In the below letter to the editor of Neurotoxicity Research, Dr. Jean Harry, lead contact researcher for the 2017 McPherson, et al NTP study, responds to criticisms made by fluoridation opponents:
July 5, 2018
Dear Dr. Kostrzewa
I am writing in response to a letter to the editor submitted to Neurotoxicity Research regarding the paper “An Evaluation of Neurotoxicity Following Fluoride Exposure from Gestational Through Adult Ages in Long-Evans Hooded Rats.” McPherson et al, Neurotox Res. 2018, Feb 5. doi: 10.1007/s12640-018-9870-x). The authors of the letter argue that results from the study are not reliable or credible because the Long-Evans Hooded rat has been found to be insensitive to fluoride-induced neurotoxicity. In their letter, it is stated that “…research previously identified the hooded rat as a species of rodent that had lower sensitivity to fluoride induced neurotoxicity which accounted for the lack of any effect of fluoride exposure on memory or learning (Elliott L, Lack of Effect of Administration of Fluoride on the Central Nervous System of Rats. Acta Pharrnacol. et Toxicol. 1967, 25, 323-328).” The authors argue that the insensitivity relative to other rat strains is related to pharmacokinetic factors and/or a lower inherent response to fluoride in the Long-Evans Hooded rat.
One main point that the letter makes is that McPherson et al (2018) did not find a significant difference in plasma levels between the 10 and 20 ppm fluoride exposure groups. It is true that plasma levels in the adult (0.036 or 0.25 g/ml) did not demonstrate a difference between the 10 and 20 ppm however, the levels in the 20 ppm group were not significantly lower than the 10 ppm dose group. It is likely that the low exposure levels used (10 and 20 ppm) simply do not allow for a reliable dose response in plasma. The levels reported in McPherson et al. (2018) may reflect timing in collecting blood from the animals since any consumption of drinking water or it may simply reflect the very transient and rapid clearance of fluoride in the blood (Whitford, 1994). When considering the more relevant tissues for drinking water exposure1, the McPherson et al (2018) study reported a dose response for fluoride levels in brain and bone of the weanling rat. In the adult, brain levels at 20 ppm fluoride were 2.5x higher than in the 10 ppm group and femur levels showed an increase with increasing dose levels. There is no evidence that Long-Evans Hooded rats are differentially sensitive to fluoride exposure, at least from a toxicokinetic perspective. Any such conclusion would require a concurrent comparison between Wistar /SD rats with Long-Evans Hooded rats.
The letter notes that Elliot (1967) did not observe any significant effect on T-maze performance in Long-Evans Hooded rats and concludes that Long-Evans rats are behaviorally insensitive to developmental fluoride exposure. Unfortunately, Elliot (1967) did not measure fluoride levels, so it is not possible to compare between studies. Elliot (1967) found no effects of exposure to fluoride on behavior using an elevated 30 choice multiple T-maze. McPherson et al (2018) observed no exposure-related differences in motor, sensory, or learning and memory performance on running wheel, open-field activity, light/dark place preference, elevated plus maze, pre-pulse startle inhibition, passive avoidance, Morris water maze acquisition, probe test, reversal learning, and Y-maze. These tests have been used extensively in neurobehavioral toxicity testing with a wide variety of rat strains. It is true that behavioral effects following fluoride exposure have been reported in Wistar and Sprague-Dawley rats but there is simply not enough data in the literature to conclude that Long-Evans Hooded rats are behaviorally insensitive to fluoride. A detailed consideration of these effects and the dose levels at which they occurred was presented in the Discussion of the McPherson et al (2018) paper. Consistent with the findings of the NTP Systematic Review, the interpretation of findings was confounded by likelihood of non-specific sensory or motor effects.
The letter also cites Varner et al. [Varner, J.A, Horvath, W.J., Huie, C.W., Naslund, H.R., Isaacson, R.L. Chronic aluminum fluoride administration: I. Behavioral observations, Behav. Neural Biol. 61 1994. 233–241] as additional evidence that Long-Evans Hooded rats are not sensitive to fluoride exposure. In this study, aluminum fluoride was used as the compound for exposure and experimental questions focused on aluminum neurotoxicity. Aluminum, at certain levels, has the capacity to decrease F uptake and reduce signs of F toxicity (Ahn et al., 1995; Lubkowska et al., 2006). In a subsequent study, Varner et al. (1998) reported neuronal abnormalities and compromised blood brain barrier in Long-Evans Hooded rats following long-term NaF exposure. Thus, data from the Varner et al. (1994, 1998) studies do not directly support the conclusion that Long Evans Hooded rats are insensitive to fluoride.
Finally, the letter avers that the selection of the Long-Evans Hooded rat was based on the need to support a “predefined outcome, or that results are biased on selection of species which misrepresent results.”
The rationale for the McPherson et al (2018) study was clearly stated: “The current study was designed to address issues identified in the NTP systematic review (NTP 2016) of determining low to moderate levels of evidence for effects of F− exposure on learning and memory and to address the paucity of quality studies conducted at exposure levels near the recommended level for community water fluoridation in the USA.”
The rationale for strain selection was based on three factors.
• For learning and memory tasks requiring detection of visual cues, rats with pigmented eyes offer better visual acuity than albino rats (Prusky et al 2002). As presented in the NTP systematic review, longer latencies for learning and memory tasks in fluoride-exposed rats raised the possibility that some deficits may also be due to compromised vision.
• The Long-Evans Hooded rat is a cross-breed of Wistar albino females with wild gray males. Thus, it allowed us to stay with the predominant Wistar rat genetic background yet have the benefit of pigmented eyes.
• Research groups such as NIDA have long employed Long-Evans Hooded rats for behavioral tests such as those used in the McPherson et al (2018) paper, and such research provided us a strong historical database for the behavior in rats maintained on the standard diet used in the McPherson et al (2018) study.
In summary, far from generating “false results” that may “misinform the public”, our data utilize an exposure level near the recommended level for human exposure and provide an extensive, systematic evaluation of sensory, motor, and cognitive function in a relevant animal model. Instead of misleading regulators and the public, the results of the McPherson et al (2018) help clarify a generally confusing database and can only facilitate decisions concerning the safety of fluoride exposure through the drinking water.
1 Quoted from McPherson et al (2018). “Fluoride is rapidly absorbed from the gastrointestinal tract with transient peak plasma concentrations of approximate 30 min half-life.” “Calcified tissues such as the bone and teeth readily incorporate fluoride (NRC 2006) most prominently during periods of rapid growth and represent approximately 99% of the body burden of F− that is not rapidly excreted by the urine (Kaminsky et al. 1990; Hamilton 1992; Whitford 1996). In humans, the average bone concentration is linearly related to water concentration and duration of exposure (Jackson and Weidmann 1958; Zipkin et al. 1958; Arnala et al. 1986; Rao et al. 1995). In rodents, F− also accumulates in the bone (Rumiantsev et al. 1988; Bucher et al. 1991; de Carvalho et al. 2006; Gui et al. 2010; Dong et al. 2015a) increasing over age and exposure (Zipkin and McClure 1952; Ekstrand et al. 1994; Dunipace et al. 1995; Whitford 1999).”
Ahn HW, Fulton B, Moxon D, Jeffery EH. Interactive effects of fluoride and aluminum uptake and accumulation in bones of rabbits administered both agents in their drinking water. J Toxicol Environ Health. 1995 Mar;44(3):337-50.]
Lubkowska A, Chlubek D, Machoy-Mokrzyniska A. The effect of alternating administration of aluminum chloride and sodium fluoride in drinking water on the concentration of fluoride in serum and its content in bones of rats. Ann Acad Med Stetin. 2006;52 Suppl 1:67-71.
Prusky G.T., Harker K.T., Douglas R.M., Whishaw I.Q. 2002. Variation in visual acuity within pigmented and between pigmented and albino rat strains. Behav. Brain Res. 136:339-348.
Varner JA, Jensen KF, Horvath W, Isaacson RL. Chronic administration of aluminum – fluoride or sodium-fluoride drinking water: alterations in neuronal and cerebrovascular integrity. Brain Res 784:284-298, 1998.
Whitford GM. 1994. Intake and metabolism of fluoride. Adv. Dent. Res. 8(1):5-14.