Publication

McPherson CA, Zhang G, Gilliam R et al. — Neurotoxicity Research. 2018 February 5. Epub ahead of print

Reviewer

Center for Fluoride Research Analysis/ Fluoride Science Editorial Board


Background: The National Toxicology Program (NTP) released a systematic review, the Effects of Fluoride on Learning and Memory in Animal Studies, in 2016 in response to reported association between high levels of naturally occurring fluoride in water and lower IQ. Of 68 studies reviewed in detail, only 32 studies were available for the analyses to generate conclusions due to serious risk of bias and incomparable measurements and designs used across the rest of the studies. These studies showed low-to-moderate confidence for a pattern of findings suggestive of fluoride’s effect on learning and memory. The NTP review group identified the following issues in the available literature and declared an intent to fill data gaps by conducting laboratory studies in rodents in the near future.

  • Very few studies assessed learning and memory effects in experimental animals at exposure levels near 0.7ppm and had information on alternative sources of fluoride (i.e. food, water supply) available, thus relevance of the findings to human exposure levels in the optimally fluoridated communities (0.7ppm fluoride concentration) is unknown.
  • The outcome endpoint in the majority of studies was a simple latency measurement of learning or memory in the final training session rather than an evaluation of the acquisition of the task to demonstrate learning. Thus, interpretation of the data is hindered by inability to exclude alterations from baseline levels or differences in motor-related performance over the training session as contributing factors.
  • In many studies, there was a lack of reporting of 1) randomization and blinding, 2) specification of test methodologies to assess the outcomes, and/or 3) controlling of confounders such as litter effects, sex, life-stage at exposure, and duration of exposure.  Studies also appeared statistically underpowered to detect a <10% or <20% change from controls for most behavioral endpoints.

 

Methods: The current study led by the Neurotoxicology Group of the NTP Laboratory addressed the previously identified methodological/design issues in the literature successfully. Specifically, the methodological improvements are notable in the following areas:

  • The fluoride exposures simulated human fluoride exposures by the use of equivalent fluoride doses and establishing a separate route of exposures from diet (20.5ppm vs. 3.24ppmF) and drinking water (0, 10, or 20ppm). Fluoride concentration of 20ppm in rat’s drinking water was equivalent to 4ppm, the US EPA’s current maximum contaminant level, based on the conventional wisdom that a 5-fold increase in dose is required in animals to achieve comparable human serum levels. The exposure levels were validated by assessing the fluoride deposition and accumulation in brain and bone (femur) in addition to fluoride levels in plasma and urine.
  • The experiment (exposure to fluoridated food and water, available ad libitum) began on gestational day 6 and continued throughout lactation. Male pups were observed through adulthood (postnatal day >90).
  • The neurobehavioral endpoint in male pups was measured in various domains: Learning, memory, motor, sensory function, depression, and anxiety. Learning and memory were also evaluated across different tests and in reversal trials and demonstrated acquisition over sessions examining a number of different aspects of performance.
  • Additional effects reported for fluoride exposure that may influence behavior were examined (i.e. thyroid hormone levels, kidney, liver, reproductive system histopathology, and neuronal and glia morphology in the hippocampus) to obtain a better understanding of observed effects.
  • To minimize biases, randomizations and blinding are sufficiently implemented and documented along with detail description of test procedures. Many of behavioral tests were video captured for detail analysis.
  • The authors statistically determined group sizes to sufficiently detect significant differences (p<0.05) between experimental and control groups.

 

Summary Findings and Public Health Implications:

  • Developmental exposure to fluoride from drinking water and diet beginning on gestational day 6 were associated with elevated internal fluoride levels in brain and femur as well as plasma and urine of male rat offspring. A differential absorption of fluoride between water and food was also demonstrated.
  • Fluoride exposure at the levels examined in this study was not found to alter motor performance or learning and memory in the test paradigms assessed or alter thyroid hormone (T3, T4, or TSH) levels or produce neuronal damage or glia reactivity in the hippocampus, or histological damage in heart, kidney, or liver. The only exposure-related effect that they found was mild hyperanalgesia and mild inflammatory response in the prostate.
  • This latest research on fluoride and neurobehavioral health overcame many limitations and weaknesses of previous studies and demonstrated 1) relationships between developmental fluoride exposures from water & diet and fluoride levels in various tissues and specimens in offspring and 2) no exposure-related differences in motor, sensory, or learning and memory performances in rats.
  • When the 2006 NRC report suggested a need for more research on neurotoxicity and neurobehavioral effects of fluoride, the committee was basing this on available data from human studies conducted in fluoride-endemic regions showing high-risk of bias but some consistencies in the findings. Meanwhile data from available molecular and cellular studies could be interpreted to suggest potential changes in nervous system functions but only a few animal studies reported unsubstantial magnitude of alterations in the behavior of rodents after fluoride treatment. Since then, more research (including epidemiological and animal studies) were published, yet the majority of studies still suffer from various sources of risk for bias and the accumulated evidence remains mixed.
  • The findings of this well-controlled animal study directly address previous concerns regarding potential biological plausibility of fluoride as a neurotoxin. The findings provide valuable information and assurance that low-level fluoride exposures from water and diet that are equivalent to the levels allowed in the US does not result in clinically adverse neurobehavioral function or pathological effects in various organs.