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Preliminary safety assessment of disodium etidronate as an additive to experimental oral hygiene products
TOXICOLOGY
AND
APPLIED
PHARMACOLOGY
22, 661-671 (1972)
Preliminary Safety Assessment Additive to Experimental
of Disodium Etidronate Oral Hygiene Products
as an
G. A. NIXON, E. V. BUEHLER AND E. A. NEWMANN The Procter & Gamble Company, Miami Valley Laboratories, P. 0. Box 39175, Cincinnati, Ohio 45239 Received November 22. 1971
Preliminary Safety Assessment of Disodium Etidronate as an Additive to Experimental Oral Hygiene Products. NIXON, G. A., BUEHLER, E. V. and NEWMANN, E. A. (1972). Toxicol. Appl. Pharmacol. 22, 661-671. Disodium etidronate is an effective anticalculus agent and is being considered for inclusion in toothpaste and mouthwash formulations. Disodium etidronate and products containing disodium etidronate were investigated for acute and subchronic toxicity and primary irritation potential. At high levels, under both acute and subchronic conditions, po toxicity was reflected by effects on the kidney, characterized by convoluted tubule dilation and degeneration and/or elevated kidney to body weight ratios. From the standpoint of possible human ingestion, these tests indicate disodium etidronate would be safe for the consumer, on either an acute or subchronic basis, when incorporated at levels of 3 % in toothpaste and 1 % in mouthwash. When included in experimental dentifrice and mouthwash products, disodium etidronate does not increase the irritant potential of the po products when compared to control formulations, and is comparable in toxicity to other commercially available dentifrices and mouthrinses.
Francis (1969) has discussed the efficacy of the disodium salt of ethane-l-hydroxy-l,ldiphosphonic acid’ (disodium etidronate) as an anti-calculus agent and has suggested its use as an additive to oral hygiene products. Because of its effects on calcium and phosphate metabolism, it also has potential in the treatment of several disorders of mineral metabolism (Bassett et al., 1969; Fleisch et al., 1969; Francis et al., 1969; Smith et al., 1971; Weiss et al., 1971). This report covers the preliminary toxicologic investigation of disodium etidronate to establish its safety when included in po products at concentrations up to 3 ‘A. The effects of disodium etidronate on the reproductive functions and embryogeny are published elsewhere (Nolen and Buehler, 1971). 1 The systemicchemical name of the parent acid, CH, .C(OH)PO(OH,),, is (l-hydroxyethylidene)diphosphonic acid, and the nonproprietary name is etidronic acid; both have been acceptedby the United States Adopted Names Council. In this paper, the term disodium etidronate will be used throughout. Copyright All rights
0 1972 by Academic Press, Inc. of reproduction in any form reserved.
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METHODS The etidronate ion2 has the structure indicated below. HxC
C P032-
OH
The experimental formulations are as follows : Ingredient Disodium etidronate Sodium fluoride Cationic Humectant Alcohol Abrasive Glycerin Binders Surfactant Flavoring agents Dyes Buffer q.s. Hz0
Dentrifrice (%) 3.00 0.221 30.00 39.00 1.70 3.87 1.29 0.22
Mouthwash (%I 1.000
0.055 0.050 16.000-20.000 lO.ooo 0.120 0.140
0.0005 0.115
There was a slight variation in the concentration of these ingredients in the various experimental products. Acute oral toxicity. Male and female Charles River CD rats weighing between 190 and 300 g were fasted 18-20 hr prior to dosing. Four dose levels of disodium etidronate, determined by using a constant geometric progression of doses, were administered by stomach tube to groups of 10 animals on the basis of an assumed toxicity and dose response curve. Dentrifrice formulations were dosed at a single level of 25 g/kg and mouthwash formulations at 25 ml/kg. LD50 values were calculated by the method of Thompson (1947) and Weil(1952). The same procedure was used for the determination of LD50 values in New Zealand rabbits of varying weight ranges. All animals were observed daily, and any changes in behavior were recorded. At the end of the 2 wk observation period, all surviving animals were necropsied and examined grossly. Blood and tissues from selected individuals were obtained for hematologic and histologic evaluation Emesis. Purebred beagle dogs ranging in age from 6 mo to 4 yr were fasted for approximately 19 hr and deprived of water for approximately 3 hr prior to treatment. Groups of 4 dogs (2 male, 2 female) were dosed po via stomach tube with disodium etidronate alone or in mouthwash or toothpaste formulations at 4 different dose levels using a constant geometric progression. The dogs were observed continuously for 4 hr after dosing, and the time and number of emetic responses were recorded. At the end of this period, food and water were offered * The pure salts of etidronic acid used in these studies were obtained from the Monsanto Company, 800 North Lindbergh Blvd., St. Louis, Missouri 63166.
DISODIUM
ETIDRONATE
SAFETY
663
ad libitum, and daily observations were continued for 2 wk. ED50 values were determined according to Weil(l952). Subchronic toxicity. Charles River CD rats were randomized according to weight, sex and litter and fed diets containing 0,0.2 and 1.O% disodium etidronate for a period of 91 days. All groups contained 20 males and 20 females; the average group weights did not differ more than 0.5 g (56.0 g for females and 60.0 g for males). Animals were housed individually in wire-bottom cages,and food and water were offered ad libitum. Body weights and food consumption were recorded weekly. Visual observations were made on individual animals daily, and all abnormal conditions were recorded. At the conclusion of the study (91 days), 5 males and 5 females from each group were randomly selectedfor necropsy. Liver and kidney to body weight ratios were calculated. Blood was taken for hematologic analyses,and organ specimenswere taken for histologic examination. All blood specimenswere examined for total red blood cell (RBC) count, total white blood cell (WBC) count, differential WBC count, hemoglobin and hematocrit. Tissues obtained for microscopic examination were liver, spleen, kidney, thyroid, trachea, esophagus, lung, heart, pancreas, adrenal, stomach, small intestine, urinary bladder, gonads and mesenteric lymph nodes. Tissues were fixed in Bouin solution, mounted in paraffin, trimmed and stained with hematoxylin and eosin. In a separatestudy, animals were fed diets containing 0 or 5.0 % disodium etidronate. The experiment was terminated after 1 wk because of severe weight loss and a large number of deaths in the test group. Five males and 5 females from both groups were necropsied, and blood and organ specimens were examined as above. Eye irritation. The potential for producing eye irritation was tested in New Zealand rabbits according to established procedures (Draize, 1959). Undiluted or appropriate dilutions of the test materials in aqueous solutions were instilled into the conjunctival sac of 1 eye in each of 3 rabbits. The untreated eye served as control. If rinsing was performed, 20 ml of lukewarm tap water was directed from a syringe into all parts of the eye within 4 set after instillation. Eyes were scored at 1 hr and at 1,2,3,4 and 7 days, and weekly thereafter for a maximum of 5 wk. The scalefor scoring ocular lesions was that of Draize (1959).The scoring method used in these studies differs from that recommended by the Federal Food and Drug Administration (1964) in that a slight dulling (bedewing) and/or stippling of the cornea is recorded as a grade 1. More severelesions of the cornea are scoredas 2 or more. In addition, anything more severe than a slight congestion of the iridial folds was scored as a grade 2 for iritis. The end result is that the scoring system used here results in slightly higher maximum averagescores(MAS) than would be expectedif one adhered to the FDA guidelines. Maximum average scores are determined by averaging the highest average score observed on any given day for the 3 animals in the test group, regardless of the time required to produce these scores. Oral mucosal irritation. The test method for detecting po mucosal irritation was developed in our laboratories to compare the irritant potential of po products. A number of years of experiencewith the rat as an experimental model had indicated that this animal is refractory to po mucosal irritation. This is not surprising since the rat oral mucosal epithelium is keratinized. In addition, experiments with a number of other species(guinea pig, hamster, rabbit) indicated that these specieswere not suitable for
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evaluating po mucosal irritation. In the beagle dog, however, by using exaggerated test conditions, it is possible to produce irritation with commercially available po products and to make comparisons among them. By combining rinse and no rinse treatments after repeated applications to the po mucosal it is possible to detect subtle differences between various test materials. Beagle dogs (3 in each group) ranging in age from 6 mo to 4 yr were used. One side of the gingiva, on the upper jaw of each dog, was treated by applying approximately 1 ml of a dentifrice or a mouthwash-saturated dental cotton roll3 approximately 1.5 in long. Immediately after applying the material, the mouth was held closed for 20-30 set to ensure good contact of the test material with the po mucosa. Two treatment schedules were used : (1) undiluted products were applied 5 times daily for 4 days, or (2) the same treatment was used and the treated areas was rinsed with 25-30 ml of tap water within 5 set after cessation of treatment. Gross observations were made prior to each treatment for signs of irritation (erythema) or epithelial sloughing. If epithelial damage was suspected, the test sites were treated with 2 % fluorescein and rinsed. Any well-defined areas of fluorescein retention indicated that epithelial damage had occurred. This response was considered to be the end point of the test, and treatment was discontinued. It is important to note that fluorescein does not stain injured tissue, but diffuses into superficial epithelial layers that are damaged. Judgments on the relative irritancy of test materials were made on the basis of the number of topical applications required to produce irritation. RESULTS
Acute Oral Toxicity (Rat) Disodium etidronate had an LD50 value of 1.34 g/kg when tested in the rat (Table 1). Lower LD50 values were found for trisodium, tetrasodium and tetrapotassium salts, LD50 values for the latter 2 compounds being significantly lower than for the disodium salt. The insoluble dicalcium salt was nontoxic (Table 1). Gross observations of the test animals during the test period failed to reveal a toxic effect. TABLE
1
ACUTE ORAL TOXICITY OF VARIOUS SALTS OF DISODIUM ETIDRONATE IN THE RAT
Concentration ( % w/w)
Salt Disodium Trisodium Tetrasodium Tetrapotassium Dicalcium
LD50 value (g/kg) 1.34 1.28 0.99 0.52
25 20 20
50 25
>ll
At necropsy, 3 of 10 surviving animals that had been dosed with high levels (1.60 and 1.14 g/kg) of disodium etidronate had pale (light gray), granular kidneys. Histologic examination revealed changes apparently involving all parts of the nephros, with as 3 Dental
Cotton
Roll, Johnson
& Johnson
Company.
665
DISODIUM ETIDRONATE SAFETY
much as 2@-40y0 of the kidney involved in all animals from the high levels. The damage was distinctly tubular, with very little involvement of the interstitium. The tubules were dilated and lined with low columnar epithelium which stained basophilic. There appeared to be a hyperplasia of tubule cells, with the nuclei larger and more numerous on cross section. The glomeruli involvement appeared to be secondary to the tubule involvement. Scattered incidences of hydropic degeneration were also observed. The responseappears to be somewhat similar to that produced by ethylenediaminetetraacetic acid. Kidneys from all animals dosed at the low level (0.814 g/kg) had m ild tubular changes as noted histologically. The only other m icroscopic finding of possible significance occurred in the stomachs of animals dosed with the 2 high levels (1.60 and 1.14 g/kg), in which mucosal irritation was noted in 4 of 10 animals examined. The kidney : body weight ratios of the 2 groups dosed with the high levels of the test material were significantly higher than those of animals receiving 0.814 g/kg. These values (2 high groups) were also higher than the normal range. All other average values were within the normal ranges established in previous studies of this type using Charles River CD rats. Experimental dentifrices (3 % disodium etidronate) and mouthwashes (1% disodium etidronate) had LD50 values of >25 g/kg and >25 m l/kg, respectively. Gross and m icroscopic observations of these animals did not reveal any abnormalities. Acute Oral Toxicity (Rabbit) Table 2 presents the data from a series of experiments to establish the LD50 value of disodium etidronate in the rabbit. Results of these tests indicate that susceptibility of the rabbit to the acute effects of disodium etidronate is a function of age, weight and sex, The stress of pregnancy apparently does not significantly alter the LD50 value of the material in this species.The LD50 value in rabbits was found to be somewhat lower in males than in females and lower in mature animals (>3300 g) as opposed to immature animals (-2500 g). TABLE 2 ACUTE ORAL TOXICITY OF DISODIUM ETIDRONATE IN THE RABBIT
No. of animals/group”
Animal condition
2F, 2M 2F, 2M 4F
Immature Immature Mature, virgin Pregnant Mature -* -*
4F 4M 4F, 4M
8F, 4M
Body weight (g> 2250-2800 2250-2800 >3300 13300 >3300 2250-2800 >3300
LD50 (g/W
Confidence lim its
1.14 1.05 0.814 0.668 0.581 1.10 0.70
0.829-1.56 0.905-l .21 0.642-l .03 0.542-0.872
0.424-0.799 0.961-1.52 0.561-0.831
u Four groupsof animalsin eachtest. b Data from all experiments combined according to body weight.
Nearly all the deaths in the test groups occurred in less than 24 hr, and none died after 4 days. All surviving animals were observed for 2 wk after dosing. The only symptom noted in these animals during this period was diarrhea. This occurred from
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approximately 4 hr through 48 hr post treatment; severity was generally in proportion to the dose level. All surviving animals were sacrificed at 2 wk; blood was taken for hematologic analyses, and tissues were taken for microscopic examination. Several animals in each test group appeared to have mild gastric irritation. No other unusual lesions were observed grossly. No unusual lesions were seen microscopically in any of the tissues. About 50 % of the surviving animals were found to have kidney lesions resembling chronic interstitial nephritis, and these were scattered throughout all groups. Because chronic interstitial nephritis is common in the rabbit, it is difficult to distinguish between this lesion and one that could be test related. Group average values (blood and organ: body weight ratios) appeared to be normal for animals of this age and species. Emesis Disodium etidronate produces an immediate emetic response in some animals, and because of this factor it was not possible to determine well defined LD50 values in the beagle dog. The ED50 value for disodium etidronate was 84.8 mg/kg with confidence limits of 31.8-226 mg/kg. When dosed at higher levels (0.25-10 g/kg) varying degrees of stomach and intestinal irritation were noted, severity being dose-dependent. A toxic response characterized by bloody emesis, diarrhea and loss of appetite was noted in male dogs at levels of 0.50 g/kg and in females at levels of 1.Og/kg. Although the LD50 value for disodium etidronate could not be calculated precisely, it was estimated to be about 1.O g/kg on the basis of early deaths and necropsy of moribund animals dosed with the higher levels (1.0-10 g/kg). Table 3 presents the data from the study to determine the ED50 value of the experimental toothpaste containing 1 or 3 % disodium etidronate and the experimental mouthwash containing 1% disodium etidronate. Disodium etidronate increased the emetic properties of the dentifrice; therefore, the ED50 values were inversely related to the concentration of disodium etidronate in the formulation. In all cases, the test animals appeared normal after cessation of vomiting. The experimental mouthwash was found to have an ED50 value of 5.10 ml/kg. TABLE
3
EMETICPROPERTIESOFANEXPERIMENTALDENTIFRICE AND MOUTHWASH CONTAINING DISODIUM ETIDRONATE
ED50
Mean onset of emesis in dogs responding
Test material
(g/kg)
Confidence limits
Dentifrice with 3 % disodium etidronate Dentifrice with 1% disodium etidronate Control dentifrice” Mouthwash with 1% disodium etidronate
0.681
0.364-1.27
19 min
1.21
0.825-l .78
28 min
6.81
4.89-5.31
5.10b
1 hr, 10 min 37 min
a Similarto experimentaldentifrice,but without disodiumetidronateand NaF. b Expressedas ml/kg.
DISODIUM
ETIDRONATE
667
SAFETY
Two beagle dogs (1 male, 1 female) were also dosed with each of the dentifrice formulations at levels of 10 g/kg. The only gross sign observed was emesis. At 1 wk postdosing, each animal was necropsied. There was no microscopic lesion that could be attributed to treatment, and all blood values were within the normal range for these animals. Subchronic Toxicity
Groups of 20 male and 20 female rats were fed diets containing varying levels of disodium etidronate (Table 4). The test group fed 5% disodium etidronate was terminated after 1 wk because of mortality and severeweight loss. Necropsy of the surviving animals from this test group revealed the presence of stomach ulcers and hyperemia. Microscopic examination revealed inflammation and dilated glands and capillaries which confirmed the gross findings of gastritis. There was also a generalized erosion of the glandular mucosal epithelium in the stomach of some animals (i.e., rough appearance with local areas of sloughing). The gastritis generally affected both the forestomach and the glandular portion of the stomach. The kidney : body weight ratio was high and may have been due to a poor growth rate. The total white blood cell count was high, probably the result of the stomach irritation. The neutrophil count was high and the lymphocyte count low, indicating the acute nature of the gastric distress. TABLE 4 91 DAY Dietary level 0
Sex F M
0.2%
F
1.0%
F M
0”
F M
M
5.0%
F
M
FEEDING STUDY IN RATS WITH DWIDIUM
ETIDRONATE
Average body weight gain Cd
Organ/body weightb Liver
Kidney
223 438 234 430 223 432
3.26 3.31 3.28 3.49 3.67 3.41
0.72 0.67 0.65 0.82c 0.70
34 48 -6 3
4.44 4.34 4.28 4.58
0.95 1.48 1.55
0.64
1.11
No. of survivors 20 20 19 20 20 20 20 20 12 16
u Groupsterminatedafter 1 wk. b Expressedas % of body weight. ’ Significantlydifferentfrom controls(P > 0.05). The survival rate in the other test groups (0.2 and 1.0% disodium etidronate) was good and gave no indication of a toxic effect. There were no gross necropsy lesions attributed to toxicity and, except for a slightly elevated kidney : body weight ratio in females on the 1 y0 disodium etidronate, all parameters in the study were normal.
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Eye Irritation
Eye irritation results are important in the safety assessmentof a dentifrice product from 2 standpoints. First, they provide data relative to the potential for actual eye irritation in case of accidental instillation into the eye during normal use. Second, they provide an indication of the irritation to a very sensitive mucosal membrane. Since the product comes in contact with po mucosa, this latter information is of value. It is necessary,however, to compare the irritation produced with other products being used safely by the consumer. Disodium etidronate instilled into the rabbit eye produced only a mild conjunctival irritation. The eyes were completely normal by day 4 (Table 5). Because the irritancy produced was quite mild, there was no apparent benefit from rinsing. Dissolving the material (10 %) in water prior to instillation did not change the irritation potential of disodium etidronate. TABLE 5 EYE IRRITATION IN THE RABBIT WITH DISODIUM ETIDRONATE AND THE EXPERIMENTAL DENTRIFRICE AND MOUTHWASH
Test material Disodium etidronate Disodium etidronate Disodium etidronate Disodium etidronate dentifrice Disodium etidronate dentifrice Fluoride dentifriceb Fluoride dentifrice High flavor dentifrice’ High flavor dentifrice Disodium etidronate mouthwash Control mouthwash
Maximum Comeal average Treatment’ scores involvement 3mgNR 3mgR 10% NR 3mgNR
No. of eyesnormal in No. of days
6.6 5.6 4.0 21.6
O/3 O/3 O/3 213
3 in 2 lin2;1in3;1in4 2inl; 1 in2 2in3;1in4
9.0
O/3
2 in 2; 1 in 3
3mgNR 3mgR 3mgNR 3mgR 100% NR
19.6 6.6 40.6 22.0 2.0
113 O/3 313 213 O/3
1 in4;2inl linl;lin2;1in3 1 in 7; 2 in 14 1 in 3; 1 in 7; 1 in 14 3 in 1
lOO%NR
2.0
O/3
3 in 1
3mgR
no rinse;R, rinse. b Stannousfluoride. c Chloroform. ’ NR,
Instillation of the undiluted experimental dentifrice (3 % disodium etidronate) into the rabbit eye produced transient alteration of the precorneal aqueous film and cornea1 epithelium and moderate degreesof conjunctival irritation that were transient, the eyes returning to normal by day 4. In this case, rinsing the eyes within 4 set effectively alleviated the mild irritative effects. Undiluted experimental mouthwash containing 1% disodium etidronate produced only slight conjunctivitis that cleared readily. Comparison of the eye irritation results with the experimental dentifrice to commercially available dental formulations indicate that it is similar to these products with regard to primary irritant properties; the disodium etidronate formulation appears to be less irritating than a dentifrice flavored with chloroform.
DISODIUM
ETIDRONATE
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Oral Mucosal Irritation
Table 6 summarizes the results of the experiments on po mucosal irritation. The experimental dentifrice containing 3 ‘A disodium etidronate was no more irritating to po mucosa than was the control formulation, but both these formulations were more irritating than fluoride dentifrice when compared under “no rinse” conditions. When rinsing was employed, the results with all formulations were similar. The chloroform flavored dentifrice was more irritating than any of the other test materials when rinsing was employed, and irritant effects were noted after only 9 applications. TABLE 6 ORAL MUCOSAL IRRITATION PRODUCED BY THE EXPERIMENTAL DENTIFRICE AND MOUTHWASH
Test material
TreatmeW
Disodium etidronate dentifrice Disodium etidronatedentifrice Control dentifrice Control dentifrice Fluoride dentifrice’ Fluoride dentifrice High flavor dentifriced High flavor dentifrice Disodium etidronatemouthwash Control mouthwash
No. of applicationsto produce irritationb
x/day, NR x/day, R x/day, NR x/day, R x/day, NR x/day, R x/day, NR x/day, R 5 x/day, NR
5 5 5 5 5 5 5 5
5 x/day, NR
4 >20 4 >20 >20 >20 4 9 20 >20
’ Maximum number of treatments = 20. NR, no rinse; R, rinse. b Epithelial sloughing detected with fluorescein. ’ Stannous fluoride. d Chloroform.
Treatment ofthe po mucosa with a mouthwash formulation containing 1% disodium etidronate (Table 6) produced results comparable to a similar commercial formulation without disodium etidronate. These results indicate that the experimental products are comparable to commercially available formulations with regard to po mucosal irritation in the dog. DISCUSSION
The acute po LDSO value of disodium etidronate in Charles River CD rats is 1.34 g/kg, and a similar po LDSO is noted in the rabbit (1.14 g/kg). Because of its potent emetic properties, it was not possible to determine the LD50 value in dogs. When incorporated into a dentifrice or mouthwash formulation, this latter property of disodium etidronate may provide an added safety factor in case of ingestion by a child. It is noted, however, that the experimental po hygiene products are nontoxic and have LD.50 values in rats of >25 g/kg (dentifrice) and ~-25 ml/kg (mouthwash). Toxicity of disodium etidronate in rats given an acute po dose of 1.14 g/kg or greater was expressed in gastric irritation and kidney lesions. The changes noted in the kidney
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were no different from those attributed to chelating agents and phosphates (Reuber and Lee, 1966; BIBRA Bulletin, 1964) and do not appear to be due to an effect that is unique to disodium etidronate. There were no lesions noted microscopically in rabbits that were attributed to disodium etidronate treatment. More definitive data on the lack of metabolic alteration of the compound are published elsewhere (Michael et al., 1972). When fed at high levels (5 %) in the diet to rats, disodium etidronate was toxic, producing morbidity and moribundity. At the next lower dietary level (lx), there was an indication that the kidney was affected (increased kidney:body weight) in female rats, but all animals in the test group appeared to be healthy. The addition of 0.2 % disodium etidronate to the rats’ diet gave no indication of a toxic effect. If a 10 kg child ingested 1 g of toothpaste per day containing 3 % disodium etidronate or 3 ml of mouthwash containing 1 ‘A disodium etidronate, the ingestion of disodium etidronate would be 30 mg/day, or 3 mg/kg/day. Since the rat ingests about 130 g/kg/day of diet during the first wk of a testing period, the level of 0.2 % (260 mg of disodium etidronatejkgjday) represents approximately a go-fold factor over the maximum probable ingestion that can be predicted. It should be recognized that as the child becomes older the probable ingestion of po hygiene products, particularly in relation to body weight, is drastically reduced, whereas the rat ingested approximately the same quantity of food throughout the entire period of rapid growth. Data obtained in these experiments and in chronic tests (to be published elsewhere) indicate that disodium etidronate would be safe for human usage in mouthwash (1%) and dentifrice (3 %). ACKNOWLEDGMENT The authors wish to thank Mr. W. R. King, who performed all hematologic evaluations and histologic examination of tissues during the various phases of the study. All development and formulation work on the experimental dentifrice and mouthwash was by Dr. J. S. Widder, whose efforts made these studies possible. REFERENCES
C. A. L., DONATH, A., MACAGNO, F., PREISIG, R., FLEISCH, H., and FRANCIS, M. D. (1969). Diphosphonates in the treatment of myositis ossificans. Luncet 7625, 845. BIBRA (1964). Toxicity studies on phosphates.Food Cosmet. Toxicol. 2, 147-154. DRAIZE, J. H. (1959). Dermal toxicity. In “Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics,” pp. 46-59, Association of Food and Drug Officials of the United States, Austin, Texas, AFDO. FLEISCH, H., RUSSELL, R. G. G., and FRANCIS, M. D. (1969). Diphosphonates inhibit hydroxyapatite dissolution in vitro and bone resorption in tissue culture and in vivo. Science 165, 1262-1264. FRANCIS, M. D. (1969). Inhibition of calcium hydroxyapatite crystal growth by polyphosphonates and polyphosphates Calcif. Tissue Res. 3, 151-162. FRANCIS, M. D., RUSSELL, R. G. G., and FLEISCH, H. (1969). Diphosphonates inhibit formation of calcium phosphate crystals in vitro and pathological calcification in vivo. Science 165, 1264-1266. Illustrated Guide for Grading Eye Irritation by Hazardous Substances (1964). U.S. Dept. of Health, Education, and Welfare, Food & Drug Administration. U.S. Gov. Printing Office, Washington, D. C. MICHAEL, W. R., KING, W. R., and WAKIM, J. M. (1972). Metabolism of disodium ethane-lhydroxy-l,l-diphosphonate (disodium etidronate) in the rat, rabbit, dog and monkey. Toxicol. Appl. Pharmacol. 21, 503-515.
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NOLEN, G. A., and BUEHLER, E. V. (1971). The effects of disodium etidronate on the reproductive functions and embryogeny of albino rats and New Zealand rabbits. Toxicol. A&. Pharmacol. 18, 548-561. REUBER, M. D., and LEE, C. W. (1966). Calcium disodium edetate nephrosis in inbred rats. Arch. Environ. Health 13, 554-557. SMITH, R., RUSSELL,R. G. G., and BISHOP,M. (May 8, 1971). Diphosphonates and Paget’s disease of bone. Lancet, 945-947. THOMPSON, W. R. (1947). Use of moving averages and interpolation to estimate median-effective dose. Part I. Bacterial. Rev. 11, 115-145. WEIL, C. S. (1952). Tables for convenient calculation of median-effective dose (LDSO or ED50) and instructions in their use. Biometrics 8, 249-263. WEISS,I. W., FISHER,L., and PHANG,J. M. (1971). Diphosphonate therapy in a patient with myositis ossificans progressiva. Ann. Int. Med. 74, 933-936.
AND
APPLIED
PHARMACOLOGY
22, 661-671 (1972)
Preliminary Safety Assessment Additive to Experimental
of Disodium Etidronate Oral Hygiene Products
as an
G. A. NIXON, E. V. BUEHLER AND E. A. NEWMANN The Procter & Gamble Company, Miami Valley Laboratories, P. 0. Box 39175, Cincinnati, Ohio 45239 Received November 22. 1971
Preliminary Safety Assessment of Disodium Etidronate as an Additive to Experimental Oral Hygiene Products. NIXON, G. A., BUEHLER, E. V. and NEWMANN, E. A. (1972). Toxicol. Appl. Pharmacol. 22, 661-671. Disodium etidronate is an effective anticalculus agent and is being considered for inclusion in toothpaste and mouthwash formulations. Disodium etidronate and products containing disodium etidronate were investigated for acute and subchronic toxicity and primary irritation potential. At high levels, under both acute and subchronic conditions, po toxicity was reflected by effects on the kidney, characterized by convoluted tubule dilation and degeneration and/or elevated kidney to body weight ratios. From the standpoint of possible human ingestion, these tests indicate disodium etidronate would be safe for the consumer, on either an acute or subchronic basis, when incorporated at levels of 3 % in toothpaste and 1 % in mouthwash. When included in experimental dentifrice and mouthwash products, disodium etidronate does not increase the irritant potential of the po products when compared to control formulations, and is comparable in toxicity to other commercially available dentifrices and mouthrinses.
Francis (1969) has discussed the efficacy of the disodium salt of ethane-l-hydroxy-l,ldiphosphonic acid’ (disodium etidronate) as an anti-calculus agent and has suggested its use as an additive to oral hygiene products. Because of its effects on calcium and phosphate metabolism, it also has potential in the treatment of several disorders of mineral metabolism (Bassett et al., 1969; Fleisch et al., 1969; Francis et al., 1969; Smith et al., 1971; Weiss et al., 1971). This report covers the preliminary toxicologic investigation of disodium etidronate to establish its safety when included in po products at concentrations up to 3 ‘A. The effects of disodium etidronate on the reproductive functions and embryogeny are published elsewhere (Nolen and Buehler, 1971). 1 The systemicchemical name of the parent acid, CH, .C(OH)PO(OH,),, is (l-hydroxyethylidene)diphosphonic acid, and the nonproprietary name is etidronic acid; both have been acceptedby the United States Adopted Names Council. In this paper, the term disodium etidronate will be used throughout. Copyright All rights
0 1972 by Academic Press, Inc. of reproduction in any form reserved.
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METHODS The etidronate ion2 has the structure indicated below. HxC
C P032-
OH
The experimental formulations are as follows : Ingredient Disodium etidronate Sodium fluoride Cationic Humectant Alcohol Abrasive Glycerin Binders Surfactant Flavoring agents Dyes Buffer q.s. Hz0
Dentrifrice (%) 3.00 0.221 30.00 39.00 1.70 3.87 1.29 0.22
Mouthwash (%I 1.000
0.055 0.050 16.000-20.000 lO.ooo 0.120 0.140
0.0005 0.115
There was a slight variation in the concentration of these ingredients in the various experimental products. Acute oral toxicity. Male and female Charles River CD rats weighing between 190 and 300 g were fasted 18-20 hr prior to dosing. Four dose levels of disodium etidronate, determined by using a constant geometric progression of doses, were administered by stomach tube to groups of 10 animals on the basis of an assumed toxicity and dose response curve. Dentrifrice formulations were dosed at a single level of 25 g/kg and mouthwash formulations at 25 ml/kg. LD50 values were calculated by the method of Thompson (1947) and Weil(1952). The same procedure was used for the determination of LD50 values in New Zealand rabbits of varying weight ranges. All animals were observed daily, and any changes in behavior were recorded. At the end of the 2 wk observation period, all surviving animals were necropsied and examined grossly. Blood and tissues from selected individuals were obtained for hematologic and histologic evaluation Emesis. Purebred beagle dogs ranging in age from 6 mo to 4 yr were fasted for approximately 19 hr and deprived of water for approximately 3 hr prior to treatment. Groups of 4 dogs (2 male, 2 female) were dosed po via stomach tube with disodium etidronate alone or in mouthwash or toothpaste formulations at 4 different dose levels using a constant geometric progression. The dogs were observed continuously for 4 hr after dosing, and the time and number of emetic responses were recorded. At the end of this period, food and water were offered * The pure salts of etidronic acid used in these studies were obtained from the Monsanto Company, 800 North Lindbergh Blvd., St. Louis, Missouri 63166.
DISODIUM
ETIDRONATE
SAFETY
663
ad libitum, and daily observations were continued for 2 wk. ED50 values were determined according to Weil(l952). Subchronic toxicity. Charles River CD rats were randomized according to weight, sex and litter and fed diets containing 0,0.2 and 1.O% disodium etidronate for a period of 91 days. All groups contained 20 males and 20 females; the average group weights did not differ more than 0.5 g (56.0 g for females and 60.0 g for males). Animals were housed individually in wire-bottom cages,and food and water were offered ad libitum. Body weights and food consumption were recorded weekly. Visual observations were made on individual animals daily, and all abnormal conditions were recorded. At the conclusion of the study (91 days), 5 males and 5 females from each group were randomly selectedfor necropsy. Liver and kidney to body weight ratios were calculated. Blood was taken for hematologic analyses,and organ specimenswere taken for histologic examination. All blood specimenswere examined for total red blood cell (RBC) count, total white blood cell (WBC) count, differential WBC count, hemoglobin and hematocrit. Tissues obtained for microscopic examination were liver, spleen, kidney, thyroid, trachea, esophagus, lung, heart, pancreas, adrenal, stomach, small intestine, urinary bladder, gonads and mesenteric lymph nodes. Tissues were fixed in Bouin solution, mounted in paraffin, trimmed and stained with hematoxylin and eosin. In a separatestudy, animals were fed diets containing 0 or 5.0 % disodium etidronate. The experiment was terminated after 1 wk because of severe weight loss and a large number of deaths in the test group. Five males and 5 females from both groups were necropsied, and blood and organ specimens were examined as above. Eye irritation. The potential for producing eye irritation was tested in New Zealand rabbits according to established procedures (Draize, 1959). Undiluted or appropriate dilutions of the test materials in aqueous solutions were instilled into the conjunctival sac of 1 eye in each of 3 rabbits. The untreated eye served as control. If rinsing was performed, 20 ml of lukewarm tap water was directed from a syringe into all parts of the eye within 4 set after instillation. Eyes were scored at 1 hr and at 1,2,3,4 and 7 days, and weekly thereafter for a maximum of 5 wk. The scalefor scoring ocular lesions was that of Draize (1959).The scoring method used in these studies differs from that recommended by the Federal Food and Drug Administration (1964) in that a slight dulling (bedewing) and/or stippling of the cornea is recorded as a grade 1. More severelesions of the cornea are scoredas 2 or more. In addition, anything more severe than a slight congestion of the iridial folds was scored as a grade 2 for iritis. The end result is that the scoring system used here results in slightly higher maximum averagescores(MAS) than would be expectedif one adhered to the FDA guidelines. Maximum average scores are determined by averaging the highest average score observed on any given day for the 3 animals in the test group, regardless of the time required to produce these scores. Oral mucosal irritation. The test method for detecting po mucosal irritation was developed in our laboratories to compare the irritant potential of po products. A number of years of experiencewith the rat as an experimental model had indicated that this animal is refractory to po mucosal irritation. This is not surprising since the rat oral mucosal epithelium is keratinized. In addition, experiments with a number of other species(guinea pig, hamster, rabbit) indicated that these specieswere not suitable for
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evaluating po mucosal irritation. In the beagle dog, however, by using exaggerated test conditions, it is possible to produce irritation with commercially available po products and to make comparisons among them. By combining rinse and no rinse treatments after repeated applications to the po mucosal it is possible to detect subtle differences between various test materials. Beagle dogs (3 in each group) ranging in age from 6 mo to 4 yr were used. One side of the gingiva, on the upper jaw of each dog, was treated by applying approximately 1 ml of a dentifrice or a mouthwash-saturated dental cotton roll3 approximately 1.5 in long. Immediately after applying the material, the mouth was held closed for 20-30 set to ensure good contact of the test material with the po mucosa. Two treatment schedules were used : (1) undiluted products were applied 5 times daily for 4 days, or (2) the same treatment was used and the treated areas was rinsed with 25-30 ml of tap water within 5 set after cessation of treatment. Gross observations were made prior to each treatment for signs of irritation (erythema) or epithelial sloughing. If epithelial damage was suspected, the test sites were treated with 2 % fluorescein and rinsed. Any well-defined areas of fluorescein retention indicated that epithelial damage had occurred. This response was considered to be the end point of the test, and treatment was discontinued. It is important to note that fluorescein does not stain injured tissue, but diffuses into superficial epithelial layers that are damaged. Judgments on the relative irritancy of test materials were made on the basis of the number of topical applications required to produce irritation. RESULTS
Acute Oral Toxicity (Rat) Disodium etidronate had an LD50 value of 1.34 g/kg when tested in the rat (Table 1). Lower LD50 values were found for trisodium, tetrasodium and tetrapotassium salts, LD50 values for the latter 2 compounds being significantly lower than for the disodium salt. The insoluble dicalcium salt was nontoxic (Table 1). Gross observations of the test animals during the test period failed to reveal a toxic effect. TABLE
1
ACUTE ORAL TOXICITY OF VARIOUS SALTS OF DISODIUM ETIDRONATE IN THE RAT
Concentration ( % w/w)
Salt Disodium Trisodium Tetrasodium Tetrapotassium Dicalcium
LD50 value (g/kg) 1.34 1.28 0.99 0.52
25 20 20
50 25
>ll
At necropsy, 3 of 10 surviving animals that had been dosed with high levels (1.60 and 1.14 g/kg) of disodium etidronate had pale (light gray), granular kidneys. Histologic examination revealed changes apparently involving all parts of the nephros, with as 3 Dental
Cotton
Roll, Johnson
& Johnson
Company.
665
DISODIUM ETIDRONATE SAFETY
much as 2@-40y0 of the kidney involved in all animals from the high levels. The damage was distinctly tubular, with very little involvement of the interstitium. The tubules were dilated and lined with low columnar epithelium which stained basophilic. There appeared to be a hyperplasia of tubule cells, with the nuclei larger and more numerous on cross section. The glomeruli involvement appeared to be secondary to the tubule involvement. Scattered incidences of hydropic degeneration were also observed. The responseappears to be somewhat similar to that produced by ethylenediaminetetraacetic acid. Kidneys from all animals dosed at the low level (0.814 g/kg) had m ild tubular changes as noted histologically. The only other m icroscopic finding of possible significance occurred in the stomachs of animals dosed with the 2 high levels (1.60 and 1.14 g/kg), in which mucosal irritation was noted in 4 of 10 animals examined. The kidney : body weight ratios of the 2 groups dosed with the high levels of the test material were significantly higher than those of animals receiving 0.814 g/kg. These values (2 high groups) were also higher than the normal range. All other average values were within the normal ranges established in previous studies of this type using Charles River CD rats. Experimental dentifrices (3 % disodium etidronate) and mouthwashes (1% disodium etidronate) had LD50 values of >25 g/kg and >25 m l/kg, respectively. Gross and m icroscopic observations of these animals did not reveal any abnormalities. Acute Oral Toxicity (Rabbit) Table 2 presents the data from a series of experiments to establish the LD50 value of disodium etidronate in the rabbit. Results of these tests indicate that susceptibility of the rabbit to the acute effects of disodium etidronate is a function of age, weight and sex, The stress of pregnancy apparently does not significantly alter the LD50 value of the material in this species.The LD50 value in rabbits was found to be somewhat lower in males than in females and lower in mature animals (>3300 g) as opposed to immature animals (-2500 g). TABLE 2 ACUTE ORAL TOXICITY OF DISODIUM ETIDRONATE IN THE RABBIT
No. of animals/group”
Animal condition
2F, 2M 2F, 2M 4F
Immature Immature Mature, virgin Pregnant Mature -* -*
4F 4M 4F, 4M
8F, 4M
Body weight (g> 2250-2800 2250-2800 >3300 13300 >3300 2250-2800 >3300
LD50 (g/W
Confidence lim its
1.14 1.05 0.814 0.668 0.581 1.10 0.70
0.829-1.56 0.905-l .21 0.642-l .03 0.542-0.872
0.424-0.799 0.961-1.52 0.561-0.831
u Four groupsof animalsin eachtest. b Data from all experiments combined according to body weight.
Nearly all the deaths in the test groups occurred in less than 24 hr, and none died after 4 days. All surviving animals were observed for 2 wk after dosing. The only symptom noted in these animals during this period was diarrhea. This occurred from
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AND NEWMANN
approximately 4 hr through 48 hr post treatment; severity was generally in proportion to the dose level. All surviving animals were sacrificed at 2 wk; blood was taken for hematologic analyses, and tissues were taken for microscopic examination. Several animals in each test group appeared to have mild gastric irritation. No other unusual lesions were observed grossly. No unusual lesions were seen microscopically in any of the tissues. About 50 % of the surviving animals were found to have kidney lesions resembling chronic interstitial nephritis, and these were scattered throughout all groups. Because chronic interstitial nephritis is common in the rabbit, it is difficult to distinguish between this lesion and one that could be test related. Group average values (blood and organ: body weight ratios) appeared to be normal for animals of this age and species. Emesis Disodium etidronate produces an immediate emetic response in some animals, and because of this factor it was not possible to determine well defined LD50 values in the beagle dog. The ED50 value for disodium etidronate was 84.8 mg/kg with confidence limits of 31.8-226 mg/kg. When dosed at higher levels (0.25-10 g/kg) varying degrees of stomach and intestinal irritation were noted, severity being dose-dependent. A toxic response characterized by bloody emesis, diarrhea and loss of appetite was noted in male dogs at levels of 0.50 g/kg and in females at levels of 1.Og/kg. Although the LD50 value for disodium etidronate could not be calculated precisely, it was estimated to be about 1.O g/kg on the basis of early deaths and necropsy of moribund animals dosed with the higher levels (1.0-10 g/kg). Table 3 presents the data from the study to determine the ED50 value of the experimental toothpaste containing 1 or 3 % disodium etidronate and the experimental mouthwash containing 1% disodium etidronate. Disodium etidronate increased the emetic properties of the dentifrice; therefore, the ED50 values were inversely related to the concentration of disodium etidronate in the formulation. In all cases, the test animals appeared normal after cessation of vomiting. The experimental mouthwash was found to have an ED50 value of 5.10 ml/kg. TABLE
3
EMETICPROPERTIESOFANEXPERIMENTALDENTIFRICE AND MOUTHWASH CONTAINING DISODIUM ETIDRONATE
ED50
Mean onset of emesis in dogs responding
Test material
(g/kg)
Confidence limits
Dentifrice with 3 % disodium etidronate Dentifrice with 1% disodium etidronate Control dentifrice” Mouthwash with 1% disodium etidronate
0.681
0.364-1.27
19 min
1.21
0.825-l .78
28 min
6.81
4.89-5.31
5.10b
1 hr, 10 min 37 min
a Similarto experimentaldentifrice,but without disodiumetidronateand NaF. b Expressedas ml/kg.
DISODIUM
ETIDRONATE
667
SAFETY
Two beagle dogs (1 male, 1 female) were also dosed with each of the dentifrice formulations at levels of 10 g/kg. The only gross sign observed was emesis. At 1 wk postdosing, each animal was necropsied. There was no microscopic lesion that could be attributed to treatment, and all blood values were within the normal range for these animals. Subchronic Toxicity
Groups of 20 male and 20 female rats were fed diets containing varying levels of disodium etidronate (Table 4). The test group fed 5% disodium etidronate was terminated after 1 wk because of mortality and severeweight loss. Necropsy of the surviving animals from this test group revealed the presence of stomach ulcers and hyperemia. Microscopic examination revealed inflammation and dilated glands and capillaries which confirmed the gross findings of gastritis. There was also a generalized erosion of the glandular mucosal epithelium in the stomach of some animals (i.e., rough appearance with local areas of sloughing). The gastritis generally affected both the forestomach and the glandular portion of the stomach. The kidney : body weight ratio was high and may have been due to a poor growth rate. The total white blood cell count was high, probably the result of the stomach irritation. The neutrophil count was high and the lymphocyte count low, indicating the acute nature of the gastric distress. TABLE 4 91 DAY Dietary level 0
Sex F M
0.2%
F
1.0%
F M
0”
F M
M
5.0%
F
M
FEEDING STUDY IN RATS WITH DWIDIUM
ETIDRONATE
Average body weight gain Cd
Organ/body weightb Liver
Kidney
223 438 234 430 223 432
3.26 3.31 3.28 3.49 3.67 3.41
0.72 0.67 0.65 0.82c 0.70
34 48 -6 3
4.44 4.34 4.28 4.58
0.95 1.48 1.55
0.64
1.11
No. of survivors 20 20 19 20 20 20 20 20 12 16
u Groupsterminatedafter 1 wk. b Expressedas % of body weight. ’ Significantlydifferentfrom controls(P > 0.05). The survival rate in the other test groups (0.2 and 1.0% disodium etidronate) was good and gave no indication of a toxic effect. There were no gross necropsy lesions attributed to toxicity and, except for a slightly elevated kidney : body weight ratio in females on the 1 y0 disodium etidronate, all parameters in the study were normal.
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Eye Irritation
Eye irritation results are important in the safety assessmentof a dentifrice product from 2 standpoints. First, they provide data relative to the potential for actual eye irritation in case of accidental instillation into the eye during normal use. Second, they provide an indication of the irritation to a very sensitive mucosal membrane. Since the product comes in contact with po mucosa, this latter information is of value. It is necessary,however, to compare the irritation produced with other products being used safely by the consumer. Disodium etidronate instilled into the rabbit eye produced only a mild conjunctival irritation. The eyes were completely normal by day 4 (Table 5). Because the irritancy produced was quite mild, there was no apparent benefit from rinsing. Dissolving the material (10 %) in water prior to instillation did not change the irritation potential of disodium etidronate. TABLE 5 EYE IRRITATION IN THE RABBIT WITH DISODIUM ETIDRONATE AND THE EXPERIMENTAL DENTRIFRICE AND MOUTHWASH
Test material Disodium etidronate Disodium etidronate Disodium etidronate Disodium etidronate dentifrice Disodium etidronate dentifrice Fluoride dentifriceb Fluoride dentifrice High flavor dentifrice’ High flavor dentifrice Disodium etidronate mouthwash Control mouthwash
Maximum Comeal average Treatment’ scores involvement 3mgNR 3mgR 10% NR 3mgNR
No. of eyesnormal in No. of days
6.6 5.6 4.0 21.6
O/3 O/3 O/3 213
3 in 2 lin2;1in3;1in4 2inl; 1 in2 2in3;1in4
9.0
O/3
2 in 2; 1 in 3
3mgNR 3mgR 3mgNR 3mgR 100% NR
19.6 6.6 40.6 22.0 2.0
113 O/3 313 213 O/3
1 in4;2inl linl;lin2;1in3 1 in 7; 2 in 14 1 in 3; 1 in 7; 1 in 14 3 in 1
lOO%NR
2.0
O/3
3 in 1
3mgR
no rinse;R, rinse. b Stannousfluoride. c Chloroform. ’ NR,
Instillation of the undiluted experimental dentifrice (3 % disodium etidronate) into the rabbit eye produced transient alteration of the precorneal aqueous film and cornea1 epithelium and moderate degreesof conjunctival irritation that were transient, the eyes returning to normal by day 4. In this case, rinsing the eyes within 4 set effectively alleviated the mild irritative effects. Undiluted experimental mouthwash containing 1% disodium etidronate produced only slight conjunctivitis that cleared readily. Comparison of the eye irritation results with the experimental dentifrice to commercially available dental formulations indicate that it is similar to these products with regard to primary irritant properties; the disodium etidronate formulation appears to be less irritating than a dentifrice flavored with chloroform.
DISODIUM
ETIDRONATE
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SAFETY
Oral Mucosal Irritation
Table 6 summarizes the results of the experiments on po mucosal irritation. The experimental dentifrice containing 3 ‘A disodium etidronate was no more irritating to po mucosa than was the control formulation, but both these formulations were more irritating than fluoride dentifrice when compared under “no rinse” conditions. When rinsing was employed, the results with all formulations were similar. The chloroform flavored dentifrice was more irritating than any of the other test materials when rinsing was employed, and irritant effects were noted after only 9 applications. TABLE 6 ORAL MUCOSAL IRRITATION PRODUCED BY THE EXPERIMENTAL DENTIFRICE AND MOUTHWASH
Test material
TreatmeW
Disodium etidronate dentifrice Disodium etidronatedentifrice Control dentifrice Control dentifrice Fluoride dentifrice’ Fluoride dentifrice High flavor dentifriced High flavor dentifrice Disodium etidronatemouthwash Control mouthwash
No. of applicationsto produce irritationb
x/day, NR x/day, R x/day, NR x/day, R x/day, NR x/day, R x/day, NR x/day, R 5 x/day, NR
5 5 5 5 5 5 5 5
5 x/day, NR
4 >20 4 >20 >20 >20 4 9 20 >20
’ Maximum number of treatments = 20. NR, no rinse; R, rinse. b Epithelial sloughing detected with fluorescein. ’ Stannous fluoride. d Chloroform.
Treatment ofthe po mucosa with a mouthwash formulation containing 1% disodium etidronate (Table 6) produced results comparable to a similar commercial formulation without disodium etidronate. These results indicate that the experimental products are comparable to commercially available formulations with regard to po mucosal irritation in the dog. DISCUSSION
The acute po LDSO value of disodium etidronate in Charles River CD rats is 1.34 g/kg, and a similar po LDSO is noted in the rabbit (1.14 g/kg). Because of its potent emetic properties, it was not possible to determine the LD50 value in dogs. When incorporated into a dentifrice or mouthwash formulation, this latter property of disodium etidronate may provide an added safety factor in case of ingestion by a child. It is noted, however, that the experimental po hygiene products are nontoxic and have LD.50 values in rats of >25 g/kg (dentifrice) and ~-25 ml/kg (mouthwash). Toxicity of disodium etidronate in rats given an acute po dose of 1.14 g/kg or greater was expressed in gastric irritation and kidney lesions. The changes noted in the kidney
670
NIXON,
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AND NEWMANN
were no different from those attributed to chelating agents and phosphates (Reuber and Lee, 1966; BIBRA Bulletin, 1964) and do not appear to be due to an effect that is unique to disodium etidronate. There were no lesions noted microscopically in rabbits that were attributed to disodium etidronate treatment. More definitive data on the lack of metabolic alteration of the compound are published elsewhere (Michael et al., 1972). When fed at high levels (5 %) in the diet to rats, disodium etidronate was toxic, producing morbidity and moribundity. At the next lower dietary level (lx), there was an indication that the kidney was affected (increased kidney:body weight) in female rats, but all animals in the test group appeared to be healthy. The addition of 0.2 % disodium etidronate to the rats’ diet gave no indication of a toxic effect. If a 10 kg child ingested 1 g of toothpaste per day containing 3 % disodium etidronate or 3 ml of mouthwash containing 1 ‘A disodium etidronate, the ingestion of disodium etidronate would be 30 mg/day, or 3 mg/kg/day. Since the rat ingests about 130 g/kg/day of diet during the first wk of a testing period, the level of 0.2 % (260 mg of disodium etidronatejkgjday) represents approximately a go-fold factor over the maximum probable ingestion that can be predicted. It should be recognized that as the child becomes older the probable ingestion of po hygiene products, particularly in relation to body weight, is drastically reduced, whereas the rat ingested approximately the same quantity of food throughout the entire period of rapid growth. Data obtained in these experiments and in chronic tests (to be published elsewhere) indicate that disodium etidronate would be safe for human usage in mouthwash (1%) and dentifrice (3 %). ACKNOWLEDGMENT The authors wish to thank Mr. W. R. King, who performed all hematologic evaluations and histologic examination of tissues during the various phases of the study. All development and formulation work on the experimental dentifrice and mouthwash was by Dr. J. S. Widder, whose efforts made these studies possible. REFERENCES
C. A. L., DONATH, A., MACAGNO, F., PREISIG, R., FLEISCH, H., and FRANCIS, M. D. (1969). Diphosphonates in the treatment of myositis ossificans. Luncet 7625, 845. BIBRA (1964). Toxicity studies on phosphates.Food Cosmet. Toxicol. 2, 147-154. DRAIZE, J. H. (1959). Dermal toxicity. In “Appraisal of the Safety of Chemicals in Foods, Drugs and Cosmetics,” pp. 46-59, Association of Food and Drug Officials of the United States, Austin, Texas, AFDO. FLEISCH, H., RUSSELL, R. G. G., and FRANCIS, M. D. (1969). Diphosphonates inhibit hydroxyapatite dissolution in vitro and bone resorption in tissue culture and in vivo. Science 165, 1262-1264. FRANCIS, M. D. (1969). Inhibition of calcium hydroxyapatite crystal growth by polyphosphonates and polyphosphates Calcif. Tissue Res. 3, 151-162. FRANCIS, M. D., RUSSELL, R. G. G., and FLEISCH, H. (1969). Diphosphonates inhibit formation of calcium phosphate crystals in vitro and pathological calcification in vivo. Science 165, 1264-1266. Illustrated Guide for Grading Eye Irritation by Hazardous Substances (1964). U.S. Dept. of Health, Education, and Welfare, Food & Drug Administration. U.S. Gov. Printing Office, Washington, D. C. MICHAEL, W. R., KING, W. R., and WAKIM, J. M. (1972). Metabolism of disodium ethane-lhydroxy-l,l-diphosphonate (disodium etidronate) in the rat, rabbit, dog and monkey. Toxicol. Appl. Pharmacol. 21, 503-515.
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NOLEN, G. A., and BUEHLER, E. V. (1971). The effects of disodium etidronate on the reproductive functions and embryogeny of albino rats and New Zealand rabbits. Toxicol. A&. Pharmacol. 18, 548-561. REUBER, M. D., and LEE, C. W. (1966). Calcium disodium edetate nephrosis in inbred rats. Arch. Environ. Health 13, 554-557. SMITH, R., RUSSELL,R. G. G., and BISHOP,M. (May 8, 1971). Diphosphonates and Paget’s disease of bone. Lancet, 945-947. THOMPSON, W. R. (1947). Use of moving averages and interpolation to estimate median-effective dose. Part I. Bacterial. Rev. 11, 115-145. WEIL, C. S. (1952). Tables for convenient calculation of median-effective dose (LDSO or ED50) and instructions in their use. Biometrics 8, 249-263. WEISS,I. W., FISHER,L., and PHANG,J. M. (1971). Diphosphonate therapy in a patient with myositis ossificans progressiva. Ann. Int. Med. 74, 933-936.