Official citation: NTP (National Toxicology Program). 2016. Systematic Literature Review on the Effects of Fluoride on Learning and Memory in Animal Studies. NTP Research Report 1. Research Triangle Park, NC: National Toxicology Program.

Conclusion (page 59):

Very few studies assessed learning and memory effects in experimental animals (rats and mice) at  exposure levels near 0.7 parts per million, the recommended level for community water  fluoridation in the United States. At concentrations higher than 0.7 parts per million, this systematic  review found a low to moderate level-of -evidence that suggests adverse effects on learning and memory in animal exposed to fluoride. The evidence is strongest (moderate level- of-evidence) in animals exposed as adults and weaker (low level- of-evidence) in animals exposed during development. Confidence in these findings was reduced primarily based on potential confounding of the learning and memory assessments by deficits in motor function or fear and risk of bias limitations. Additional research is needed, in particular to address potential effects on learning and memory following exposure during development to fluoride at levels nearer to 0.7 parts per million. NTP is conducting laboratory studies in rodents to fill data gaps identified by this systematic review of the animal studies. The findings from those studies will be included in a future systematic review to evaluate potential neurobehavioral effects from exposure to fluoride during  development with consideration of human, experimental animal and mechanistic data.

Limitations of the Systematic Review(pages 57-58):

• Evidence synthesis was restricted to potential learning and memory effects of fluoride. Additional analyses could be conducted to understand patterns of findings between learning and memory and other behavioral responses, such as motor and sensory function, and general systematic toxicity.

• This review focused on selected behavioral measures. Studies examining fluoride exposure on endpoints assessing brain-related cellular, morphometric, or histological endpoints were considered beyond the scope of this analysis. Similarly, effects on thyroid function, which might alter specific neurobehavioral measures, were considered beyond the scope of this review. In the NRC report (2006), the histological, chemical, and molecular studies were considered to provide evidence that fluorides can interfere with the functions of the brain and the body by direct and indirect means. Potential mechanisms described included reduced brain content of lipids and phospholipids, phosphohydrolases and phospholipase D, and other proteins; inhibition of cholinesterase activity (including acetylcholinesterase); reduction in acetylcholine receptors in the brain; activation of Gp, a protein of the G family, which mediates the release of  many central nervous system transmitters; and increased production of free radicals in the  brain. Cellular effects of the neocortext [sic, neocortex], hippocampus, and amygdala also were described in rat  studies.

• Dose levels were converted to fluoride equivalents (F) expressed as mg/kg-d and ppm (e.g., 100 ppm sodium fluoride = 45.3 ppm fluoride). Most studies reported sodium fluoride as the form of fluoride administered. In several studies, the specific form of the chemical administered to animals was reported simply as “fluoride” with no details on counter ion, source, or purity, and no conversions to ionic fluoride equivalents were made. Dose levels expressed in fluoride equivalents would overestimate dose levels if, in fact, sodium fluoride were the test compound administered.

• Values for dose conversion (i.e., body weight, food and water consumption) were based on EPA dosimetry guides (US EPA 1988 ; US EPA 1994 ). The European Food Safety Authority has developed more recent guidance on default values (EFSA 2012). Use of different default values can influence the dose levels as expressed in mg/kg.

• A meta-analysis was not performed in the current review, but quantitative approaches could be one way to understand sources of heterogeneity more completely and how they might affect confidence ratings, that is, heterogeneity for dose level could lead to greater confidence in dose- response assessment, while heterogeneity due to risk of bias might lead to reduced confidence.

• The ability to address potential publication bias was limited due to (1) challenges of having to select one outcome per study using approaches such as funnel plots when multiple endpoints related to the primary outcome were reported; (2) three or fewer studies being available for certain behavior tests; and (3) a lack of reporting on funding and conflict of interest in papers. In addition, analytical tools, such as funnel plots or trim-and-fill approaches, could be useful to assess publication bias but also have substantial limitations and should be interpreted with caution (Guyatt et al. 2011c ). These limitations are especially pertinent for studies having small sample sizes, as is the case in the fluoride literature. In the current analysis, publication bias was described as “undetected.” This determination is based on GRADE guidance to use ratings of either “undetected” or “strongly suspected,” acknowledging the difficulty in having confidence that publication bias is absent and identifying a threshold to rate down the quality of evidence based on its presence (Guyatt et al. 2011c).

Contributors to report:
Kristina Thayer, PhD NIEHS/NTP, Office of Health Assessment and Translation, co-project lead
Jean Harry, PhD NIEHS/NTP, Neurotoxicology Group Leader, NTP Laboratories, co-project lead
Andrew Shapiro, MS NIEHS/NTP, NTP Program Operations Branch
Stephanie Holmgren, MS NIEHS/NTP, Office of Scientific Information Management
Mamta Behl, PhD NIEHS/NTP, NTP Toxicology Branch
Mary Wolfe, PhD NIEHS/NTP, Office of Liaison, Policy, and Review
John Bucher, PhD NIEHS/NTP, Associate Director’s Office

External technical advisors
Malcolm Macleod, PhD, University of Edinburgh, Department of Neurology and Translational  Neuroscience Coordinator of the Collaborative Approach to Meta-Analysis and Review of Animal Data in Experimental Studies

Carlijn Hooijmans PhD, Radboud University Medical Centre Departments of Systematic Review Centre for Laboratory animal Experimentation (SYRCLE) and Anesthesiology

Contract support: Assisted in protocol development, literature screening, data extraction, risk of  bias assessment and document production
Robyn Blain, PhD – ICF International
Canden Byrd, BS – ICF International
Greg Carter, MEM – ICF International, co-project lead
Susan Goldhaber, MPH – ICF International
Pamela Hartman, MEM – ICF International, technical lead
Cara Henning, PhD – ICF International, co-project lead
Tao Hong, PhD – ICF International
Penny Kellar, MS – ICF International
Bryan Luukinen, MSPH – ICF International
Maureen Malloy, BA – ICF International
Whitney Mitchell, BS – ICF International
Johanna Rochester, PhD – ICF International

Protocol peer reviewers
Emily Sena, PhD – University of Edinburgh, Department of Neurology and Translational  Neuroscience Coordinator of the Collaborative Approach to  Meta-Analysis and Review of Animal Data in Experimental Studies

Anonymous – National Industrial Chemicals Notification and Assessment Scheme

Report peer reviewers

Charles Vorhees, PhD – Cincinnati Children’s Hospital Medical Center Division of Neurology

Russell Carr, PhD – Center for Environmental Health Sciences, Department of Basic Sciences College of Veterinary Medicine Mississippi State University