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The mutagenicity of fluoride supports the conclusion that fluoride is a probable human carcinogen

The biological plausability of fluoride and bone cancer cannot be denied

"The mutagenicity of fluoride supports the conclusion that fluoride is a probable human carcinogen. An important toxicologic consideration is that a toxic substance stores at the same place it exerts it toxic activity." (Dr. William Marcus, Environmental Protection Agency Scientist)

What is the NTP (National Toxicology Program)?
  • Calabrese E, PhD., Evaluation of the National Toxicology Program (NTP) Cancer Bioassay on Sodium Fluoride, 1991 Jun (Amherst), commissioned by the East Bay Municipal Utility District (provides water for Oakland and area)
This independent evaluation was completed before the epidemiological data from New Jersey found a statistically significant association between fluoridation and osteosarcoma rates in young males. Professor Calabrese's report will be available on line in the future. Portions are now available at cancer2.htm. For a short synopsis of this 17-page report see the summary by Dr. John R. Lee below.

SUMMARY by John R. Lee, M.D.

Professor Calabrese was requested by the East Bay Municipal Utility District (water provider for Oakland and environs) to conduct an independent appraisal of the 1990 NTP report (NTP Technical Report Series 393). Of the NTP findings, professor Calabrese stated:

"The principal finding of the NTP study was the occurrence of a significant dose response trend in male rats of osteosarcoma (malignant bone cancer)." (emphasis added)

Concerning this, he found the NTP's choice of the word "equivocal" in classifying the evidence for fluoride's carcinogenicity to be "confusing", "inappropriate", and "not consistent with what most people would call equivocal." The reasons he gave are the following:

  • (1) Its own definition of equivocal is in disagreement with the generally accepted definition of equivocal.
  • (2) The findings with the male rat clearly exceeded marginal increases and are biologically plausible given the capacity for fluoride to both concentrate and be biologically active in bone. (emphasis added)
  • (3) The statistical analysis for trend effects is stronger than pair wise comparisons since it uses all available data not just data from two comparison groups; yet this point is never acknowledged.
  • (4) The basic reality is that humans can be exposed in critical target tissue to as much fluoride as the high dose rats while consuming water at the EPA maximum contaminant level 1 of 4 mg/liter. (emphasis added)

In his "Overview of NTP Bioassay Procedure", Dr. Calabrese points out that, in the NTP test, the fluoride exposure covered only the time after weaning and not during the embryonic, fetal and neonatal stages. contrary to the experience of humans reared in a fluoridated community. This lack of exposure during the critical developmental stages is an important limitation of the NTP bioassay which must be taken into consideration when interpreting the findings of such studies. (emphasis added).

Dr. Calabrese also points out that the NTP decision to use only 50 animals per sex per dosage level represents a "practical compromise" between scientific requirements and practical (e.g. cost) constraints. He states, "If a tumor incidence were to occur in one out of 250 animals, it would not be detectable in this study. Yet such an incidence in the U.S. population would be of considerable public health concern."

Thirdly, Dr. Calabrese points out that the common, practice of using historical controls to determine "natural" tumor incidence is not applicable in this NTP test since such data is derived over different time intervals, often at different locations, using different diets and possibly different histological criteria for diagnosing tumors. He adds that "past historical control diets often had appreciable amounts of fluoride thereby providing fluoride ingestion levels between the low (25 ppm) and mid (100 ppm) group range (of the NTP study)". Furthermore, "the most appropriate historical control is the experience of the Battelle Columbus Laboratory where the actual NTP NaF study took place." Of these, only 0.6% displayed osteosarcoma. [compare to 5% in the high dose NTP male rats] Dr. Calabrese also notes that the issue of "sex specific response" (osteosarcoma in male rats, osteofluorosis in females) can not be used to"discount the significant trend effect in the males". He points out that "the only previous chemical (i.e. acromycin) determined by NTP to be a bone carcinogen (osteosarcoma) likewise displayed its response in male but not female rats (NCI. 1978)".

Lastly, Dr. Calabrese reminds us "of particular concern and in great need of emphasis is that the bone fluoride levels observed in the NTP bioassay at the high dose group were similar to human bone samples taken from people who lived for at least 10 years in an area with average fluoride level of 4 ppm in drinking water, the U.S. EPA national primary drinking water standard for fluoride. (emphasis added) This information is striking since most NTP bioassays would be expected to use dosage levels that grossly exceed expected human exposures. For example, exposure to TCE in the NTP bioassay exceeded typical human exposures by about 100,000-fold!"

Dr. Calabrese concludes that the word "equivocal" should not be applied to findings (such as the NTP study) where statistically significant trend relationships exist and where the response has substantial biological plausibility. He states that, "the fact that fluoride is a potential mutagen/clastogen, concentrates in the bone and is known to stimulate bone development, suggests that bone alterations need to be carefully considered. The linkage of the site of deposition and biological activity with bone cancer outcome all speak to the plausibility issue." (emphasis added) Several times, he states that "these collective findings indicate that the decision by the NTP to classify the male F344 rat osteosarcoma findings as equivocal is inappropriate." He notes that all members of the task force convened to review the findings (and who concurred with the interpretation of the NTP) were from PHS agencies.

In discussing the human epidemiologic bone cancer data from NCI. Dr. Calabrese cautions that the negative appraisal, admittedly highly debated and contentious, "is not particularly re-assuring that a problem is not present." He points out that "the entire cadre of available studies are ecological in nature", i.e., that "the unit of analysis was the community and not the individual." In no study is it known which people drank the community water or how much, whether they smoked cigarettes or how often, had X-rays and how many, how long specific residents lived in the community, or had other risk factors. Such studies many be useful for hypothesis generation but "not for determining cause-effect relationship." The assertion by PHS that "optimal fluoridation of drinking water does not pose a detectable cancer risk to humans as evidenced by extensive human epidemiological data ... does not reflect the limitations of ecological epidemiological methodology used to address the hypothesis."

Despite the impossibility of determining to what extent the NTP rat model can be extrapolated to humans, it is possible that "the animal bioassay in male rats would predict that exposure to NaF at normally fluoridated levels in drinking water would pose a highly significant risk in males." Since other agents (e.g. bone seeking radioisotopes, radiotherapy, alkylating agents, familial factors, and loss or inactivation of a tumor suppressor gene) can induce or cause osteosarcoma, the NTP male rat cannot be literally used to estimate human cancer risk. That is, "while the information available indicates that NaF is a bone carcinogen for the male rats, it is not possible to confidently use this qualitative judgement to define what fraction of the annual cases of osteosarcomas, if any, can be attributed to the consumption of NaF." (emphasis added) What is needed now, Dr. Calabrese states, is "more powerful epidemiologic protocols."

See cancer2.htm for Professor Calabrese's report.

Caspary WJ; Myhr B; Bowers L; McGregor D; Riach C; Brown A, Mutagenic activity of fluorides in mouse lymphoma cells, Mutat Res, 187: 3, 1987 Mar, 165-80

The L5178Y mouse lymphoma cell forward-mutation assay was used to test for the mutagenic activity of sodium and potassium fluoride at the thymidine kinase locus. Mutants were detected by colony formation in soft agar in the presence of trifluorothymidine. Mutagenic and toxic responses were observed in the concentration range of 300-600 micrograms/ml with both sodium and potassium fluoride. Approximately 3-fold increases in mutant frequency were observed for concentrations in the 500-700 micrograms/ml range that reduced the relative total growth to approximately 10% in the absence or presence of a rat-liver S9 activation system. A sample of 30% sodium fluoride-70% sodium bifluoride (NaHF2) induced a similar mutagenic response but was more toxic with respect to the fluoride concentration. A specificity for fluoride ions in causing mutagenesis was indicated by the fact that much higher concentrations of sodium or potassium chloride were necessary to cause toxicity and increases in the mutant frequency. The possible involvement of chromosomal changes was signaled by the predominant increase in the small colony class of mutants.

Cohn PD, A Brief Report On The Association Of Drinking Water Fluoridation And The Incidence of Osteosarcoma Among Young Males, NJ Depart. of Health, Environ. Health Service, 1992, 1- 17

still to come

Did the Hoover et al., study, discounted by the USPHS find increased rates of bone cancer in fluoridated areas?

  • Hoover RN, Devesa S, Cantor K, Fraumeni JF, et al., Time Trends For Bone And Joint Cancers And Osteosarcomas in the Surveillance, Epidemiology and End Results (SEER) Program national Cancer Institute, 1990 Aug, in: Review of Fluoride Benefits and Risks, report of the Ad Hoc Subcommittee on Fluoride, 1991 Feb, DHHS, USPHS.

Jones CA, Callaham MF, Huberman E, Sodium fluoride promotes morphological transformation of Syrian hamster embryo cells, Carcinogenesis, 1988, 9:12, 2279-2284

Sequential treatment of Syrian hamster embryo (SHE) cells with a chemical carcinogen followed by sodium fluoride (NaF) resulted in a higher yield of morphologically transformed cell colonies than treatment of the cells with carcinogen alone [in other words, fluoride acts as a cancer promoter rather than as a causative agent]. For example, cells treated with benzo[a]pyrene (B[a]P; 3 micrograms/ml) for 3 days, then with NaF (25 micrograms/ml) for 4 days, exhibited a transformation frequency more than six times greater than that obtained by summing the transformation frequencies from cells treated with either B[a]P or NaF alone. This enhancement/promotion of cell transformation by NaF was only expressed after the cells had been pretreated with either direct-acting carcinogens or procarcinogens. Pretreatment of the cells with noncarcinogens or weakly-acting carcinogens or administration of NaF prior to treatment with the carcinogen failed to enhance the yield of transformation. Transformation was enhanced even when the NaF treatment was delayed for several days after the carcinogen treatment. However, the continued presence of NaF was necessary for maintenance of the increased level of transformation. Removal of NaF prior to termination of the assay resulted in a reversal of the transformed clonal morphologies to a normal phenotype such that the final yield of transformants was decreased, but was still greater than that observed after carcinogen treatment alone. A similar pattern for reversibility of the transformation enhancement also occurs for the widely recognized tumor promoter phorbol 12-myristate 13-acetate (PMA). Seven different SHE cell pools were tested for sensitivity to NaF promotion following carcinogen treatment. Although the response was heterogeneous, no carcinogen-treated cell pool was refractory to the NaF-induced enhancement. A second fluorocompound, sodium monofluorophosphate (NaMFP), was also found to enhance carcinogen-induced cell transformation in a manner resembling that of NaF. [emphasis & comment aded]

Lee JR, Fluoride Exposure and Childhood Osteosarcoma A case-control study (A report by Gelberg KH et al., of the N.Y. State Department of Health, in The American Journal of Public Health, 1995, 85, 1678-1683), Fluoride,1996, 29:4, 237-240

Does fluoride increase the risk of osteosarcoma in young men? This case control study by the New York Department of Health tested this hypothesis by comparing the estimated fluoride intake of 130 osteosarcoma victims with that of an equal number of presumed healthy sex- and age-matched surrogates. The authors report finding little difference, and concluded that fluoride does not increase the risk, and may even be protective. Differing conclusions are not uncommon in science, and especially in medical science since underlying causes are often exceedingly complex, subtle and heterogeneous. It is important, therefore, to examine this report's results, its test design, and the assumptions on which the test and the conclusions are based.

Several lines of investigation suggest that fluoride intake increases the risk of cancer in general and, in particular, the incidence of osteosarcoma in males. As the authors admit, in vivo studies show fluoride to be mutagenic, inducing chromosome aberrations, sister chromatid exchanges, cytotoxicity, and neoplastic transformation in cultured mammalian cells. The authors also agree that fluoride accumulates primarily in bones; and that children, who are actively forming bone, have a higher uptake of fluoride into bone than adults. Further, bone in knees, ankles, shoulders, and wrists, where childhood osteosarcoma most often occurs, shows a high response to fluoride.1

In 1990, a two-year carcinogenicity study by the National Toxicology Program (NTP) found a statistically significant, dose-related increase of osteosarcoma rates in male rats, but not in mice.2 That the so-called peer review members at the time quixotically chose to call this fluoride/osteosarcoma correlation "equivocal" (as reported by the authors of this present study) does not change the facts. This same study revealed a strong correlation of fluoride intake with nasal and oral cancer and precancerous lesions in test rats and mice. A coincidental Proctor and Gamble study reported an increased incidence of cancer in rats but this was discounted later on the basis of a concomitant viral contamination in the test rodents.3 Time trends for bone and joint cancer and osteosarcoma derived from the Surveillance, Epidemiology and End Results (SEER) data of the National Cancer Institute (NTP) revealed a positive association of osteosarcoma incidence and water fluoridation among males under 20 years of age.4 In 1993, an ecological study performed by the New Jersey Department of Health found a strong statistical association between fluoridation and osteosarcoma among young men.5 It would appear that the fluoride/osteosarcoma hypothesis is credible and convincing, if not yet "conclusive" to fluoridation proponents.

Testing for the fluoride/osteosarcoma link is a daunting prospect. Osteosarcoma is quite rare, the incidence being only 2.9 cases per million people in the US. Test design always follows from assumptions made. The susceptibility of a cell to be cancer-prone may stem from a variety of subtle influences. In the case of xenobiotics (petrochemical compounds such as pesticides and various plastics), it is now known that exposure during embryonic tissue differentiation is far more toxic than later in life; yet the effects show up much later in life as an increased susceptibility to cancer of urogenital tissues such as the vagina, cervix, ovary, or testes.6

Fluoride readily crosses the placenta. Maternal fluoride intake determines whether baby's bones are fully fluoridated or not. It is likely that fluoride intake later in life will be more toxic to the child whose bones are already fully fluoridated than to one whose bones are not so fluoridated. Thus, one might well assume that exposure to fluoride during embryo life could be a factor in developing bone cancer later in life, regardless of whatever other factors may also play a role. Oncology researchers often make the distinction between cancer initiators and cancer promoters. Whether fluoride is considered a cancer initiator or a promoter, one's test design must include the fluoride intake by the mother prior and during the time of her pregnancy. This factor is missing in this present study.

In testing the fluoride/osteosarcoma link, one must be able to calculate total fluoride intake at various stages of life preceding the onset of the cancer. This is more difficult than it might at first seem. In calculating total fluoride intake, the study included fluoride tablets used, mouth rinses, toothpaste used, dental treatments, and water fluoridation levels. Missing from this list are calculations of differences in water actually consumed based on differences in ambient temperature, individual work or athletic exercise that greatly increases water consumption, and dietary habits such as processed beverages versus "plain" water. It is not difficult to understand that commercially processed beverages made from fluoridated water are sold in unfluoridated communities. Likewise, it is not difficult to understand that some children drink more processed beverages than water from the tap. Thus, knowing the fluoride concentration of the tap water is not the same as knowing the fluoride intake from one's drinking of fluids.

Further, it is well established that much of our US diet choices are canned or processed foods rather than fresh, unprocessed foods. Community water fluoridation adds fluoride not only to one's drinking water but to foods processed with the fluoridated water. It is for this reason that processed foods of different brands can differ greatly in their fluoride content, and this difference is not recognized when making food purchase choices. It is likely that one family will routinely choose one brand while the next family always uses another brand. Estimating averages does not help since the "average" does not exist; one brand will be fluoridated and the other is not. Fluoride from processed foods comprise a major portion of one's total fluoride intake, often equalling or exceeding that obtained from tap water.' This calculation, too, is missing from the fluoride exposure variables listed in this study.

In the present study, something is odd about the case subjects. While it is routinely found that osteosarcoma is more common in young men than in young women, this study's list of 130 cases included only 42 males, or 32% of the total. Thus, the osteosarcoma cases used were not typical of the disease in question. Did the males go elsewhere for treatment? Did some male cases of osteosarcoma slip through undetected in the study's case selection method? Did the young women with osteosarcoma drink more fluoridated beverages and less unfluoridated water than the young men? From the information given, no clue is found. The authors seem unconcerned over this discrepancy.

Finally, one must question the case-control method of the study. In the case control method, patients with the disease in question are compared to similar appearing, same-age people without the disease. In effect, patients susceptible to osteosarcoma were selected controls, i.e. those without evident osteosarcoma. Given the rarity of osteosarcoma, and the fact that the sources of fluoride exposure are so ubiquitous, it would be no problem to find an equal group of healthy people living in the same communities and using the same toothpaste as those with osteosarcoma.

The fact that the two groups' drinking water and toothpaste choices are the same does not invalidate the conclusion that fluoride was a factor in the development of osteosarcoma. The study's authors apparently assume that osteosarcoma victims require higher fluoride exposure than those without the disease. An equally plausible assumption is that variable individual susceptibility exists such that equal fluoride exposure will affect only those with the requisite susceptibility. Given the rarity of the disease, this seems more probable. The susceptibility for osteosarcoma may stem from early prenatal fluoride exposure or from factors not yet known. The later occurrence of the cancer may require only the level of fluoride exposure common to fluoridated communities. If this assumption was correct, as case-control study such as this comparing only post-natal fluoride exposure between osteosarcoma victims and controls would find no difference.

When polio "epidemics" were common, it was clear that only a small percentage of children in any given community developed clinically apparent poliomyelitis while well over 90% of the children showed an equal rise in polio antibodies. That is, despite equal exposure, only a few children were sufficiently susceptible to be stricken with polio. A similar scenario might well apply to the osteosarcoma problem. Since we often do not know all the factors that "cause" or "promote" a given cancer, we do not know what factors are important in selecting comparison groups. Case-control study designs are not appropriate for all illnesses and this, one might suspect, is one of them.

This present study, while being used to cast doubt on the relationship of fluoride to osteosarcoma, is flawed by: 1) disregard of prenatal fluoride exposure; 2) inadequate calculation of postnatal total fluoride intake; and 3) inappropriate choice of study design. Thus, the study carries little weight in negating the fluoride/osteosarcoma connection or in any consideration of continuing fluoridation as a public policy.

[all emphasis added]

  • 1 Gelberg KH, Fitzgerald, EF, Hwang S. Dubrow R Fluoride exposure and childhood osteosarcoma a case control study. American Journal of Public Health 85 1678-1683 1995
  • 2 Maurer JK, Cheng MC. Boysen BG, Anderson RI Two-year carcinogenicity study of sodium fluoride in rats Journal, National Cancer Institute 82 1118-1126 1990
  • 3 As presented at the NTP peer review conference at Research Triangle Park. North Carolina. 1990, attended by the author
  • 3 Hoover RN, Devesa SS. Canter KP, Lubin JH, Fraumeni JF Jr .Time trends for bone and joint cancers and osteosarcomas in the Surveillance, Epidemiology and End Results (SEER) Program. National Cancer Institute In: Review of Fluoride: Benefits and Risks Report of the Ad Hoc Committee on Fluoride of the Committee to Coordinate Environmental Health and Related
  • Programs U S Public Health Service 1 991 pp F1 -F7
  • 5 Cohn PD A brief report on the association of drinking water and the incidence of osteosarcoma among young males New Jersey Department of Health. Trenton NJ November 8 1992
  • 6 Colborn T, vom Saal FS, Soto AM Developmental effects of endocrine-disrupting chemical in wildlife and humans Environmental Health Perspectives 101 378-384 1993
  • 7 Lee JR Optimal fluoridation - the concept and its application to municipal water fluoridation Western Journal of Medicine 122 431-436
Published by the International Society for Fluoride Research

Dr John R Lee, 9620 Bodega Hwy, Sebastopol CA 95472, USA.

Liu YQ, Promotive action of sodium fluoride on precancerous lesions of hepatocellular carcinoma induced by diethylnitrosamine (DEN) in rats--stereologic study of enzyme histochemistry, Chung-hua Ping Li Hsueh Tsa Chih, 1993 Oct, 22(5), 299-301 (Article in Chinese)

In 10 Wistar male rats, partial hepatectomy was performed and followed by intraperitoneal injection of DEN (30 mg/kg) once and giving drinking water containing 80 ppm sodium fluoride (36 ppm F) for 14 weeks. By stereologic method of enzyme histochemistry, it was observed for the first time that the foci of hepatocytic precancerous enzyme alterations (gamma-GT positive, ATPase negative and G-6-Pase negative) were significantly increased in number and size compared with the negative control group. The results suggest that sodium fluoride promoted the growth of precancerous lesions of the liver induced by DEN in rats, and this has provided some data to the understanding of the relationship between fluorosis and neoplasms [emphasis added]




DATE: May 1, 1990
SUBJECT: Fluoride Conference to Review the NTP Draft Fluoride Report
FROM: Wm L. Marcus, Ph.D., Senior Science Advisor, Criteria & Standards Division, ODW (WH-550D)
TO: Alan B. Hais, Acting Director, Criteria & Standards Division, ODW (WH-550D)

The conference was held in RTP at the NIEHS headquarters on April 26, 1990. The subject of the conference was a peer review of the NTP draft report on the toxicology and carcinogenesis studies of Sodium Fluoride in F344/N Rats and B6C3F Mice (Drinking Water Studies) NTP Report Number 393. Dr. Robert Scala was to chair this meeting but was unable to attend because of ill health. Dr. Michael Gallo appointed acting Chairperson. One of the attenders seated with the panel members was David Rall, Ph.D., M.D., Director of NIEHS. Dr. Rall took an extremely active interest in the proceedings and remained seated for the entire proceedings with only two minor interruptions.

The most disturbing part of the report was the continual reference to the historical controls as having the same or higher cancers as the test groups. On pages 89 - 90 of the report starting with the last paragraph the authors state the following:

An important consideration which limits the usefulness of the historical control data base in the current studies is that the diet used in all other NTP studies had not been closely monitored for fluoride content. Fluoride concentrations in typical batches of NHI-07 diet range between 28 and 47 ppm (.7 and 1.2 mg/kg/day)(Rao and Knapka1, 1987). Assuming a minimum bioavailability of 60% (Tests show 66% absorption page I-18), the historical database animals actually constitute a group receiving sufficient fluoride to place them between the low- and mid-concentration group in the current (the studies reviewed at RTP at the conference). The fact that this fluoride is available for absorption from the standard diet is supported by the levels of fluoride found in the bones of animals maintained on this diet in the six months studies (Appendix I). (The levels in the bones of the rats on the standard NHI chow was ten [10] times the levels of those fed the semisynthetic diet and deionized water, 0.922 vs 0.0901). If the fluoride [is] in fact influencing the "spontaneous " or background incidence of osteosarcoma in male rats, comparisons with those in the historical database maybe misleading. This forces an even greater reliance on the within-study comparisons, ie., the incidences of the dosed groups compared with the concurrent control, in the interpretation of the results of the sodium fluoride studies. [italics & emphasis in memo]

When I plotted a bar graph of osteosarcoma in male rats and placed the historical controls on the graph 0.6% is just where expected. This helps demonstrate a relationship between osteosarcoma and fluoride. The purpose of such graphs is to predict occurrence. Since the historical controls comprise some 6,000 animals, this data point is extremely significant compared to the other three. Osteosarcoma is an extremely rare animal tumor and may be the result of the variable high fluoride content in the feed. In order to demonstrate this, all that need be done is require that the fluoride content of animal chow be lowered dramatically and that fluoride be removed from the water given to the animals under study.

The dose of fluoride to which the concurrent controls were exposed is 0.2 mg/kg/day. A 70 kg man who drinks 2 liters daily is exposed to 0.03 mg/kg/day. The "control" animals were exposed to an amount of fluoride six to seven (6-7 X) greater. Lois Gold, Ph.D. of the review panel concluded that, "this group of animals therefore, can hardly be termed a control group. It can best be described as a lowest dosed group." This is an important consideration because as the document reports on page 9, the levels of fluoride in bone are linearly dependent upon dose and length of exposure ("depend upon total intake") in people. The level of fluoride in ashed samples of bone of 20-30 year old people is 200 - 800 mg/kg compared to 70 to 80 year old people of 1,000 - 2.500 mg/kg. In the document, the authors cited Zipkin2 who reported on bone fluoride concentrations in four groups of individuals with average ages of 56 to 76 who lived in areas with fluoride concentrations in drinking water of 0. 1, 1, 2.6, or 4 ppm The relationship to bone fluoride concentrations and water fluoride content was linear; bone fluoride ranged from about 800 to 7,000 ppm ash with increasing water fluoride."

In the animal studies the levels of fluoride (Appendix I) found in the bones of the animals were the same as or lower than those found in people. The highest dosed level of rats had lower levels of fluoride in their bones (5,470 ppm) compared to people (7,000 ppm) at the MCL of 4 ppm. This can be interpreted as people who ingest drinking water at the MCL have 1.3 times more fluoride in their bones than male rats who get osteosarcoma This is the first time in my memory that animals have lower concentrations of the carcinogen at the sight of adverse effect than do humans. An important toxicologic consideration is that a toxic substance stores at the same place it exerts it toxic activity. This is true of benzene and now for fluoride. Fluoride however, is at twice the concentration in human bones compared to benzene which is 10 to 100 [times] greater in animal marrow. This portends a very serious problem. One would expect to be able to discern a carcinogenic effect in the exposed population when compared to the unexposed population especially if data exist on the populations before fluoridation.

Yiamouyiannis and Burk published epidemiology studies that have since been revised twice3, by Burk (former head of the Cytochemistry section at NIH). In these extensively peer reviewed papers, the authors found that about 10,000 deaths a year are attributable to fluoride water treatment. The U.S. Public Health Service (U.S.PHS) criticized the original studies by erroneously asserting that the results reported by the authors were a result of changes in the age, race and sex composition of the sample. The U.S.PHS made mathematical errors and did not include 90% of the data. U.S.PHS method of analysis when applied to the database, confirmed that 10,000 excess cancer deaths yearly were linked to fluoridation of water supplies. This evidence has been tested most recently in the Pennsylvania Courts and round scientifically sound after careful scrutiny.

There were three different short term in vitro tests performed on fluoride and all these tests proved fluoride to be mutagenic. An Ames test was performed and reported to be negative. Bruce Ames, in a letter to Arthur Upton introduced in the Congressional Record, stated that his test system was inappropriate for fluoride testing based on a number of technical considerations. EPA's own guidelines require that in vitro tests be taken into consideration when found positive. In this case, the mutagenicity of fluoride supports the conclusion that fluoride is a probable human carcinogen. [emphasis added]

Melvin Reuber, M.D, a board certified pathologist and former consultant to EPA and part time EPA employee, reviewed some of pathology slides and the Battelle report. Dr. Reuber has had his pathologic diagnoses questioned several times in the past. When an independent board together with Dr. Reuber went over the Slides his opinion was always upheld. He first published the work that identified hepatocholangiocarcinoma as a pathologic entity. The report changed Battelle's board certified veterinary pathologists diagnoses from hepatocholangiocarcinoma to hepatoblastoma and finally to hepatocarcinoma. Dr. Reuber reviewed the pathology slides and stated that these lesions are indeed hepatocholangiocarcinoma. Because Dr. Reuber first identified and published his findings on this tumor, I trust his opinion in this matter. These tumors are extremely rare. Dr. Reuber's diagnoses would make the liver cancers significant because of their rarity. This changes the equivocal finding of the board to at least some evidence or clear evidence of carcinogenicity. In addition, the oral changes in the report were down-graded from dysplasia and metaplasia to degeneration. Dr. Reuber said that this. change should also be reviewed. The report also down-graded adrenal pheochromocytomas and tumors to hyperplasia. This needs to be reviewed by an independent board. The other liver carcinomas were down-graded to foci by artificially defining a need for 75% compression in the tumor before it was no longer a foci. Using this changed definition carcinomas were down-graded to adenomas and adenomas downgraded to eosinophilic foci. In almost all instances, the Battelle board certified pathologists' findings were down-graded. It is my suggestion that a board independent of NIEHS should be assembled by ODW consisting of human pathologists (for their experience in diagnosing osteosarcoma), the Battelle pathologist (to defend his original diagnoses), Dr. Melvin Reuber, Dr. Thomas Squires and two other well known independent board-certified animal pathologists. The charge to this board is to meet as a body, review the slides, agree on a pathologic diagnoses and prepare a report to be submitted to ODW for incorporation in our docket for the fluoride regulation. [emphasis added]

The report talks about the efficacy of fluoride and tooth decay. Since the studies were performed to determine the carcinogenicity of fluoride this should not have been addressed. There appear to be at least four different publications from the U.S., Canada, and New Zealand that have reported similar or lower tooth decay rates in nonfluoridated areas as compared to fluoridated areas4,5,6,7 Therefore, the entire question of the efficacy of fluoridation based on extensive and multiple studies has been called into question. Our job is to set safe levels for fluoride in drinking water based on the scientific evidence.

The problem with this meeting was the inability of independent reviewers to get to see the slides prior to the meeting. We must perform our own scientific review of the slides and write our conclusions for use in the development of the revised fluoride regulation.

(1) Roa, G.N., and Knappa, J.J. 1987. Contaminant and nutrient concentrations of natural ingredient rat and mouse diet used in chemical toxicology studies. Fundam. Appl. Toxicol. 9, 329-338.
(2) Zipkin, L., McClure, F.J., Leone, H.C., and Lee, W.A. 1958. Fluoride deposition in human bones after prolonged ingestion of fluoride in drinking water. Public Health Rep. 73, 732-740.
(3) Graham, J.R., Burk, O., and Morin, P. 1987. A current restatement and continuing reappraisal concerning demographic variables in American time-trend studies an water fluoridation and human cancer. Proc Pennsylvania Academy of Sci. 61:138-146.
(4) Colquhoun, J. 1987. Comm. Health Studies. 11:85. (5) Gray, a. 1987. J. Canadian Dental Assoc. 53:763. (6) Hildebolt, C.F. et al. 1989. Amer J, Physiol. Anthropol. 78:79-92. (7) Diesendorf, M. 1986. Nature. 321:125.

Maurer JK, Cheng MC, Boysen BG, Anderson RL, Two-year carcinogenicity study of sodium fluoride in rats, J Natl Cancer Inst, 1990 Jul 4, 82:13, 1118-26

To determine the carcinogenic potential of sodium fluoride (NaF), we fed Sprague-Dawley rats a diet containing NaF for up to 99 weeks. Rats receiving NaF at a dose of 4, 10, or 25 mg/kg per day added to a low-fluoride diet were compared with controls receiving either a low-fluoride diet or laboratory chow. Each treatment group consisted of 70 rats of each sex. A 30% decrement in weight gain occurred at an NaF dose of 25 mg/kg per day. Evidence of fluoride toxicity was seen in the teeth, bones, and stomach, and the incidence and severity of these changes were related to the dose of NaF and the duration of exposure. Despite clear evidence of toxicity, NaF did not alter the incidence of preneoplastic and neoplastic lesions at any site in rats of either sex. Results from this study indicate that NaF is not carcinogenic in Sprague-Dawley rats. [see comments by E. Calabrese]

Meng Z, Zhang B, Chromosomal aberrations and micronuclei in lymphocytes of workers at a phosphate fertilizer factory, Mutat Res, 1997 Oct 24, 393(3), 283-288

The frequencies of chromosomal aberrations (CA) and micronuclei (MN) in peripheral blood lymphocytes of 40 workers at a phosphate fertilizer factory in North China, were studied. HF and SiF4 are the main air pollutants and small amounts of dust containing fluoride, NH3 and SO2 were also present in the factory. It was shown that the chemicals caused an increase in both CA and MN. The mean frequencies per 100 metaphase of major CA type (chromosome rings, translocations, and dicentrics) of the workers and the non-exposed controls were 0.91 and 0.24 (p < 0.01), respectively. The average percentages of lymphocytes with MN of the workers and the controls were 1.55 ± 0.71 and 0.62 ± 0.54 (p < 0.01), respectively. Both CA frequency and MN frequency of the workers increased with length of the chemical exposure period up to 10 years.

NTP, National Toxicology Program Technical Report TR 393: Toxicology and Carcinogenesis Studies of Sodium Fluoride (CAS No. 7681-49-4) in F344/N Rats and B6C3F1 Mice (NIH, U.S. Department of Health and Human Services)

a note about the above: the bioassay was first contracted out to the Battelle Institute of Columbus, Ohio. Pathologists found significant dose-related occurrences of osteosclerosis (abnormal bone density); oral tumors; a rare bone cancer (osteosarcoma); and, a rare liver cancer called hepatocholangiocarcinoma (this diagnosis was confirmed by Dr. Reuber, the first pathologist to discover and classify the rare tumor). Battelle's results were systematically down-graded by another contractor, and then again by an NTP panel. Congress first ordered the U.S. PHS to conduct the NTP animal study in 1977. [see NTP Introduction by E. Calabrese for more info]

Smith GE, Is fluoride a mutagen?, Sci Total Environ, 68:1988 Jan, 79-96

Recent studies suggest that fluoride may be genotoxic. While the concentration of fluoride in artificially fluoridated water (1 mg Fl-1) is generally considered to be "safe", levels of fluoride present in a number of widely used dental health products, such as fluoride-containing toothpaste, appear to be potentially mutagenic. Since fluoride is increasingly being used as a drug, and contamination of the total environment by fluoride emissions and solid wastes from industry is a growing problem, a review of the evidence regarding the potential mutagenicity of fluoride may be in order.

Tohyama E, Relationship between fluoride concentration in drinking water and mortality rate from uterine cancer in Okinawa prefecture, Japan., J Epidemiol, 1996 Dec, 6 (4), 184-191.

The Okinawa Islands located in the southern-most part of Japan were under U.S. administration from 1945 to 1972. During that time, fluoride was added to the drinking water supplies in most regions. The relationship between fluoride concentration in drinking water and uterine cancer mortality rate was studied in 20 municipalities of Okinawa and the data were analyzed using correlation and multivariate statistics. The main findings were as follows. (1) A significant positive correlation was found between fluoride concentration in drinking water and uterine cancer mortality in 20 municipalities (r = 0.626, p < 0.005). (2) Even after adjusting for the potential confounding variables, such as tap water diffusion rate, primary industry population ratio, income gap, stillbirth rate, divorce rate, this association was considerably significant. (3) Furthermore, the time trends in the uterine cancer mortality rate appear to be related to changes in water fluoridation practices.

Tsutsui T, Suzuki N, Ohmori M., Sodium fluoride-induced morphological and neoplastic transformation, chromosome aberrations, sister chromatid exchanges, and unscheduled DNA synthesis in cultured syrian hamster embryo cells. Cancer Res 1984 Mar;44(3):938-41

The effects of exposure of early-passage Syrian hamster embryo cells in culture to sodium fluoride have been studied with respect to induction of morphological and neoplastic transformation, chromosome aberrations, sister chromatid exchanges, and unscheduled DNA synthesis. Exposure of Syrian hamster embryo cells to NaF concentrations between 75 and 125 micrograms/ml for 24 hr caused approximately 90 to 40% cell survival and resulted in a dose-dependent increase in the frequency of morphological transformation of the cells. Mass cultures of cells treated with NaF (75 or 100 micrograms/ml) for 24 hr, followed by continuous cultivation for 35 to 50 passages, developed the ability to grow in soft agar and to produce anaplastic fibrosarcomas when injected into newborn hamsters. In contrast, no morphological and neoplastic transformation was observed in untreated cells. Furthermore, a significant increase in chromosome aberrations at the chromatid level, sister chromatid exchanges, and unscheduled DNA synthesis was induced by NaF in a dose- and time-dependent manner. These results indicate that NaF is genotoxic and capable of inducing neoplastic transformation of Syrian hamster embryo cells in culture. A potential for carcinogenicity of this chemical, which is widely used by humans, is suggested. However, the carcinogenic risk of this chemical to humans may be reduced by factors regulating in vivo dose levels.

Tsutsui T, Suzuki N, Ohmori M, Maizumi H, Cytotoxicity, chromosome aberrations and unscheduled DNA synthesis in cultured human diploid fibroblasts induced by sodium fluoride. Mutat Res 1984 Apr;139(4):193-8.

The effects of exposure of cultured human diploid fibroblasts (JHU-1 cells) to sodium fluoride have been studied with respect to cytotoxicity and induction of chromosome aberrations and unscheduled DNA synthesis (UDS) Cytotoxicity of NaF on JHU-1 cells, as determined by a decrease in colony-forming ability, linearly increased with increasing dose of NaF (50-150 micrograms/ml) or exposure time (1-24 h). Treatment of the cells with 50 micrograms/ml NaF for 24 h resulted in a lethality (approximately 70%) similar to that obtained with 100 micrograms/ml for 12 h. A linear increase in cytotoxicity was observed as a fraction of the product of NaF treatment time and dose. JHU-1 cells treated with 20-50 micrograms/ml NaF for 12 or 24 h were analyzed for chromosome aberrations. A significant increase in the frequency of chromosome aberrations at the chromatid level was observed in treated cells in a dose-dependent manner. For detection of UDS, confluent JHU-1 cells were cultured with medium containing low serum and then exposed to NaF in the presence of 10 mM hydroxyurea. Treatment with 100-400 micrograms NaF/ml for 4-24 h reproducibly elicited UDS in a dose-related fashion as determined by direct scintillation counting of [3H]thymidine incorporated into DNA during repair synthesis. These results suggest that NaF causes DNA damage in human diploid fibroblasts in culture.

Zeiger E, Shelby MD; Witt KL, Genetic Toxicity of Fluoride, Environmental and Molecular Mutagenesis, 1993, 21:4, 309-318 (Experimental Carcinogenesis and Mutagenesis Branch, National Institute of Environmental Health Sciences)

F is not mutagenic in standard bacterial systems, but produces chromosome aberrations and gene mutations in cultured mammalian cells. Although there is disagreement in the literature concerning the ability of F- to induce chromosome aberrations in cultured human and rodent cells, the weight of the evidence leads to the conclusion that F- exposure results in increased chromosome aberrations in these test systems. NaF induced primarily chromatid gaps and chromatid breaks, indicating that the rodent cells are responsive in the G2 stage of the cell cycle. In contrast, studies with synchronized human cells indicated that the S phase was the most sensitive. If F does have a cell cycle-specific effect, it could be expected that differences in the cell treatment and harvest protocols could lead to conflicting results for the induction of chromosome aberrations. Gene mutations were produced in cultured rodent and human cells in the majority of the studies. Unfortunately, a number of the in vitro and in vivo cytogenetic studies are of questionable utility because of the protocols used, the quality of the responses reported, or the interpretations of the data. The conflicting results in the in vivo cytogenetic studies are difficult to reconcile. There are reports of increased chromosome aberrations in rat bone marrow and testes, but other studies, using similar protocols and dose ranges, have reported no induced chromosome damage. Although some of the studies were performed at toxic levels of F, other studies, including those that showed positive results, were at F concentrations (1-5 ppm) equivalent to human exposure levels. In the majority of studies that were reported to be positive, there were high background frequencies, or the investigators reported categories of nuclear or chromosome damage that are difficult to interpret. Interestingly, many of the positive results were obtained when anaphase cells were scored, whereas similar treatment protocols in other laboratories yielded negative results when metaphase cells were the only cell type examined. It is difficult, without additional data, to determine the reasons for finding chromosome breaks in anaphase, but not metaphase, cells. Other reports have presented insufficient information to allow adequate evaluations. Therefore, at this time, the question of whether F produces chromosome damage in vivo should be considered unresolved. [emphasis added]