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* The following are 2 sections from the World Health Organization's 2002 report "FLUORIDES" which dicsuss the National Toxicology Program's Fluoride Bioassay. To access a pdf file of the full WHO report, click here.



From Section 7.3: Long-term exposure and carcinogenicity (pp. 86-91)

In early carcinogenicity bioassays conducted by Tannenbaum & Silverstone (1949), Taylor (1954) and Kanisawa & Schroeder (1969), the incidence of tumours in mice administered sodium fluoride (in either their diet or drinking-water) was, in general, not markedly greater than that observed in the unexposed controls. However, the documentation and protocols of these studies were inadequate. Deficiencies in design included small groups of animals of single sexes or various ages exposed to single dose levels for short periods of time with inadequate examination of possible target tissues.

In a comprehensive study of the carcinogenicity of sodium fluoride in laboratory animals (NTP, 1990), groups of male and female F344/N rats were administered drinking-water containing 0, 25, 100 or 175 mg sodium fluoride/litre (0, 11, 45 and 79 mg fluoride/litre) for a period of 2 years. There were 100 rats in each of the control and 175 mg/litre groups, while 70 rats per group were administered drinking-water containing 25 or 100 mg sodium fluoride/litre. Groups of 10 rats of each sex at each level of exposure were sacrificed after 27 and 66 weeks. In the high-dose group, 42 males and 54 females survived until terminal sacrifice. The estimated total intakes of fluoride from food and drinking-water by the male and female F344/N rats administered drinking-water containing 0 (control), 25, 100 or 175 mg sodium fluoride/litre were approximately 0.2, 0.8, 2.5 and 4.1 mg/kg body weight per day and 0.2, 0.8, 2.7 and 4.5 mg/kg body weight per day, respectively. At the end of the 2-year study, the levels of fluoride in the humeral bone of the control, low-, mid- and high-dose male and female rats were approximately 0.44, 0.98, 3.65 and 5.26 µg/mg bone ash and 0.55, 1.34, 3.72 and 5.55 µg/mg bone ash, respectively (NTP, 1990).

In male F344/N rats receiving 0.2, 0.8, 2.5 or 4.1 mg fluoride/kg body weight per day, the incidence of osteosarcomas (three tumours in the vertebra and one in the humerus) was 0/80, 0/51, 1/50 and 3/80, respectively (NTP, 1990). A pairwise comparison of the incidence in the high-dose group versus controls was not statistically significant (P = 0.099); if an extraskeletal osteosarcoma, located in the subcutis of the flank of one high-dose male rat, was included in the total tumour incidence in this group of animals, the pairwise comparison with the control group remained statistically insignificant (P = 0.057). However, the osteosarcomas occurred with a statistically significant (P = 0.027, by logistic regression) dose-response trend (NTP, 1990). The incidence of osteosarcoma at any site was within the range of historical controls; however, the amount of fluoride in the diets in previous National Toxicology Program (NTP) studies of other chemicals was uncontrolled and was estimated to be approximately 3.5- to 5.9-fold higher than in the NTP sodium fluoride study (NTP, 1990). In male F344/N rats receiving 0.2, 0.8, 2.5 or 4.1 mg fluoride/kg body weight per day, the incidence of oral cavity lesions (squamous papillomas or squamous cell carcinomas) was 0/80, 1/51, 2/50 and 3/80, respectively; the incidence in the fluoride-exposed groups was not significantly different from the controls and was not considered to be compound-related. The incidence of (thyroid gland) follicular cell tumours (adenomas and carcinomas) was 1/80, 1/51, 1/50 and 4/80 in the control, low-, medium- and high-dose males, respectively; the incidence in the high-dose group was not significantly different from controls and was not considered to be compound-related. There was no increase in the incidence of osteosarcomas in female F344/N rats receiving fluoride; the incidence of oral cavity neoplasms (squamous papillomas or squa-mous cell carcinomas) was 1/80, 1/50, 1/50 and 3/81 in female F344/N rats receiving 0.2, 0.8, 2.7 and 4.5 mg/kg body weight per day, respectively; the incidence in the high-dose group was not significantly different from the controls (NTP, 1990).

In the NTP carcinogenicity bioassay with male and female F344/N rats, no significant compound-related effects upon survival, body weight or weights of major internal organs were observed, compared with controls (NTP, 1990); however, the incidence of osteosclerosis in female rats administered drinking-water containing 175 mg sodium fluoride/litre (18/81) was significantly (P = 0.04) increased, compared with controls (6/80) (NTP, 1990). It was concluded (NTP, 1990) that the osteosarcoma finding was “equivocal evidence of carcinogenic activity” in male rats, and that there was “no evidence of carcinogenic activity” in the female rats exposed to fluoride under these conditions.

In a carcinogenicity bioassay conducted with Sprague-Dawley rats, groups of 70 animals of each sex were administered diets supple-mented with various amounts of sodium fluoride for a period of 95-99 weeks (Maurer et al., 1990). The estimated total intake of fluoride by these groups of animals was 0.1, 1.8, 4.5 or 11.3 mg/kg body weight per day, respectively. After 26 weeks on study, as many as 10 animals of each sex per group were sacrificed, and after 53 weeks, 10 animals of each sex per group were sacrificed (Maurer et al., 1990). At terminal sacrifice (i.e., after 95 and 99 weeks on study for males and females, respectively), there were 26 males and 12 females remaining in the high-dose groups. At study termination, the concentration of fluoride in the bone (radius and ulna) of the males and females receiving 0.1, 1.8, 4.5 or 11.3 mg fluoride/kg body weight per day was 0.5, 5.0, 8.8 and 16.7 µg/mg bone ash and 0.5, 4.5, 8.3 and 14.4 µg/mg bone ash, respectively.

In this study, the incidence of bone tumours was 0/70, 0/58, 2/70 (one chordoma and one chondroma) and 1/70 (fibroblastic sarcoma with areas of osteoid formation) in male rats and 0/70, 2/52 (one osteosarcoma and one chondroma), 0/70 and 0/70 in female rats receiving 0.1, 1.8, 4.5 and 11.3 mg fluoride/kg body weight per day, respectively (Maurer et al., 1990). However, detailed information on the incidence of tumours in tissues or organs other than the bone and stomach were not presented, and histological examination of bone from both mid-dose groups was limited. The cranium, femur, premaxilla, maxilla, mandible, cervical vertebra, stomach, liver, kidney, incisors, adrenals, brain, heart, lungs, ovaries, uterus, pancreas, pituitary, pros-tate, seminal vesicles, spleen, bladder, testes, epididymides, thyroids and parathyroids obtained at the interim and terminal sacrifices from all animals receiving 0.1 or 11.3 mg fluoride/kg body weight per day were examined microscopically. The stomach, bones and teeth from animals receiving 4.5 mg fluoride/kg body weight per day and sacrificed after 26 weeks and from animals receiving 1.8 or 4.5 mg fluoride/kg body weight per day and sacrificed after 53 weeks were also examined microscopically. Not all of the bones (i.e., cranium, femur, premaxilla, maxilla, mandible, cervical vertebra) from each of the remaining animals (i.e., those alive after the interim sacrifices) receiving 1.8 or 4.5 mg fluoride/kg body weight per day were examined microscopically; however, tissues with gross lesions obtained from dead and moribund animals were evaluated.

Sprague-Dawley rats receiving 11.3 mg fluoride/kg body weight per day (administered in the diet) over a period of 95-99 weeks had reduced weight gain; animals receiving 4.5 or 11.3 mg fluoride/kg body weight per day had an increased incidence of subperiosteal hyperostosis in the skull and hyperkeratosis and acanthosis in the stomach, compared with controls receiving 0.1 mg/kg body weight per day (Maurer et al., 1990).

In the NTP (1990) carcinogenicity bioassay, male and female B6C3F 1 mice were also administered drinking-water containing 0, 25, 100 or 175 mg sodium fluoride/litre (0, 11, 45 and 79 mg fluoride/litre) for 2 years. There were 100 animals in each of the control and 175 mg/litre groups, and 70 mice per group were administered drinking-water containing 25 or 100 mg sodium fluoride/litre; the total intake of fluoride from water and the diet for the males and females in these groups was estimated to be approximately 0.6, 1.7, 4.9 and 8.1 mg/kg body weight per day and 0.6, 1.9, 5.7 and 9.1 mg/kg body weight per day, respectively. At the end of the 2-year study (groups of 10 animals of each sex at each level of exposure were sacrificed after 24 and 66 weeks), the levels of fluoride in the humeral bone of the control, low-, mid- and high-dose male and female mice were approximately 0.72, 1.61, 3.58 and 5.69 µg/mg bone ash and 0.92, 1.52, 4.37 and 6.24 µg/mg bone ash, respectively (NTP, 1990).

In male B6C3F1 mice receiving 0.6, 1.7, 4.9 or 8.1 mg fluoride/kg body weight per day, the incidence of hepatoblastoma was 0/79, 1/50, 1/51 and 3/80, respectively (NTP, 1990). The overall incidence of hepatic neoplasms (adenoma, carcinoma, hepatoblastoma) was similar among all groups, and the incidence of liver tumours in all groups (control and exposed) of male mice (73-78%) was higher than observed (16-58%) in previous NTP carcinogenicity bioassays (NTP, 1990). In female B6C3F1 mice receiving 0.6, 1.9, 5.7 or 9.1 mg fluoride/kg body weight per day, the incidence of hepatoblastoma was 0/80, 1/52, 0/50 and 2/80, respectively. The overall incidence of hepatic neoplasms (adenoma, carcinoma, hepatoblastoma) was similar among all groups, and the incidence of liver tumours in all groups (control and exposed) of female mice (52-69%) was higher than observed (3-20%) in previous NTP carcinogenicity bioassays (NTP, 1990). The incidence of malignant lymphoma was 11/80, 5/52, 11/50 and 19/80 (P = 0.051), respectively. The incidence of malignant lymphoma in the control and low-dose groups was less than the lowest incidence observed in nine other investigations conducted at the study laboratory. In addition, the incidence in the high-dose group was less than the average incidence observed in historical controls (35%) and within the range of incidence observed in historical controls (10-74%) (NTP, 1990).

The administration of drinking-water containing 25, 100 or 175 mg sodium fluoride/litre to male or female B6C3F 1 mice over a period of 2 years had no significant compound-related adverse effects upon survival, body weight or weights of major internal organs, compared with controls (NTP, 1990). It was concluded (NTP, 1990) that there was “no evidence of carcinogenic activity” in male and female mice exposed to fluoride under these conditions.

In a carcinogenicity bioassay in which sodium fluoride was administered in the diet to groups of 60 male and 60 female CD-1 mice over a period of 95 and 97 weeks, respectively (10 mice of each sex per group were sacrificed after 40 weeks), the incidence of osteomas in male and female controls and mice receiving 1.8, 4.5 or 11.3 mg fluoride/kg body weight per day was 1/50, 0/42, 2/44 and 13/50 and 2/50, 4/42, 2/44 and 13/50, respectively (no statistical analysis provided) (Maurer et al., 1993). The incidence of this type of tumour was increased in the high-dose groups compared with the controls. These animals were infected with a Type C retrovirus, and the role of fluoride in the etiology of these tumours is not clear (Maurer et al., 1993). Moreover, there is some controversy concerning whether they should be classified as neoplasms (US NRC, 1993). The concentration of fluoride in the bone (radius or ulna) of male controls and mice receiving 1.8, 4.5 or 11.3 mg fluoride/kg body weight per day was approximately 1.5, 4.4, 7.2 and 13.2 µg/mg bone ash, respectively; the concentration of fluoride in the (radius or ulna) bone of female controls and mice receiving 1.8, 4.5 or 11.3 mg fluoride/kg body weight per day was approximately 1.0, 3.4, 6.2 and 10.6 µg/mg bone ash, respectively.

From Section 10.1.2: Hazard identification (pp. 167-170)

In early carcinogenicity bioassays conducted by Kanisawa & Schroeder (1969), Taylor (1954) and Tannenbaum & Silverstone (1949), the incidence of tumours in mice administered sodium fluoride (in either the diet or drinking-water) was, in general, not markedly greater than that observed in controls. Owing to inadequate documentation and to numerous methodological shortcomings, however, the results of these investigations do not contribute meaningfully to an assessment of the weight of evidence of the carcinogenicity of (sodium) fluoride. The administration of drinking-water containing sodium fluoride (in amounts estimated to provide intakes ranging from 0.6 to 9.1 mg fluoride/kg body weight per day) to male and female B6C3F 1 mice over a period of 2 years produced a marginal (statistically insignificant) increase in the incidence of hepatoblastoma, compared with the incidence in groups of controls administered drinking-water without added fluoride; however, this minor increase was not considered “biologically significant,” since the overall incidence of hepatic tumours (adenoma, carcinoma, hepatoblastoma) was not increased in animals receiving sodium fluoride, and the incidence of all hepatic tumours in these groups of mice was higher than that in previous NTP carcinogenicity bioassays (NTP, 1990). The marginal increase in the incidence of malignant lymphoma in female B6C3F 1 mice administered drinking-water containing sodium fluoride was considered not to be compound-related, since the incidence in the high-dose group was similar to that observed in historical controls (NTP, 1990). No other increases in tumour incidence were observed; however, failure to attain the maximum tolerated dose may have reduced somewhat the sensitivity of this study in mice. In a less extensive carcinogenicity bioassay in which the incidence of osteomas in male and female CD-1 mice receiving 11.3 mg fluoride/kg body weight per day in the diet was increased compared with controls (Maurer et al., 1993), the specific role of fluoride in the etiology of the tumours cannot be determined with certainty, owing to the infection of these animals with Type C retrovirus (US DHHS, 1991; Maurer et al., 1993; US NRC, 1993).

The administration of drinking-water containing sodium fluoride (in amounts estimated to provide intakes ranging from 0.2 to 4.5 mg fluoride/kg body weight per day) to F344/N rats over a period of 2 years produced a marginal (not statistically significant) increase in the incidence of oral cavity neoplasms (in males and females) and tumours in the thyroid gland (in males) (NTP, 1990). The squamous cell tumours of the oral cavity were not considered to be compound-related, since the incidence of tumours in the high-dose group was not significantly different from the controls, the incidence of these neo-plasms was within the range observed in historical controls and there was no supporting evidence of focal hyperplasia of the oral mucosa (NTP, 1990). The marginal increase in thyroid (follicular cell) tumours was also considered not to be compound-related, since the incidence of these tumours in the high-dose group was not significantly different from the controls, the incidence of these neoplasms was within the range observed in historical controls and the incidence of follicular cell hyperplasia was not increased in fluoride-exposed animals (NTP, 1990). The incidence of osteosarcoma in rats with intakes ranging from 0.8 to 4.5 mg fluoride/kg body weight per day was not significantly increased, compared with controls receiving approximately 0.2 mg fluoride/kg body weight per day (NTP, 1990); however, for the male F344/N rats, it was reported that “the osteosarcomas occurred with a significant dose response trend (P = 0.027, by logistic regression)” (NTP, 1990). In a more limited carcinogenicity bioassay conducted by Maurer et al. (1990), the administration of diets containing sodium fluoride (in amounts estimated to provide intakes ranging from 1.8 to 11.3 mg fluoride/kg body weight per day) to male and female Sprague-Dawley rats over a period of 95–99 weeks produced no significant increase in the incidence of any types of tumours, compared with groups of controls receiving approximately 0.1 mg fluoride/kg body weight per day, although a small number of malignant tumours of the bone were observed.

In assessing the evidence for the carcinogenicity of fluoride derived from studies conducted with laboratory animals, some significance might be attributed to the observation of a dose–response trend in the occurrence of osteosarcomas in male F344/N rats administered sodium fluoride in drinking-water (NTP, 1990). Such a trend associated with the occurrence of a rare tumour in the tissue in which fluoride is known to accumulate cannot be casually dismissed. Moreover, the level of fluoride in the bones of the high-dose group of male rats in the NTP carcinogenicity bioassay, in which a non-significant increase in osteosarcomas was observed, is similar to that measured in humans with a preclinical phase of skeletal fluorosis. However, the biological significance of this dose–response trend is tempered by the lack of statistical significance of the observed excess in the high-dose males in comparison with controls, as well as by the absence of a comparable statistically significant trend in the incidence of osteosarcomas in female F344/N rats or male and female B6C3F 1 mice receiving comparable amounts of inorganic fluoride (NTP, 1990). Indeed, the levels of fluoride in the bone of (male and female) F344/N rats and B6C3F 1 mice administered sodium fluoride in drinking-water were similar (NTP, 1990). No dose–response trend in the incidence of osteosarcomas was observed in groups of male and female Sprague-Dawley rats administered diets containing sodium fluoride (Maurer et al., 1990), even though the levels of fluoride in the bone in the high-dose animals were greater than those in the male F344/N rats in which there was an increase in osteosarcomas in the NTP (1990) carcinogenicity bioassay; however, there may be variations sensitivity of the two strains to the effects of fluoride. There is controversy concerning whether or not the osteomas in male and female CD-1 mice observed in the carcinogenicity bioassay conducted by Maurer et al. (1993) should be classified as neoplasms, and a retrovirus (in addition to fluoride) has been implicated in their etiology; however, the significant increases in (the highest-dose) fluoride-exposed versus control groups (in animals infected with retrovirus), in a tissue in which fluoride is known to accumulate, adds some weight, albeit weak, to the evidence of carcinogenicity. Overall, the evidence regarding the carcinogenicity of fluoride in laboratory animals is inconclusive.