Participants Six healthy volunteers (4 females, mean age 36.2, range 23-52 yr) were recruited among staff members of the Universidad Católica de Santa María, Arequipa, Peru. Participants were taking no medications and had most permanent teeth present. Participants were asked to avoid fish or tea for 12 h before the study and not use fluoridated toothpaste 4 h prior. The Comité de Etica de Investigacion, Universidad Católica de Santa María approved the study and informed consent was obtained.

Study Agent DSF \[Ag(NH3)2F, CAS RN 33040-28-7, Saforide, Toyo Seiyaku Kasei Co. Ltd. Osaka, Japan\] was used. DSF is clear and colorless, with a weak odor of ammonia. The 38% solution contains between 24.4-28.8% (w/v) silver (Ag), and 5.0-5.9% fluoride (F). Concentrations of Ag and F in the DSF used for study were 24.9% and 5.5%, respectively, and the specific gravity was 1.253 at 25°C (Certificate of Analysis Lot #155060). Diammine silver fluoride is also referred to in the literature as silver diammine fluoride, silver diamine fluoride or silver fluoride.

Procedures Three maxillary teeth (cuspid and premolars) in the same quadrant were treated. Three teeth were treated because this was the average number of sensitive teeth reported in an epidemiological study of adults \[5\]. The teeth were free of restorations or cavitation. The facial surface was dried with cotton gauze and then DSF was applied to the cervical area of each tooth. The teeth were isolated in order to avoid wetting the brush with saliva. Each tooth was coated using a separate microbrush, which was dipped in the DSF and weighed before and after the application (Model S2000, Kern \& Sohn GmbH, Balingen, Germany). The sum of the differences in weight of material applied was calculated for each participant.

Blood Collection Immediately before application of DSF and then again at approximately 30 min, 1, 2,3 and 4 h after application, blood was obtained from a peripheral vein. The time periods were picked based on a previous pharmacokinetics study of fluoride varnish \[6\]. The blood was transferred to a fluoride-free plastic Vacutainer tube with clot activator (BD Vacutainer Plus Plastic Serum Tube, BD Diagnostics, New Jersey). The tubes were spun at 1100 g for 10 min and serum transferred by plastic pipette to plastic cryogenic tubes. The samples were frozen and transported to the University of Washington for analysis.

Analysis Fluoride. Samples were thawed and analyzed in duplicate or triplicate using the diffusion and detection method with hexamethyldisiloxane (HMDS) and Orion F-sensitive electrode \[7-8\]. To improve detection, the pre-diffusion step was omitted \[9\], and seals were completed before mixing 1 mL serum with 1 mL HMDS, saturated 3 M H2SO4 \[10\]. The trapping solution was 20 µmoles 1 M NaOH. It was dried down to eliminate variability of evaporation and residual HMDS before adding 100 µL 3 M acetic acid to a final pH 3.4. Standards were 10 and 100 µM fluoride in the same final concentration of NaOH and acetic acid corresponding to 1 and 10 µM F in the samples. ORION F and sleeve type reference electrodes were coupled with an Accumet Basic 0.1 mV meter (Thermo Fisher Scientific, Milwaukee WI).

Vacutainers were tested for fluoride by adding bovine plasma to 2 tubes and letting them sit overnight before combining the 2 samples and comparing them to plasma directly placed in diffusion dishes. Average concentrations (μM±SD) were 0.34±0.014 and 0.32±0.012, respectively. Therefore, less than 2% F in the serum samples came from the Vacutainers and clot enhancer. Recovery (mean±SD) of 1 nmole/ml F added to bovine serum was 101±4%. Fluoride levels were averaged and are expressed as µM.

Silver. Samples were thawed and analyzed using Inductively Coupled Plasma-Mass Spectrometry (EPA 6020a Rev.1 2007). Serum (0.5 mL) was brought to 2 mL with dilute acid (final concentration: 2% HCl, 1% HNO3; trace metal grade, Fisher, Fairlawn, NJ). Samples were centrifuged (1290 g) for 10 min and filtered (0.45 µm, PTFE; all polypropylene syringe) into 15 mL polypropylene centrifuge tubes. The samples were analyzed on an Agilent 7500CE (Santa Clara, CA) inductively-coupled mass spectrometer (ICPMS) with He as the collision cell gas and an ASX-510 autosampler (CETAC, Omaha, NB). A Micromist nebulizer (Glass Expansion, Pocasset, MA) was used (1.15 L/min carrier gas; no makeup gas). Spray chamber temperature was 2°C. RF power was 1500 W. Ag was quantified using Y as internal standard. Calibrants were diluted with 2% HCl and 1% HNO3 prepared from commercial ICPMS grade solutions (Ultra Scientific, N. Kingstown, RI; BDH Aristar, VWR, Radnor, PA) and ranged from 0.5-50 ng/mL. Calibration was by weighted (1/cps) linear regression. Data was corrected by procedure blank. Values of silver less than 2 ng/mL were recoded to 2. Silver concentrations are expressed as ng/mL.

Soft Tissue Assessment The gingiva adjacent to the application of DSF and the mucosa generally were observed at baseline and 24 h after treatment. Erythema, bleeding, white changes, ulceration and pigmentation were assessed using methods developed for an earlier study \[1\].