K., D. B. Crane & J. C. Muhler: Role of skeleton and kidney in fluoride absorption in rat. Proc. SOC. Exp. Biol. Med. 1963, 113, 366-370. Taylor, 1. M., J. K. Scott, E. A. Maynard, F. A. Smith & H. C. Hodge: Toxic effects of fluoride on the rat kidney. I. Acute injury from single large doses. Toxicol. Appl. Pharmacol. 1961, 3, 278-289.
From the Department of Oral Histopathology, Faculty of Odontology, University of Lund, Malmo, Sweden
Acute Sodium Fluoride Toxicity in Rats in Relation to Age and Sex BY HPkan Mlrnstad (Received April 29, 1975; Accepted May 5, 1975)
Abstract: The 24 hour LD50 dose for intraperitoneally injected sodium fluoride was determined in developing and young adult rats of both sexes. The developing animals tolerated two and a half times as much fluoride as the young adults when expressed as mg F-/kg of body weight. There were no significant differences between the two sexes. The 24 hour LD50 values were: 4 day old females 45 (39-52), males 40 (34-48);20 day old females 47 (39-57), males 51 (47-55); 60 day old females 30 (21-43),males 28 (17-42); 90 day old females 21 (17-26), males 20 (17-24) mg F-Ag of body weight.
Key-words: Age - fluoride
- toxicity.
The beneficial effects of fluoride in the prophylaxis of dental caries are well established as well as its adverse effects on bone and teeth following chronic overdosage. In experimental studies it is sometimes valuable to study the tissue response to a single high dose of fluoride. In such experiments, it is important to take into account whether the effects on the tissues are caused by a local effect of the fluoride per se or secondarily to a general intoxication. For this reason it is of importance to know the LD50 values for the experimental animals. The 24 hour LD50 dose of intraperitoneally injected sodium fluoride in adult rats has been estimated at 15-25 mg F / k g of body weight (STOKINGER 1949; SHOURIE et al. 1950; MAYNARD et al. 1951a & b; DYBING & LOE 1956; TAYLOR et al. 1961). MAYNARD et al. (1951a) considered young rats more resistant than older rats, and males more resistant than females, but did not present any LD50 values for developing rats. The purpose of this study was to obtain LD50 values of developing and young adult rats of both sexes.
HAKAN MURNSTAD
426
Materials and methods Developing and young adult rats (Sprague-Dawley, randomly bred) of both sexes were used in the following age groups: 4, 20, 60 and 90 days. The body weights are as shown in table 1. Aqueous solutions of sodium fluoride were injected intraperitoneally using a constant volume of 0.01 mug of body weight by means of a microsyringe. The solutions were freshly prepared from analytical grade sodium fluoride and contained 0.15, 0.225, 0.30, 0.45, 0.525, 0.60 and 0.75 % F- respectively. The administered amount of F- was thus 15, 22.5, 30, 45, 52.5, 60 and 75 mg F-/kg of body weight. All injections were given in the morning. Groups of at least 10 animals of each age and sex were tested at four or five dose levels. After the injection the animals were observed for two hours. All mortalities up to 24 hours were counted and considered as being caused by fluoride toxicity. The 24 hour LD50 values and the 95 % confidence limits were calculated according & WILCOXON (1949). to LITCHFIELD
Results The LD50 values, including the 95 % confidence limits, expressed as mg F/kg of body weight for the different age groups and sexes are given in table 1 and fig. 1. The tolerated amount of injected fluoride decreased with increasing age of the animals. During the first three weeks of life the animals survived two and a half times as much fluoride as the young adults when expressed as mg F/kg of body weight. There were no significant differences between the two sexes. Symptoms of intoxication appeared five to ten minutes after the injec-
Table 1. LD50 values, including the 95 % confidence limits, for intraperitoneally injected sodium fluoride into developing and young adult rats. Age in days
Sex
Weight in gram
LD50 mg M k g
4
9 8
10.5 f 1.5 11.5 k 1.9
45 (39-52) 40 (34-48)
20
9
42 f 10 48 f 10
47 (39-57) 51 (47-55)
6
30 (21-43) 28 (17-42)
90
9 6
232 f 21 342 f 38
21 (17-26) 20 (17-24)
AGE AND SEX RELATION IN FLUORIDE TOXICITY
427
4 70
-
M
Y
'IM
Ei
F:
50-{L
...I
0
Qo 30
-
1
' ; I
10
*
e
tions. Doses around the LD50 values caused decreased motor activity, ataxia and occasionally marked tetanic contractions. The normally pink colour of uncovered skin turned cyanotic. A reddish brown exudate was evacuated from the eyes. In the majority of the cases death occurred in a comatous state half an hour after injection. Only a few animals died during the remainder of the 24 hour period. Gross autopsy revealed intestinal congestion and in some cases a moderate ascites.
Discussion
This study confirms the statement by MAYNARD et al. (1951a&b) that developing rats have a higher tolerance to injected fluoride than adult rats, but does not support the suggestions of a lower susceptibility for males than for females. The LD50 values obtained for the young adult rats are et al. (1950), consistent with those presented by STOKINGER(1949), SHOURIE MAYNARD et al. (1951a & b), DYBING & LOE (1956) and TAYLOR et al. (1961). The higher tolerance levels for the younger animals might be explained by a rapid transfer of fluoride from the blood and soft tissues into miner-
428
HAKAN MORNSTAD
alizing tissues, where fluoride is strongly bound to the forming apatite crystals (CARLSON et al. 1960; STOOKEY et al. 1963; ERICSSON 1966). Other possible explanations for this tolerance may be the lower absolute amount of injected fluoride in relation to the functions of excreting organs, mainly the kidneys, or the fact that some enzymatic functions are not fully developed in the younger age groups. Fluoride has been reported to interact with enzyme function, calcium homeostasis, circulation and, finally, with specific organ functions (SIMONS 1965). The mechanism of death caused by high doses of fluoride is still unknown, but a general inhibition of cellular respiration has most often been held responsible (TAYLOR et al. 1961).
REFERENCES Carlson, C. H., L. Singer & W. D. Armstrong: Radiofluoride distribution of normal and nephrectomized rats. Proc. SOC. Exp. Biol. Med. 1960, 103, 418420. Dybing, 0.& L. V. Loe: Fluoride poisoning and cholinesterases in rats. Acta pharmacol. et toxicol. 1956, 12, 364-368. Ericsson, Y.: Blood fluoride clearence in rats differing in age or previous fluoride exposure. Acta Odont. Scand. 1966, 24, 393-404. Litchfield, J. T.& F. Wilcoxon: A simplified method of evaluating dose-effect experiments. J. Pharmacol. 1949,96, 99-113. Maynard, E. A., W. L. Downs & M. F. LeSher: Univ. Rochester At. Energy Proj. Quart. Tech. Rept. 1951a, UR-164, 73-77. Maynard, E. A., W. L. Downs & M. F. LeSher: Univ. Rochester At. Energy Proj. Quart. Tech. Rept. 1951b, UR-200. Shourie, K. L., J. W. Hein & H. C. Hodge: Preliminary studies of the caries inhibiting potential and acute toxicity of sodium monofluorophosphate. J . Dent. Res. 1950, 29, 529-533. Simons, J. H.: Fluorine chemistry. Academic Press, New York and London, 1965. Stokinger, H. E.: Toxicity following inhalation of fluorine and hydrogen fluoride. In: Pharmacology and toxicity of uranium compounds. Eds.: C. Voegtlin & H. C. Hodge. McGraw-Hill, New York, 1949. chapt. 17. Stookey, G. K., D. B. Crane & J. C. Muhler: Role of skeleton and kidney in fluoride absorption in rat. Proc. SOC. Exp. Biol. Med. 1963, 113, 366-370. Taylor, 1. M., J. K. Scott, E. A. Maynard, F. A. Smith & H. C. Hodge: Toxic effects of fluoride on the rat kidney. I. Acute injury from single large doses. Toxicol. Appl. Pharmacol. 1961, 3, 278-289.
