Fluoride

Health Effects*

Essentiality

Although Health Canada has classified fluoride as an essential element in the past,⁴⁷ it now recommends that fluoride requirements should "only be based on the beneficial effect on dental caries" and notes that "attempts to demonstrate its essentiality for growth and reproduction in experimental animals have not been successful."⁴⁸ Similarly, the U.S. National Research Council considers fluoride to be a "beneficial element for humans."⁴⁹

Absorption, Distribution and Excretion

Ingested NaF is rapidly absorbed from the gastrointestinal tract.^50-53 The extent of dietary fluoride absorption was greater than 90% in balance studies with human volunteers.^54,55 In both adults and children, peak plasma levels were reached 30-60 minutes after the ingestion of doses ranging from 0.5 to 10 mg fluoride (as NaF).^56-58 The water solubility of fluoride compounds can influence their absorption; NaF is more readily ^11,59 absorbed than the less soluble CaF₂and MFP. The plasma half-life of fluoride in humans and rabbits ranges from 2 to 11 hours following single or multiple oral doses of NaF (3.0-40 mg fluoride).^57,60 Up to 75% of absorbed fluoride may be deposited in calcified tissues, with the highest deposition found in children with active bone growth or individuals consuming non-fluoridated drinking water.⁶¹ Approximately 99% of total body fluoride is localized in calcified tissues (i.e., bones and teeth), where it is substituted for hydroxyl ions (OH^-) in hydroxyapatite, forming fluorapatite.^62-65 The dose, duration of exposure and turnover rate of skeletal components all affect calcified tissue fluoride concentrations.^54,66 Although bone fluoride concentrations increase with age, the amount retained on a daily basis is inversely related to age; this is due to the greater surface area for fluoride uptake in hydrated young bone and the increased rate of resorption over formation in the elderly.⁶⁷ Fluoride can be mobilized from bone through a relatively rapid interstitial ion-exchange mechanism or a much slower bone remodelling process.⁶⁸ Many of the factors affecting the uptake and retention of fluoride in bone also affect fluoride concentrations in teeth, with the exception that tooth enamel and dentin do not undergo continuous remodelling.¹¹ Enamel fluoride concentrations decrease with distance from the tooth surface and also vary with location, surface wear, age and degree of exposure to systemic and topical fluorides.^69,70

Mean iliac bone fluoride concentrations recorded for adults (60 years of age) consuming non-fluoridated (<0.1 ppm) and fluoridated (0.97 ppm) drinking water were 351 mg/kg (106-790 mg/kg) and 1090 mg/kg (347-2360 mg/kg), respectively.⁷¹ Surface enamel fluoride concentrations were reported to be 740-1400 mg/kg and 1351-2100 mg/kg for adults 20 years of age or older from communities with drinking water fluoride concentrations of 0.1 and 1 ppm, respectively.⁷² The concentration of fluoride in dentin is generally 2-3 times higher than that in enamel.⁷³ Fluoride is excreted primarily via the urine, with perspiration, saliva, breast milk and faeces making smaller contributions to daily body clearance.^67,74-76 In adult humans, approximately 50-75% of an oral dose of fluoride appears in the urine within 24 hours after ingestion.^57,77,78 Under conditions of relatively constant exposure, urinary excretion correlates well with drinking water fluoride levels and is often used as an indicator of exposure.¹¹ Fluoride is readily transferred from mother to foetus across the placenta.¹¹

Acute and Chronic Toxicity

In humans, acute ingestion of fluoride can result in nausea, vomiting, abdominal pain, diarrhoea, fatigue, drowsiness, coma, convulsions, cardiac arrest and death.^{10,11,63,67,79} Effects are most severe following ingestion of the more soluble fluoride salts.¹¹ The LD₁₀₀ for fluoride in the average adult has been estimated to be 32-64 mg/kg bw (as NaF), and deaths in children have been reported after ingestion of as little as <5-30 mg/kg bw.^11,67 Oral fluoride LD₅₀s in rats and mice range from 25.5 to 45.7 mg/kg bw for stannous fluoride 80-82 (SnF₂), from 31 to 101 mg/kg bw for NaF^82-86 and from 54 to 102 mg/kg bw for MFP.^82,84,86,87 In a comprehensive National Toxicology Program (NTP) chronic toxicity/carcinogenicity bioassay, groups of male and female F344/N rats and B6C3F₁mice (70-100 per sex per dose) were exposed to drinking water containing 0, 25, 100 or 175 ppm NaF for 2 years (estimated intakes 0.2, 0.8, 2.5 and 4.1 mg/kg bw per day for male rats, 0.2, 0.8, 2.7 and 4.7 mg/kg bw per day for female rats; 0.6, 1.7, 4.9 and 8.1 mg/kg bw per day for male mice, 0.6, 1.9, 5.7 and 9.1 mg/kg bw per day for female mice). Bone ash fluoride content increased in both species during the course of the study, with terminal concentrations ranging from 0.44 (controls) to 5.26 (high-dose group) µg/mg in male rats, from 0.55 to 5.55 µg/mg in female rats, from 0.72 to 5.69 µg/mg in male mice and from 0.92 to 6.24 µg/mg in female mice. The high-dose female rats had a significantly higher incidence of osteosclerosis and a slight but significant increase in brain to body weight ratio compared with controls. Serum alkaline phosphatase activity was increased in high-dose male mice after 66 weeks and in high-dose female mice after 27 and 66 weeks.⁸⁸ Estimated no-observed-adverse-effect levels (NOAELs) were 2.7 and 4.1 mg/kg bw per day for the female and male rats, respectively, and 5.7 and 4.9 mg/kg bw per day for the female and male mice, respectively.³⁹ In another chronic toxicity/carcinogenicity bioassay, NaF was administered via the diet to groups of male and female Sprague-Dawley albino rats (70 per sex per dose) and CD-1 mice (60 per sex per dose) for 95 weeks (male rats and mice), 99 weeks (female rats) or 97 weeks (female mice). Estimated fluoride intakes for both rats and mice were 0.1 (low-fluoride diet control), 1.8, 4.5 and 11.3 mg/kg bw per day. At the end of the study, bone ash fluoride content ranged from 0.5 (controls) to 16.7 (high-dose group) µg/mg in male rats, from 0.5 to 14.4 µg/mg in female rats, from 1.5 to 13.2 µg/mg in male mice and from 1.0 to 10.6 µg/mg in female mice. Increased subperiosteal hyperostosis in the medium-and high-dose rats was the most notable non-neoplastic skeletal effect observed in the study. Other effects included reduced weight gain in the high-dose rats and hyperkeratosis and acanthosis in the stomachs of medium- and high-dose rats.^89,90 For the rats, a NOAEL was estimated at 1.8 mg/kg bw per day.³⁹ Most of the available studies of potential non-neoplastic human health effects from chronic fluoride ingestion have focused on adverse effects on the skeleton, principally skeletal fluorosis and fractures. The data consist primarily of epidemiological studies of populations exposed to various concentrations of fluoride in drinking water, case reports of individuals exposed to drinking water containing elevated concentrations of fluoride and clinical studies of osteoporosis patients treated with NaF.

Skeletal fluorosis is an excessive accumulation of fluoride in bone associated with increased bone density and outgrowths (exostoses).¹⁰ Fluoride incorporated into bone (i.e., as fluorapatite) produces a crystal lattice that undergoes less resorption (i.e., less soluble; more stable) and has an increased compression strength, but is more brittle and has a decreased tensile strength.^8,65 Characteristic signs and symptoms of skeletal fluorosis range from asymptomatic radiographic enlargement of spinal trabeculae in the preclinical form (stage I) to the severe calcification of ligaments, spine and joint deformities, muscle wasting and neurological defects observed in crippling skeletal fluorosis (stage III).⁸ The more severe symptoms tend to be associated with the vertebral column in the lower, weight-bearing parts of the body.⁹¹ Ashed bone fluoride concentrations may range from 3500 to 5500 mg/kg in stage I skeletal fluorosis to >8400 mg/kg in stage III.⁸ Age, nutritional deficiencies, renal insufficiency, bone remodelling and the dose and duration of fluoride exposure can all influence the occurrence of the disease.^{8,10,11,63,64} Studies from the United States showed no evidence of skeletal fluorosis following the consumption of drinking water containing fluoride concentrations of 1.2 and 3.3-6.2 mg/L for 10 years and a lifetime, respectively.^92-95 In an older study conducted in Texas, radiographic evidence of osteosclerosis but no clinical signs of skeletal fluorosis were reported for 18% (n = 89) of people from a small town (Bartlett) who consumed drinking water containing 8 mg/L fluoride for an average of 37 years, whereas the incidence was only 4% (n = 101) for the population of a control town (Cameron) where the drinking water contained 0.4 mg/L fluoride.⁹⁶ X-rays of residents of Texas and Oklahoma who had consumed drinking water containing 4-8 mg/L fluoride indicated 23 cases of osteosclerosis "due to fluoride" but no cases of skeletal fluorosis.⁹⁷ In a recent review of available radiographic studies, Kaminsky et al.⁶³ concluded that for individuals in the United States who consumed drinking water containing -4.0 mg/L fluoride, there was no evidence of the skeletal changes associated with skeletal fluorosis.

Endemic crippling skeletal fluorosis has been reported in adults and children from areas of India, Africa and China where the fluoride concentrations in drinking water ranged from 3 to >20 mg/L.^{8,11,63,98,99} As not all residents of these areas show signs of the disease, other factors, such as dietary deficiencies (e.g., protein, calcium, etc.) and other sources of daily fluoride intake, may be contributing to the development of the disease.^11,99,100 A recent North American case report of stage I skeletal fluorosis involved a 54-year-old woman from Oklahoma who consumed drinking water containing 7-8 mg/L fluoride for 7 years.¹⁰¹ Crippling skeletal fluorosis has been described in only five North American case reports over the past 40 years, all from the southwestern United States: three were associated with long-term consumption (40->60 years) of drinking water containing elevated concentrations of fluoride (2.4-7.8 mg/L),^102-104 one involved a history of geophagia (eating soil)¹⁰⁵ and one gave no details on fluid or food consumption.¹⁰⁶ Although a recent review estimated the total fluoride intake for some of these patients to be 15-20 mg/d for 20 years⁸ (estimated fluoride intake: 215-285 µg/kg bw per day³⁹), only one case report, that of a 40-year-old woman with a history of geophagia, gave an estimate of daily fluoride intake (1.4 mg/d from drinking water, 4.2 mg/d from tea and 10.0 mg/d from soil consumption).¹⁰⁵ Several of the cases were complicated by pre-existing or associated renal disease, polydipsia and the daily consumption of large quantities of tea.^102-105 Radiographic signs of stage I skeletal fluorosis were observed for 8/25 post-menopausal women treated for osteoporosis with a combination of 40-60 mg/d NaF (estimated fluoride intake: 260-389 µg/kg bw per day³⁹), calcium and vitamin D₂for a period of 18 months. Pa- tients treated only with calcium and vitamin D₂showed no evidence of skeletal fluorosis.¹⁰⁷ Kleerekoper and Balena¹⁰⁸ reported that, depending on whether there is concurrent supplementation with calcium and vitamin D₂, mild, asymptomatic osteomalacia may occur in osteoporotic patients administered NaF doses above 40 mg/d (estimated fluoride intake: 260 µg/kg bw per day³⁹).

Possible associations between the occurrence of skeletal fractures (predominantly hip fractures in elderly persons) and the exposure of populations to fluoridated drinking water have been examined in a number of epi-demiological studies of the ecological or geographical correlation type. Studies of this type can suffer from a number of weaknesses, such as a lack of information on individual fluoride intake (i.e., intake from other sources, such as food, dental products, etc.) within the fluoridated and control communities; geographical differences in various factors (e.g., smoking, lifestyles, environmental and occupational exposures, genetics, etc.) that could affect the occurrence of fractures; uncontrolled migration between fluoridated and control areas; and geographical variations in the quality of disease diagnosis and reporting.^8,39,109 In a U.S. study, 216 counties with drinking water containing 0.7-1.2, 1.3-2.0, 2.1-3.9 or >4.0 mg/L fluoride (fluoridated) were compared with 95 counties where the drinking water contained <0.4 mg/L fluoride (non-fluoridated) for hip fracture hospitalization rates (1985-1986) in men and women over 65 years of age. The ratios comparing hip fracture rates in the fluoridated versus non-fluoridated counties increased from 1.016 for the 0.7-1.2 mg/L counties to 1.224 for the >4.0 mg/L counties, but they were significant (p < 0.01) only for counties with fluoride concentrations at or above 1.3-2.0 mg/L.¹¹⁰ May and Wilson¹¹¹ found that the hip fracture rate in men and women older than 65 years from each of 438 U.S. counties with populations greater than 100 000 was positively correlated with the fraction of the population receiving fluoridated drinking water (up to 1.0 mg/L fluoride). However, a further analysis of 51 counties showed no relationship between hip fracture rate and duration of exposure, as the fracture rate was highest in counties with up to 10 years of exposure to fluoridated water, 20% lower for counties with 11-18 years of exposure and intermediate for counties with more than 18 years of exposure.¹¹¹ Jacobsen et al.,¹¹² employing a time trend analysis approach, reported a slightly but significantly higher relative risk (RR) of hip fracture for white men (RR = 1.08, 95% confidence interval [CI] = 1.06-1.1) and white women (RR = 1.17, 95% CI = 1.13-1.22) older than 65 who resided in fluoridated counties (n = 129) in which the percentage of the population receiving fluoridated (1.0 mg/L) drinking water increased from <10% to >66% over 3 years compared with counties (n = 194) where >90% of the population was served with non-fluoridated (<0.3 mg/L) drinking water. Counties starting fluoridation less than 5 years before the study had the highest fracture rates, whereas the rates declined for communities with progressively longer exposure periods.¹¹² A second study compared hip fracture rates in Rochester, Minnesota, 10 years prior to the initiation of drinking water fluoridation in 1960 and 10 years after. A total of 651 incident hip fractures were recorded in the 20-year period, 268 in men and 383 in women. Fluoridation was not associated with a risk of hip fracture in men and women 50 years of age or older (RR = 0.6, 95% CI = 0.42-0.85 in women; RR = 0.78, 95% CI = 0.73-1.66 in men).¹¹³ A comparison of hospital discharge rates between 1984 and 1990 for hip fracture in 65-year-olds between a fluoridated (1.0 mg/L) community and two non-fluoridated (<0.3 mg/L) communities in Utah gave age-adjusted risk ratios of 1.41 (95% CI = 1.00-1.81) for men (fluoridated: n = 19; non-fluoridated: n = 32) and 1.27 (95% CI = 1.08-1.46) for women (fluoridated: n = 65; non-fluoridated: n = 130) in the fluoridated versus non-fluoridated communities.¹¹⁴ Suarez-Almazor et al.¹¹⁵ conducted a study of hip fracture hospitalization rates in Alberta between 1981 and 1987 in men and women (-45 years of age) from Edmonton, where fluoridation (to 1 mg/L) was initiated in 1967, and Calgary, where the drinking water contained 0.3 mg/L fluoride. Men from Edmonton in the age groups -45 or -65 years of age had significantly higher hip fracture rates (i.e., 12 and 13%, respectively; n = 827) than the corresponding age groups from Calgary (n = 700). However, there were no significant differences between the two cities when all age groups of women were considered or when both sexes were combined.¹¹⁵ A more powerful analytical approach was used to study the incidence of skeletal fractures in women from Iowa who lived for at least 5 years in either a fluoridated (i.e., 1 mg/L fluoride) or a naturally elevated fluoride (i.e., 4 mg/L fluoride) community.^93,116 Individual interviews and examinations were conducted in 1983-1984 to obtain detailed information on such factors as fracture history, water consumption, oestrogen use and bone mass and density. The estimated mean intake of fluoride from water-based beverages in the elevated-fluoride community was 72 µg/kg bw per day. For post-menopausal women (age 55-80 years), the incidence of skeletal fractures over the previous 10 years was significantly greater (p = 0.0001) in the elevated-fluoride (57/200) versus the fluoridated community (20/151). However, there was no significant difference between the two communities for fracture incidence in the 20-35 year age group (pre-menopausal).⁹³ Based on follow-up interviews and examinations 5 years later (1988-1989), the relative risk (age and body size adjusted) for any fracture in the post-menopausal group from the elevated-fluoride community (31/163) compared with the fluoridated community (11/121) was 2.11 (95% CI = 1.0-4.4). Hip fracture incidence for the post-menopausal group in the elevated-fluoride community was 5/163 compared with 0/121 for the fluoridated community. The 5-year (1983-1984 to 1988-1989) relative risk of fractures specifically in the hip, wrist or spine in the elevated-fluoride versus the fluoridated community was 2.7 (95% CI = 0.16-8.28) for the pre-menopausal and 2.2 (95% CI = 1.07-4.69) for the post-menopausal groups. The pre-menopausal women from the elevated-fluoride community experienced a greater loss of radial bone mass during the 5-year period than women in the same age group from the fluoridated community (i.e., 3.6% vs. 2.1%; p = 0.08).¹¹⁶ Cauley et al.¹¹⁷ reported no association between exposure duration and bone mineral density (with or without adjustment for age and body mass) or fracture history in a study in which they obtained details on the source of drinking water, bone mineral density and hip and wrist fracture histories for 1878 white women, ages 65-93, from the Pittsburgh, Pennsylvania, area. Less than half the women were exposed to fluoridated water (1.0 mg/L); for those exposed, the mean duration was 6 years (range 0-38 years).¹¹⁷ The incidence of skeletal fractures has been examined in several clinical case studies of osteoporosis patients undergoing treatment with NaF for extended periods of time. Inkovaara¹¹⁸ found a 7.5% incidence of hip fracture in male and female geriatric patients (n = 146) administered NaF at 25 mg/d (estimated fluoride intake: 162 µg/kg bw per day³⁹) for 5 months or 25 mg NaF twice weekly for 3 months, compared with a 3.0% incidence in controls (n = 169; p < 0.1). Inkovaara¹¹⁸ also commented on an additional study that reported an incidence of hip fracture of 5/16 in osteoporosis patients (mean age 70) receiving 40-80 mg/d NaF (estimated fluoride intake: 260-520 µg/kg bw per day³⁹) supplemented with calcium and vitamin D for 4 years compared with an incidence of 0/8 for controls. Post-menopausal women (mean age 67 years) treated with 40-60 mg/d NaF (estimated fluoride intake: 260-389 µg/kg bw per day³⁹) supplemented with calcium and vitamin D for 18 months were observed to have an increased incidence of hip fracture compared with patients receiving only calcium and vitamin D (6/25 vs. 1/24; p > 0.05).¹⁰⁷ Mamelle et al.¹¹⁹ found no significant difference in the occurrence of hip fractures between a group of 257 osteoporosis patients of both sexes (mean age 70.1 years) treated with 50 mg/d NaF (estimated fluoride intake: 324 µg/kg bw per day³⁹), calcium and vitamin D for 2 years and a group of 209 control patients treated with a variety of non-fluoride regimes during the same period. The administration of 50 mg/d NaF combined with calcium or calcitriol to 35 women (68 years of age) for 12 or 13 months resulted in a hip fracture incidence of 5/35 compared with an incidence of 0/43 in patients administered only calcitriol or a placebo (p = 0.015).¹²⁰ In a more recent study, women of median age 68 years who were administered 75 mg/d NaF (estimated fluoride intake: 486 µg/kg bw per day³⁹) supplemented with calcium for 4 years experienced significantly (p < 0.01) more non-vertebral fractures than controls treated with calcium alone. However, the difference in hip fracture incidence between the two groups was not significant.¹²¹

Reproductive Toxicity and Teratogenicity

Several researchers have examined the effects of relatively high doses of NaF administered in drinking water or in the diet on reproductive function in experimental animals. In a study in which weanling Swiss-Webster female mice were fed a low-fluoride diet (0.1-0.3 ppm) and administered drinking water containing up to 200 ppm fluoride (approximately 40 mg/kg bw per day from drinking water) for 5 weeks prior to and during breeding, maternal growth, survival and litter production were reduced or inhibited.¹²² A multigeneration mouse study showed no significant difference in reproductive function for females fed a diet containing <0.5, 2 or 100 ppm fluoride.¹²³ No pregnancies or embryo implantations were reported in groups of Swiss albino mice orally dosed with fluoride at either 5.2 or 17.3 mg/kg bw per day on days 6-15 after mating.¹²⁴ Male rabbits fed NaF at doses of 20 or 40 mg/kg bw per day for 30 days had decreased body weights and significantly lowered sperm motility, sperm counts and fertility rates.¹²⁵ Similarly, male Swiss mice fed NaF at doses of 10 or 20 mg/kg bw per day for 30 days exhibited sperm abnormalities, significantly decreased sperm motility and counts and a loss of fertility.¹²⁶ In a study in which male and female pastel mink were exposed for 7 months to a diet containing 35 ppm fluoride supplemented with additional fluoride in doses ranging from 33 to 350 ppm, survival was reduced in offspring from dams fed the highest dose of supplemental fluoride, and body weights were increased in offspring of dams receiving 60 and 108 ppm fluoride; litter sizes and gestation periods were not altered by supplemental fluoride.¹²⁷ The oral administration (oral dosing or administration in drinking water) of fluoride at approximately 4.5-200 mg/kg bw per day has also been reported to produce a number of adverse effects on reproductive organs, including a cessation of spermatogenesis and decreases in sperm in the vas deferens and the density of epididymal epithelial cilia in rabbits,¹²⁸ increases in seminal vesicle and prostate weights and decreases in the height of the testicular germinal epithelial cells and the cauda and caput epididymis epithelial cells in mice^129,130 and an absence of spermatocyte maturation and degeneration and necrosis of the testicular tubules in mice.¹³¹ Human studies of the reproductive and developmental effects of ingested fluoride have included a number of case-control and ecological studies examining possible associations between exposure to fluoridated drinking water or fluoride supplements during pregnancy and adverse effects on reproductive function or foetal development. In three case-control studies, no associations were found between fluoride intake and increases in spontaneous abortions,¹³² congenital cardiac disease¹³³ or late adverse pregnancy outcomes, including congenital anomalies, stillbirths and deaths.¹³⁴ A recent ecological study that examined total annual fertility rate (TFR) in women aged 10-49 years from 30 regions of the United States reported that twice as many regions containing counties with at least 3 ppm fluoride in their drinking water showed significant negative associations between TFR and fluoride exposure as positive associations. Although meta-analysis of the region-specific results gave a combined negative TFR/exposure association, the authors cautioned that the measures of exposure and outcome may differ between individual women and that the occurrence of significant positive TFR/exposure associations in some regions indicates the possibility of confounding by unknown factors.¹³⁵ In a clinical study, children from mothers who had been exposed to fluoridated drinking water and had received a fluoride supplement (1 mg/d) during pregnancy (n = 117) were found to be slightly but significantly heavier and longer at birth and suffered from fewer birth defects than those whose mothers had consumed fluoridated water but had received no supplement (n = 375).¹³⁶

Genotoxicity

Fluoride (as NaF) has generally given negative results in gene mutation assays using Escherichia coli WP2 hcr¹³⁷ and various strains of Salmonella typhi-murium.^88,137-141 In addition, NaF was not mutagenic and did not induce gene conversion or aneuploidy in Saccharomyces cerevisiae D4.^141,142 NaF induced the "morphological transformation" of Syrian hamster embryo cells in vitro, but only at cytotoxic concentrations.^143-146 NaF and potassium fluoride (KF) increased the frequency of gene locus mutations in cultured mammalian^147-149 and human cell lines.^149,150 The preferential increases in "small mutant colonies"^147,148 and negative results obtained for the ouabain locus¹⁴⁷ in these studies are believed to indicate a mechanism based on chromosomal damage rather than point mutations.^39,151 Also, the negative results observed with sodium chloride (NaCl) and potassium chloride (KCl) controls^147,147,148 suggest that the genotoxic effects are due to a specific effect of the fluoride ion rather than the cations.³⁹ Although NaF increased unscheduled DNA synthesis in Syrian hamster embryo cells, human foreskin fibroblasts, human keratinocytes^143,152,153 and rat hepatocytes,¹⁵⁴ these results were not confirmed using more rigorous methods of quantifying DNA repair synthesis.^{39,138,154,155} Although NaF has generally demonstrated clasto-genic activity (primarily breaks, deletions and gaps with few exchanges) in chromosomal aberration assays using a variety of mammalian and human cell lines, some inconsistencies have been observed.^39,156-158 Inconsistent results have also been reported for in vitro sister chromatid exchange assays in human peripheral blood lymphocytes,^138,159,160 Chinese hamster ovary cells^88,138,161 and Syrian hamster embryo cells.¹⁴³ NaF exposure increased micronuclei formation in human foreskin fibro-blasts¹⁵⁷ and Chinese hamster lung cells.¹⁶² Based on the in vitro test results, it has been suggested that fluoride-induced clastogenicity involves the inhibition of DNA synthesis and/or repair and has a threshold concentration of approximately 10 µg/L.³⁹ High dietary concentrations of NaF or SnF₂have been shown to induce recessive lethal mutations in male Drosophila melanogaster.^163,164 In most in vivo studies with rodents, oral administration of NaF produced no significant effects on the frequency of sister chromatid exchange^161,165,166 or DNA strand breaks ⁸⁵ or on the incidences of chromosomal aberrations, ^141,167 bone marrow micronuclei^140,168-170 or abnormal sperm.^169,171 However, the increased incidences of the latter three end points generally observed following intraperitoneal injection of NaF^172-174 may indicate differential toxicity based on route of administration.³⁹

Carcinogenicity

In 1990, the NTP completed a comprehensive study on the carcinogenicity of NaF administered in drinking water (0, 25, 100 or 175 ppm) to male and female 88  Osteosarcomas were F344/N rats and B6C3F₁mice. not induced in the female F344/N rats; in male rats, the incidence was 0/80, 0/51, 1/50 and 3/80 for the four respective dose groups. The incidence in the high-dose male rats was not significantly different from the control group incidence (p = 0.099), although a significant dose-response trend was observed (p = 0.027). One high-dose male had a subcutaneous osteosarcoma, but no primary bone tumour; although this tumour increased the significance of the trend test (p = 0.010), the pairwise comparison with controls remained non-significant (p = 0.057). Although the incidence in the high-dose male rats was significantly higher than the average rate for male control rats in the NTP historical data base, the investigators concluded that it was more appropriate to use concurrent controls for comparison purposes because more extensive gross and histopathological examinations of bone and other tissues were made in the current study and because the fluoride content of the standard diet used in the older studies (28-47 ppm) was equivalent to a total fluoride intake between the low-and medium-dose groups in the current study. No other observed tumours (squamous cell neoplasms of the oral mucosa, thyroid gland follicular cell neoplasms, hepatoblastoma, malignant lymphoma) in mice or rats were considered to be significant by the NTP investigators. Based on the study results, the NTP concluded that there was "equivocal evidence of carcinogenic activity" (defined as a marginal increase in neoplasms that may be related to chemical administration) of NaF in male F344/N rats, but no evidence of carcinogenic activity in female F344/N rats or male or female 88 B6C3F₁mice. In another carcinogenicity bioassay, Sprague-Dawley rats and CD-1 mice received NaF in doses of 0, 4, 10 or 25 mg/kg bw per day in the diet for 95-99 weeks.^89,90 The incidences of bone tumours (chordoma, chondroma, fibroblastic sarcoma and osteosarcoma) in rats (0/70, 0/58, 2/70 and 1/70 for the males and 0/70, 2/52, 0/70 and 0/70 for the females) were not statistically significant compared with controls. Osteomas were found to occur with a statistically significant dose-response trend in both the male and female mice (2/50, 0/42, 5/44 and 26/50 for males and 4/50, 10/42, 5/44 and 26/50 for females), and statistically significant increases were observed for high-dose males and females compared with controls. However, after reviewing the osteoma data, the U.S. Armed Forces Institute of Pathology commented that none of these tumours advanced beyond the benign state or showed pre-cancerous morphology, many were multicentric (i.e., most primary bone cancers are unicentric) and a human counterpart to this type of tumour is not known.¹⁷⁵ The U.S. Food and Drug Administration examined the results of the study, noted a number of problems affecting the interpretation of the results (e.g., high levels of minerals, ions and vitamins in the diet and water; inappropriate dose determination in the preliminary studies; low survival rate for experimental animals and infection of the mice with a retrovirus) and concluded that "under the conditions of the studies, malignant tumours related to dietary fluoride exposure in rodents were not observed."⁸ Since the introduction of water fluoridation to North America in the 1950s, more than 50 epidemiological studies have been conducted to examine possible associations between the ingestion of fluoridated drinking water and the occurrence of human cancer.⁸ One of the more recent studies was a time trend analysis by Freni and Gaylor,¹⁷⁶ which examined the change in cumulative risk of bone cancer for persons 10-29 or 0-74 years of age in 1958-1987 in fluoridated (i.e., fluoride in drinking water serving at least 50% of the population increased to 1 mg/L during or before 1960) versus non-fluoridated areas of the United States, Canada and Europe. The mean change with time in the cumulative bone cancer risk was not significantly different for fluoridated versus non-fluoridated areas.¹⁷⁶ Hoover et al.¹⁷⁷ analysed fluoridated counties (i.e., percentage of population receiving fluoridated water increased from <10% to >60% within a 3-year period) compared with control counties (i.e., <10% of population exposed to fluoridated water) from two National Cancer Institute Surveillance, Epidemiology and End Results (NCI SEER) Program registries (Iowa and Seattle, Washington) for the occurrence of cancer and cancer mortality in Caucasians. The observed incidences of osteosarcoma (91 cases), generalized bone and joint cancer (290 cases), cancer of the oral cavity (2693 cases) and renal cancers (2583 cases) in the fluoridated counties were compared with the expected incidences in the control counties. No consistent differences in the observed versus expected cases were noted for any of the cancer types. There was a significant trend (p = 0.04) towards increased risk of renal cancer for both sexes combined as the duration of water fluoridation increased from <5 to 15-19 years in the Seattle area (i.e., the relative risk increased from 0.9 [95% CI = 0.7-1.1] to 1.0 [95% CI = 0.9-1.2]). However, no significant trends were observed when males and females were analysed separately or when the data were broken down according to diagnoses made from 1973 to 1980 and from 1981 to 1987.^177,178 An examination of the entire NCI SEER incidence data base showed that for all ages combined, the incidence of osteosarcoma increased 18% in males and decreased 11% in females between the above two periods. For males <20 years of age in fluoridated communities, the incidence rate of osteosarcoma increased 53% between 1973 and 1980 (88 cases) and between 1980 and 1987 (100 cases), but further analyses showed that the increase was not related to the duration of water fluoridation.^177,178 Hoover et al.^177,178 also recently analysed information on more than 2.3 million U.S. cancer deaths occurring between 1950 and 1985 and found no consistent association between exposure to fluoridated drinking water (i.e., counties >50% urbanized in 1980 where the proportion of the population receiving water containing >0.3 ppm fluoride increased from <10% to >66% within a 3-year period) and deaths due to any type of cancer.

These epidemiological studies are of the ecological or geographical correlation type, which suffer from a number of weaknesses, including variations in fluoride intake, geographical differences in variable factors affecting the occurrence of cancer, migration between areas and variations in the quality of mortality and morbidity data.^8,39,109 Although it is generally accepted that ecological studies cannot provide conclusive proof for or against causality, the U.S. Department of Health and Human Services noted that "most epidemiologists consider studies of geographic correlations valuable in indicating the likelihood that positive links do or do not exist or in demonstrating the feasibility of hypotheses."⁸ Many of these studies were reviewed by three major working groups (i.e, the British Working Party on the Fluoridation of Water and Cancer, the International Agency for Research on Cancer and the U.S. National Academy of Sciences), and all three concluded that the available body of evidence shows no consistent association between the consumption of fluoridated drinking water and the risk of cancer morbidity or mortality.^109,179,180

Dental Effects

Dental fluorosis is a permanent hypomineralization of tooth enamel due to a fluoride-induced disruption of tooth development.^181-184 In the mildest forms, only the outermost layer of enamel is affected, producing diffuse white lines across the tooth surface.¹⁸² As the severity increases, deeper layers are affected and the porosity increases, leading to a chalky white appearance.^175,182 Eventually, chewing and other forces erode the surface enamel, producing pits that can become stained by various food constituents.¹⁸² Generally, the milder forms of dental fluorosis do not alter tooth function and are considered to represent an aesthetic rather than a health effect,⁸ although significant enamel erosion could lead to tooth pain and impairment of chewing ability and require complex restorative procedures.¹⁷⁵ Epidemiological studies of various age cohorts of children exposed to different fluoride concentrations in drinking water have identified the later maturation stage rather than the earlier secretory stage as the period of enamel development most sensitive to the occurrence of dental fluorosis.¹⁸¹ As the milder forms of dental fluorosis are the ones most often seen in North America,¹⁸⁵ the anterior teeth, particularly the maxillary central incisors (MCI), are believed to be the most important ones for judging the risk of dental fluorosis.^181,175,186 An analysis of small groups of Hong Kong school children before and after a reduction in the fluoride concentration in the community drinking water supply concluded that there is a minimal risk to the MCI before 18 months of age, but 22-26 months of age represents the period of greatest risk.¹⁸⁷ After tooth development is complete (i.e., 5-6 years of age for the MCI), there is no longer a risk of dental fluorosis.^32,186,187 An Advisory Review Panel of dental researchers recently examined the available data on the relationship between total daily fluoride intake and the prevalence of dental caries and dental fluorosis in children.¹⁸⁸ The data reviewed ranged from the studies of Dean et al.,^189,190 conducted in the 1940s with children 12-14 years of age who were lifelong residents of 21 U.S. communities and whose drinking water contained naturally occurring fluoride, to more modern studies using children of various ages from fluoridated and non-fluoridated communities in Canada, the United States, Australia and other countries.^191,192 The critical studies for defining the dose-response relationship between total daily fluoride intake and dental caries/fluorosis were found to be those of Dean et al.,^189,190 supplemented with data compiled in 1958 on 12- to 14-year-old children from 20 U.S. communities.¹⁹³ The panel concluded that these older dose-response studies were more applicable than modern ones because the age ranges of the children in the modern studies varied; the modern studies have relatively few data on dental fluorosis and caries prevalence in communities with fluoride concentrations in drinking water below 1.0 ppm; and most of the modern studies have not accounted for the confounding effect of sources of fluoride that were not available in the 1940s (e.g., toothpaste, mouth rinses, gels).¹⁸⁸ With respect to the occurrence of dental fluorosis, analysis of the original data of Dean et al.^189,190 showed that 1940s children consuming drinking water containing -1.6 ppm fluoride experienced low rates of very mild (22%) and mild (4%) dental fluorosis, but no moderate or severe dental fluorosis. The total daily fluoride intake for these children (i.e., from air, soil, food and water) can be considered to represent the maximum daily fluoride intake that is unlikely to result in moderate to severe dental fluorosis.

The maximum daily fluoride intake can be estimated from modern data (Table 1) if it is assumed that the daily fluoride intakes from air, soil and food have not changed significantly since the 1940s and the daily drinking water consumption (i.e., number of litres per day) has remained relatively constant.** Using the 7 month to 4 year age group (Table 1) as a surrogate for the period of greatest risk of dental fluorosis (22-26 months of age), the estimated maximum daily fluoride intake is the sum of the maximum daily intakes from air (0.01 µg/kg bw per day), soil (1.19 µg/kg bw per day), food (22.3 µg/kg bw per day) and drinking water (0.8 L/day × 1.6 mg/L^189,190 ÷13 kg bw = 98.5 µg/kg bw per day), which is 122 µg/kg bw per day.

Dental caries result from the localized dissolution of tooth enamel by acids produced by bacterial deposits (plaque). The period of greatest susceptibility to caries is believed to extend from the time teeth first emerge to full eruption for both the primary and permanent dentition.¹⁹⁵ Initially, the ability of fluoride to prevent the formation of clinically detectable caries was thought to be primarily due to pre-eruptive incorporation, producing improved crystal stability and reduced enamel solubility.^196,197 Fluoride was also shown to inhibit plaque bacterial acid production.¹⁹⁸ However, reviews of clinical studies of water fluoridation and fluoride-s effects on mineralization indicate that fluoride-s major anticariogenic effect is post-eruptive, through the inhibition of demineralization and the enhancement of remineralization of early caries lesions.^195,197 Consistent with this post-eruptive mechanism are observations of significantly less decayed and filled tooth surfaces in adults exposed to fluoridated drinking water from ages 15 to 34¹⁹⁸ and significantly lower coronal and root caries incidences for adults >65 years of age residing in fluoridated communities for at least 30-40 years compared with lifelong residents of non-fluoridated communities.¹⁹⁹ The dose-response data of Dean et al.^189,190 and Eklund and Striffler¹⁹³ suggest a relatively small decline in dental caries incidence in 12- to 14-year-olds when fluoride concentrations in drinking water increased from 0.8 to 1.2 ppm compared with much larger declines for fluoride concentrations below 0.8 ppm. This decrease in the slopes of the dose-response curves and the fact that Dean et al.^189,190 observed the occurrence of only the very mild to mild forms of dental fluorosis at concentrations of 0.8-1.2 ppm led the Advisory Review Panel¹⁸⁸ to select this range as an optimal range of fluoride concentrations. The panel concluded that at the time of Dean et al.'s^189,190 research, children who consumed drinking water containing 0.8-1.2 ppm fluoride combined with intakes of fluoride from air, food and soil were obtaining an optimal daily fluoride intake for the prevention of dental caries.¹⁸⁸ However, as fluoride prevents dental caries through both pre- and post-eruptive mechanisms^195-197 in children and adults,^198,199 it is likely that there is a relatively wide range of optimal daily fluoride intakes, depending on the age group considered.