In vitro assessment of a toothpaste range specifically designed for children

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1 ORIGINAL ARTICLE International Dental Journal 2013; 63 (Suppl. 2): doi: /idj In vitro assessment of a toothpaste range specifically designed for children David Churchley 1 and Bruce R. Schemehorn 2 1 GlaxoSmithKline Consumer HealthCare, Weybridge, UK; 2 Therametric Technologies Inc, Noblesville, IN, USA. Objective: To evaluate the ability of a range of low abrasivity experimental toothpastes designed for use by children at different stages of their development (typically ages 0 2 years, 3 5 years and 6+ years) to promote fluoride uptake and remineralisation of artificial caries lesions. Methods: ph cycling study: demineralised human permanent enamel specimens were subjected to a daily ph cycling regime consisting of four 1-minute treatments with toothpaste slurries, a 4-hour acid challenge and remineralisation in pooled whole human saliva. Surface microhardness (SMH) was measured at baseline, 10 days and 20 days, and the fluoride content determined at 20 days. Enamel Fluoride Uptake (EFU): these studies were based on Method #40 described in the US Food and Drug Administration (FDA) testing procedures. Abrasivity: relative enamel abrasivity (REA) and relative dentine abrasivity (RDA) were measured using the Hefferren abrasivity test. Bioavailable fluoride: the bioavailable fluoride was determined for all experimental toothpastes from slurries of one part toothpaste plus 10 parts deionised water. Results: Enamel remineralisation measured by changes in SMH correlated with enamel fluoride content. A statistically significant fluoride dose response was observed for all toothpastes tested across all age groups (P < 0.05). The fluoride content of specimens in the ph cycling model correlated with the EFU testing results. The enamel and dentine abrasivities were low and the level of bioavailable fluoride was high for all experimental toothpastes. Conclusion: A series of low abrasivity experimental toothpastes were developed which were effective at promoting fluoride uptake and remineralisation of artificial caries lesions. Key words: Remineralisation, fluoride, children, toothpaste INTRODUCTION In children, the deciduous or primary teeth typically begin to erupt at 6 8 months of age. However, from around the age of 6 years, these teeth start to be replaced by permanent teeth. At this stage, the mouth will contain both deciduous and permanent teeth until the last deciduous tooth is lost at around 12 years of age 1,2. There are a number of important differences between deciduous and permanent enamel. Specifically, deciduous enamel contains more organic material 3, is generally less mineralised in the outer enamel layers 4 and has a higher porosity 5. This makes deciduous enamel more susceptible to acid-mediated dissolution than permanent enamel 6 8. In addition, deciduous enamel is softer and therefore less mechanically resistant than permanent enamel. Consequently, tooth wear owing to abrasion can be more pronounced in deciduous than in permanent teeth 9. Childhood caries is recognised as being a major public health problem among the general population. According to the National Health and Nutrition Examination Survey carried out from 1999 to 2004 in the USA, 42% of children aged 2 11 years have had dental caries in their primary teeth and 21% of children aged 6 11 years have had dental caries in their permanent teeth 10. A number of comprehensive reviews of caries clinical trial data in children and adolescents concluded that there was strong evidence that daily use of fluoride (F)-containing toothpastes can reduce the incidence of caries compared with a placebo or with non-brushing 11,12. These effects were also improved with supervised brushing and increased frequency of brushing 11. Mechanistic studies have shown that the anti-caries effect of fluoride is through the prevention of demineralisation and enhancement of remineralisation 13.In addition, fluoride has been shown to interfere with bacterial metabolism in vitro, which may inhibit plaque acid production 14. A series of guidelines have been established by the US Food and Drug Administration (FDA), the American FDI World Dental Federation

2 In vitro assessment of a toothpaste range Dental Association (ADA) and FDI World Dental Federation to ensure that marketed toothpastes are safe and effective Caries clinical trials are considered to be the ultimate proof of anti-caries effectiveness, however, because of the long duration and high costs associated with these types of studies, a number of preclinical methods known as bioequivalence studies have been developed to evaluate fluoride efficacy 18. In these studies, the experimental toothpaste is tested against a clinically proven control toothpaste containing the same active ingredient at the same nominal concentration. The maximum permitted concentrations of fluoride for toothpastes in different markets are governed by regulatory requirements; however, fluoride concentrations for specific child age ranges are generally set by national guidance via health authorities or dental associations. A new range of toothpastes have been developed for use by children at different stages of their development (typical ages are 0 2 years, 3 5 years and 6+ years) containing different concentrations of fluoride and for different markets. The aim of the present studies was to evaluate the efficacy of these toothpastes to (1) promote lesion remineralisation under dynamic demineralising/remineralising conditions simulating in vivo caries formation, and (2) promote enamel fluoride uptake. The enamel and dentine abrasivities of these toothpastes were also determined relative to the ADA reference abrasive. MATERIALS AND METHODS Materials The experimental toothpastes and commercial toothpaste controls evaluated are shown in Tables 1 and 2, respectively. The placebo toothpastes were fluoride-free variants of the corresponding experimental toothpastes. ph cycling The method used was based on the model of White 19,20 which was subsequently modified by Schemehorn et al Specimen preparation. Enamel specimens (3 mm diameter) were removed from extracted human permanent teeth and mounted on rods. The specimens were initially ground by hand on a lapidary wheel (600 grit wet/dry paper). Following hand grinding the specimens were mounted on a counter rotational grinding/polishing apparatus and finally ground for 3 minutes with the same paper. The specimens were then polished on the same apparatus with 0.05 lm Gamma Alumina (Buehler, Lake Bluff, IL, USA) against a urethane pad using a force of 0.1 N for 1 hour. Baseline surface microhardness. The initial baseline surface microhardness (SMH) of the sound enamel Table 1 Typical age ranges, fluoride concentrations, abrasive, pack type and intended markets for each experimental toothpaste Age range Fluoride concentration (ppm) and source Abrasive Pack type Intended market 0 2 years 0*, 500 or 1,000 (as sodium fluoride (NaF)) Silica Tube Non-US 3 5 years 0*, 250, 500, 1,000 or 1,450 (as NaF) Silica Tube Non-US 6+ years 0*, 500, 1,450 (as NaF) Silica Tube Non-US 2 5 years 1,150 (as NaF) Silica Tube and pump US 6+ years 0*, 250, 1,150 (as NaF) Silica Tube US *Matched placebo formulation. Matched dose response control. Table 2 Age ranges, fluoride concentrations, abrasive and current market for each commercial toothpaste control Toothpaste Age range Fluoride concentration (ppm) and source Abrasive Market Manufacturer Odol-Med3 Milchzahn 0 6 years 500 (as NaF) Silica Germany GlaxoSmithKline Aquafresh Milk Teeth 0 3 years 1,000 (as sodium Silica UK GlaxoSmithKline monofluorophosphate (SMFP)) Aquafresh Little Teeth 4 6 years 1,000 (as NaF) Silica South Africa GlaxoSmithKline Aquafresh Kids Bubble Fresh 2 years and older 1,100 (as SMFP) Chalk US GlaxoSmithKline Aquafresh Big Teeth 6+ years 1,400 (as NaF) Silica UK GlaxoSmithKline Aquafresh Little Teeth 4 6 years 1,400 (as NaF) Silica UK GlaxoSmithKline Aquafresh Fresh and Minty Not specified 1,450 (as NaF) Silica UK GlaxoSmithKline USP Reference Toothpaste N/A Concentration not specified (as NaF) Silica N/A Not specified 2013 FDI World Dental Federation 49

3 Churchley and Schemehorn specimens was determined using a Vickers hardness indenter at a load of 200 g for 15 seconds. The average baseline specimen SMH was determined from four indentations. Only specimens with a sound SMH range of Vickers hardness numbers (VHN) were accepted. Initial demineralisation. Artificial lesions were formed in the enamel specimens by a 68-hour immersion into a solution of 0.1 M lactic acid and 0.2% w/v Carbopol C907 which had been 50% saturated with hydroxyapatite and adjusted to ph 5.0 with potassium hydroxide. The initial SMH of the demineralised specimens was determined using the procedure described above. Only specimens with a lesion SMH range between 25 and 45 VHN were accepted into the studies. Specimens were balanced into groups and subgroups based on their post-demineralisation SMH values. Thirty specimens per treatment group were used in this study. Treatment slurries. During the treatment period, the specimens were immersed in toothpaste slurries to simulate daily exposure to toothpaste. The slurries were prepared by adding 5.0 g of toothpaste to 10.0 g of pooled human saliva in a beaker with a magnetic stirrer bar. All treatments were stirred at 350 rpm. A fresh slurry was prepared immediately before each treatment period. Treatment regimen. The ph cycling regimen consisted of a 4 hour per day acid challenge in the lesion forming solution and minute treatment periods with toothpaste slurries. After the treatments, the specimens were rinsed with running deionised water. During remineralisation periods, specimens were stored in pooled human saliva. This regimen was repeated for 20 days. The treatment schedule is shown in Table 3. Post-treatment surface microhardness. After 10 days and 20 days of treatment, the average specimen SMH Table 3 ph cycling regimen Time of day Treatment 08:00 08:01 Toothpaste treatment* 08:01 09:00 Saliva treatment 09:00 09:01 Toothpaste treatment 09:01 10:00 Saliva treatment 10:00 14:00 Acid challenge 14:00 15:00 Saliva treatment 15:00 15:01 Toothpaste treatment 15:01 16:00 Saliva treatment 16:00 16:01 Toothpaste treatment 16:01 08:00 Saliva treatment *On the first day this treatment was not given; the study started with the saliva treatment to allow a pellicle to form. was determined from four indentations on each specimen, next to the baseline indentations. The difference between the SMH following treatment and the initial lesion SMH indicated the ability of that treatment to enhance remineralisation after 10 days and 20 days of treatments. Fluoride analysis. At the end of the 20-day treatment regimen, the fluoride content of each enamel specimen was determined using the microdrill technique to a depth of 100 lm. The diameter of the drill hole was also determined. The enamel powder from the drill hole was collected and dissolved in 20 ll of perchloric acid (HClO 4 ) followed by the addition of 40 ll of citrate/ethylenediaminetetraacetic acid (EDTA) buffer and 40 ll of deionised water. Solutions were analysed for fluoride using an ion-selective electrode by comparison with a similarly prepared standard curve. Fluoride content was calculated as lg F/cm 3. Enamel fluoride uptake (EFU) The methodology was identical to the one identified as Method 40 in the US Anti-Caries Monograph 18 except that the lesions were formed using a solution of 0.1 M lactic acid containing 0.2% w/v Carbopol 907 and 50% saturated with hydroxyapatite (ph 5.0). Sound, upper, central, bovine incisors were selected and cleaned of all adhering soft tissue. A core of enamel 3 mm in diameter was prepared from each tooth by cutting perpendicular to the labial surface with a hollow-core diamond drill bit. Each specimen was embedded in the end of a Plexiglas rod using methyl methacrylate and polished with 600 grit wet/ dry paper followed by 0.05 lm Gamma Alumina (Buehler, Lake Bluff, IL, USA). Twelve specimens per group were used in this study. Each enamel specimen was etched by immersion into 0.5 ml of 1 M HClO 4 for 15 seconds with continuously agitation. A sample of each solution was then buffered with a total ionic strength adjustment buffer (TISAB) to a ph of 5.2 (0.25 ml of sample, 0.5 ml of TISAB and 0.25 ml of 1 M sodium hydroxide) and the fluoride content determined by comparison to a similarly prepared standard curve (1 ml standard and 1 ml TISAB). This data formed the baseline fluoride concentration of each specimen before treatment. The specimens were once again ground and polished as described above. An incipient lesion was formed in each enamel specimen by immersion into 0.1 M lactic acid containing 0.2% w/v Carbopol 907 and 50% saturated with hydroxyapatite (ph 5.0) for 24 hours at room temperature. These specimens were then rinsed with deionised water and stored in a humid environment until used FDI World Dental Federation

4 In vitro assessment of a toothpaste range The treatments were performed using supernatants of the toothpaste slurries. The slurries were prepared by adding 9.0 g of toothpaste to 27.0 ml of deionised water. The slurries were mixed well and then centrifuged. The specimens were then immersed into 25 ml of their assigned supernatant with constant stirring (350 rpm) for 30 minutes. Following treatment, the specimens were rinsed with deionised water and etched with 0.5 ml of 1 M HClO 4 for 15 seconds with continuous agitation. The etch solution was analysed for fluoride as outlined above. The baseline fluoride concentration of each specimen was then subtracted from the post-treatment value to determine the change in enamel fluoride due to the test treatment. Relative dentine abrasivity (RDA) The procedure used in this study was the Hefferren abrasivity test 24 recommended by the ADA and described in ISO 11609: for determination of toothpaste abrasiveness in dentine. Eight human dentine specimens were subjected to neutron bombardments resulting in the formation of radioactive phosphorus ( 32 P) 15. The specimens were mounted in methyl methacrylate and placed into a V- 8 cross-brushing machine (Sabri Dental Enterprises, Inc., Downers Grove, IL, USA). The specimens were brushed for a 1,500 stroke, precondition run using a slurry consisting of 10 g ADA reference material [calcium pyrophosphate (Ca 2 P 2 O 7 )] in 50 ml of a 0.5% w/v carboxymethylcellulose (CMC)/10% w/v glycerine solution. The brushes used were those specified by the ADA (Oral-B P-40) with a brushing force of 1.5 N. Following the precondition run the test was performed using the above parameters (1.5 N and 1,500 strokes) in a sandwich design. Before and after brushing with the test toothpaste (25 g product/40 ml deionised water) each tooth set was brushed with the ADA reference material. This dilution produces a final slurry volume and a concentration similar to those of the reference abrasive slurry. The procedure was repeated so that each toothpaste was assayed on each tooth set. RDA calculations. One millilitre samples were taken, weighed and added to 5 ml of Ultima Gold scintillation cocktail. The samples were mixed well and immediately put on a liquid scintillation counter for radiation detection. The counts per minute (CPM) values for the test and reference products were measured. The RDA of the test toothpaste is calculated using Equations 1 and 2: G mr ¼ G pre þ G post 2 ð1þ where G mr = mean reference net CPM per mass of slurry (g) G pre = pre-net CPM per mass of slurry (g) G post = post-net CPM per mass of slurry (g) RDA ¼ 100 G mt G mr ð2þ where G mt = mean test toothpaste net CPM per mass of slurry (g) G mr = mean reference net CPM per mass of slurry (g) 100 = Dentine abrasivity of the ADA reference material Relative enamel abrasivity (REA) The procedure was identical to that used for determination of RDA except that human enamel was used instead of dentine. In addition, specimens were brushed for 5,000 strokes. In this test, the ADA reference material was assigned a value of 10. Determination of bioavailable fluoride Toothpaste slurries were prepared by homogenisation for 10 minutes of one part test toothpaste with 10 parts (w/w) deionised water. The slurries were centrifuged at ca g to obtain the supernatant. The supernatants were diluted with water such that the range of the working standard bracketed the concentration of the fluoride ion. These solutions were analysed by Dionex Ion Chromatography (Dionex Corporation, Camberley, UK) with suppressed conductivity detection. Fluoride concentrations were determined by external standardisation. Statistical analysis Statistical analyses were conducted using an analysis of variance model (ANOVA) (Sigma Stat Software, Version 3.1, Systat Software, Chicago, IL, USA). Where significant differences were found, additional pair-wise comparisons were performed using a Student Newman Keuls test (P < 0.05). RESULTS ph Cycling Studies The change in hardness (DVHN), fluoride content and slurry ph data from the ph cycling studies are shown in Tables 4 7. The ph of all toothpaste slurries tested 2013 FDI World Dental Federation 51

5 Churchley and Schemehorn as one part test product with two parts (w/w) pooled human saliva ranged from Ages 0 2 years (Non-US toothpastes) All fluoride-containing toothpastes were significantly more effective at promoting remineralisation compared with the fluoride-free placebo after 10 days and 20 days (Table 4). A significant difference in DVHN between the 500 and 1,000 ppm F toothpastes was only observed after 20 days. All differences in the enamel fluoride content between toothpaste treatments were significant. Ages 3 5 years (non-us toothpastes) All fluoride containing toothpastes were significantly more effective at promoting remineralisation compared with the fluoride-free placebo after 10 days and 20 days (Table 5). After 10 days, all DVHN treatment differences were significant with the exception of the 250 versus 500 ppm F and 1,000 versus 1,450 ppm F toothpastes. After 20 days, a statistically significant dose response in DVHN was only observed up to 500 ppm F. While there were no statistically significant differences between the 500, 1,000 and 1,450 ppm F toothpastes at this timepoint, the differences were directional. All differences in the enamel fluoride content between toothpaste treatments were significant. Ages 2 5 years and 6+ years (US toothpastes) All fluoride containing toothpastes were significantly more effective at promoting remineralisation compared with the fluoride-free placebo after 10 days and 20 days (Table 6). At both timepoints, a statistically significant dose response was observed for DVHN, however, there were no statistically significant differences between the 250 ppm F dose response toothpaste, the current US Aquafresh Kids Bubble Fresh toothpaste and the USP reference toothpaste. After 20 days, there were no significant differences between the two experimental toothpastes containing 1,150 ppm F and the USP reference toothpaste. The enamel fluoride content data show a statistically significant dose response; however, the differences between the two experimental toothpastes containing 1,150 ppm F and the USP reference toothpaste were not significant. Table 4 Summary of DVHN and enamel fluoride content after ph cycling with experimental toothpastes (ages 0 2 years/non-us) toothpastes (n = 30). The ph of the toothpaste slurry is also reported Treatment DVHN 10 day DVHN 20 day Enamel fluoride content (lg F/cm 3 ) ph of toothpaste slurry 0 ppm F 1 (0.7) a 3.6 (1.0) a 585 (19) a ppm F 12 (1.0) b 17.3 (1.0) b 2,259 (101) b ,000 ppm F 14.2 (0.9) b 24.2 (1.2) c 3,181 (131) c 7.20 Table 5 Summary of DVHN and enamel fluoride content after ph cycling with experimental toothpastes (ages 3 5 years/non-us) toothpastes (n = 30). The ph of the toothpaste slurry is also reported Treatment DVHN 10 day DVHN 20 day Enamel fluoride content (lg F/cm 3 ) ph of toothpaste slurry 0 ppm F 0.6 (0.7) a 3.2 (0.9) a 352 (12) a ppm F 11.2 (0.7) b 14.3 (0.8) b 1,484 (56) b ppm F 13.3 (0.8) b 19.0 (0.9) c 1,692 (62) c ,000 ppm F 15.7 (0.9) c 20.1 (1.1) c 1,886 (86) d ,450 ppm F 17.3 (0.9) c 20.7 (0.8) c 2,301 (68) e 7.21 Table 6 Summary of DVHN and enamel fluoride content after ph cycling with experimental toothpastes (ages 2 5 years and 6+ years/us) toothpastes (n = 30). The ph of the toothpaste slurry is also reported Treatment DVHN 10 day DVHN 20 day Enamel fluoride content (lg F/cm 3 ) ph of toothpaste slurry 0 ppm F ( 6+ years) 5.9 (0.9) a 9.5 (0.8) a 365 (15) a ppm F (6+ years) 11.7 (1.2) b 18.5 (1.1) b,c 1,062 (56) b ,150 ppm F (2 5 years) 15.3 (1.1) b 23.9 (1.5) d 1,539 (59) c ,150 ppm F (6+ years) 22.7 (1.5) c 22.6 (1.4) c,d 1,655 (66) c 7.11 USP Reference Toothpaste 14.9 (1.3) b 20.7 (2.5) b,c,d 1,633 (67) c 7.09 Aquafresh Kids Bubble Fresh 15.5 (0.7) b 16.8 (0.9) b 1,048 (39) b FDI World Dental Federation

6 Table 7 Summary of DVHN and enamel fluoride content after ph cycling with experimental toothpastes (ages 6+ years/non-us) toothpastes (n = 30). The ph of the toothpaste slurry is also reported Treatment DVHN 10 day DVHN 20 day Enamel fluoride content (lg F/cm 3 ) ph of toothpaste slurry 0 ppm F 8.0 (0.8) a 5.8 (0.7) a 582 (20) a ppm F 14.7 (0.9) b 17.6 (0.9) b 2,029 (51) b ,450 ppm F 20.2 (1.2) c 23.2 (1.4) c 3,074 (146) c 7.20 Letter superscripts represent the different statistical groupings. Standard error in brackets In vitro assessment of a toothpaste range Ages 6+ years (Non-US toothpastes) At both the 10-day and 20-day time-points, all differences in DVHN and enamel fluoride content between the 0, 500 and 1,450 ppm F toothpastes were statistically significantly (Table 7). EFU The EFU results for the US and non-us experimental toothpastes with commercially available controls are shown in Tables 8 and 9 respectively. All fluoride containing toothpastes promoted significant fluoride uptake into demineralised enamel compared with the fluoride free placebo. The fluoride uptake data in Table 8 show that there were no significant differences between the three US experimental toothpastes. However, these toothpastes were significantly more effective at promoting fluoride uptake than the USP reference toothpaste. The order of fluoride uptake was 0 ppm F < Aquafresh Kids Bubble Fresh < USP reference toothpaste < 1,150 ppm F (ages 2 5 years (Tube) = 1,150 ppm F (ages 2 5 years (Pump) = 1,150 ppm F (ages 6+ years). For all non-us toothpastes, the fluoride uptake data showed a dose response (Table 9). The fluoride uptake from the experimental toothpastes was either equivalent to or statistically higher than the commercial controls containing the same fluoride concentration. For the 500 and 1,000 ppm F experimental toothpastes, fluoride uptake from toothpastes for ages 3 5 years was significantly higher than from toothpaste for ages 0 2 years. RDA & REA The RDA and REA data for the experimental toothpastes and the commercial controls are shown in Table 10. The products evaluated in this study have a wide range of RDA values (from ) Table 8 Enamel fluoride uptake data for US experimental and commercial toothpastes using FDA method #40 (n = 12) Treatment Pack fluoride concentration (ppm) Mean enamel fluoride concentration (ppm) Experimental Toothpaste (6+ years) 0 8 (2) a Experimental Toothpaste (2 5 years/tube) ,779 (59) b Experimental Toothpaste (2 5 years/pump) ,730 (37) b Experimental Toothpaste (6+ years) ,818 (53) b USP standard N/A 1,296 (51) c Aquafresh Kids Bubble Fresh (17) d Table 9 Enamel fluoride uptake data for non-us experimental and commercial toothpastes using FDA Method #40 (n = 12) Treatment Pack fluoride concentration (ppm) Mean enamel fluoride concentration (ppm) Experimental Toothpaste (3 5 years) 0 40 (6) a Aquafresh Milk Teeth 1, (20) b Odol-med 3 Milchzahn 500 1,000 (21) c Experimental Toothpaste (0 2 years) 500 1,383 (27) d Experimental Toothpaste (3 5 years) 500 1,521 (42) e Aquafresh Little Teeth 1,400 1,540 (72) e Experimental Toothpaste (0 2 years) 1,000 1,648 (46) e,f Aquafresh Little Teeth 1,000 1,758 (36) f,g Experimental Toothpaste (3 5 years) ,860 (35) g,h Aquafresh Big Teeth 1,450 1,919 (54) h,i Experimental Toothpaste (3 5 years) 1,450 1,959 (41) h,i Experimental Toothpaste (6+ years) 1,450 2,035 (53) i 2013 FDI World Dental Federation 53

7 Churchley and Schemehorn Table 10 Relative enamel and dentine abrasion results for experimental and commercial toothpastes (n = 8) Treatment REA RDA Aquafresh Fresh and Minty 4.58 (0.23) a (2.13) a Aquafresh Kids Bubble Fresh 2.49 (0.11) b (1.90) b Experimental Toothpaste 2.26 (0.17) b,c (1.60) d (6+ years/non-us) Experimental Toothpaste 2.25 (0.18) b,c (1.88) d (0 2 years/non-us) Aquafresh Big Teeth 2.03 (0.12) b,c,d (1.20) c Experimental Toothpaste 2.02 (0.12) b,c,d (1.21) d (2 5 years/us tube) Experimental Toothpaste 2.00 (0.17) b,c,d (1.65) d (6+ years/non-us) Experimental Toothpaste 1.76 (0.21) c,d (1.13) d (2 5 years/us pump) Experimental Toothpaste 1.64 (0.18) c,d (1.35) d (6+ Years/US) Aquafresh Milk Teeth 1.49 (0.08) d (1.87) a Odol-Med 3 Milchzahn 0.93 (0.19) e (2.96) d Letter superscripts represent the different statistical groupings. Standard error in brackets. Table 11 Bioavailable fluoride results for experimental toothpastes Age range Pack fluoride concentration (ppm) Bioavailable fluoride (ppm) 0 2 years years years 1, years 0 None detected 3 5 years years years 1, years 1,150 (Tube) 1, years 1,150 (Pump) 1, years 1,450 1, years years years years 1,150 1, years 1,450 1,335 whereas the range of REA values is considerably smaller (from ). The statistical analysis showed that while there were no differences between the experimental toothpastes for both RDA and REA, there were significant differences between the commercial control toothpastes. Bioavailable fluoride The bioavailable fluoride data for the experimental toothpastes is shown in Table 11. All placebo formulations contained 4 ppm fluoride. For all other toothpastes, the concentration of bioavailable fluoride was high and similar to that of the on-pack fluoride concentration. DISCUSSION The range of experimental toothpastes evaluated in this study have been developed for use by children at the different stages of their development. The formulations for the three age groups are different; specifically, in the level of surfactant and the type of thickening gums used. In addition, flavour and colour variations are present across the three formulations. The rationale for these differences is discussed by Stovell et al. 25 These toothpastes were designed to maximise fluoride availability, minimise abrasivity and incorporate levels and types of surfactant that will minimise interference with fluoride delivery. In vitro ph cycling models are frequently used to replicate the dynamics of demineralisation and remineralisation involved in caries lesion formation and daily toothpaste usage 26. The ph cycling model described in this paper has been previously used to investigate the effect of fluoride containing toothpastes on remineralisation of artificial caries lesions 27,28. The cycling studies described here used permanent human teeth because of the difficulty in obtaining sufficient numbers of deciduous teeth. Furthermore, deciduous teeth are smaller and so there is less surface area for experimental manipulation 26. While there are a number of distinct differences between deciduous and permanent enamel, it has been reported that remineralisation of initial caries lesions is similar in both substrates 29. This means that permanent enamel can be used as a surrogate for deciduous enamel in these studies. The lesions used in this study represented early stage lesions where the mineral loss was confined to the outer regions of the enamel where fluoride is considered most effective 30. The changes in specimen hardness following ph cycling were less that those reported by Newby et al. using the same experimental protocol 28. These differences may be explained by compositional variations in the saliva used as the remineralisation medium. As saliva varies from person to person, and the composition of an individual s saliva can vary depending on the time of collection 31, the saliva used in these studies was pooled from at least five individuals in an attempt to mitigate this variability. In this paper, the enamel remineralisation neared a plateau in the range of ppm F, however, in most cases the dose response continued to be significant up to higher fluoride concentrations. Similar remineralisation plateaux have been observed by other authors for shallow lesions 32. In addition, while net remineralisation increased from 10 days to 20 days for all fluoride-containing toothpastes, the changes were small. A plausible explanation could be a surface-zone blocking effect, which reduces the number of diffusion pathways to the lesion body. This FDI World Dental Federation

8 In vitro assessment of a toothpaste range effect has been demonstrated during mechanistic studies in vitro 33,34. The fluoride source in all experimental toothpastes and the majority of the commercial control toothpastes is NaF. Only the Aquafresh Kids Bubble Fresh and Aquafresh Milk Teeth Toothpastes use SMFP as a fluoride source. To be effective in the mouth, the fluoride ion needs to be freely available. While this is the case for NaF, the SMFP must be initially hydrolysed by salivary or microbial phosphatases in order to release the fluoride ion 35. As a result, the toothpaste slurries used in the ph cycling studies were prepared in human saliva in order to initiate hydrolysis. It is worth noting that whilst the in vitro ph cycling data has shown that treatment with both experimental toothpastes (US) produced greater lesion remineralisation and a higher enamel fluoride content than the Aquafresh Kids Bubble Fresh toothpaste, a number of caries clinical trials have shown no significant differences in the anti-caries effectiveness of toothpastes containing fluoride as either NaF or SMFP 36. Fluoride uptake has long been accepted as a positive indicator of the anti-caries activity of fluoride toothpastes. All experimental toothpastes contain a high level of bioavailable fluoride and were effective at delivering this fluoride to demineralised enamel. However, not all toothpaste containing an equivalent fluoride source and concentration produced the same fluoride uptake. This demonstrates that fluoride uptake can be influenced by different toothpaste excipients 25. In both EFU studies, the fluoride uptake was higher from NaF than from SMFP toothpastes containing equivalent fluoride ion concentrations. For comparison, Arends et al. 37 and de Rooij et al. 38 reported similar findings using sound enamel. For the US experimental toothpastes, the fluoride uptake was either equivalent to or greater than the USP reference toothpaste and therefore satisfies this part of the monograph testing requirements 18. However, as the EFU method does not incorporate biological factors that in vivo would promote SMFP hydrolysis, it is not possible to make any inference from this study as to the efficacy of the Aquafresh Kids Bubble Fresh toothpaste relative to the USP reference toothpaste. Although not reported in this paper, the Aquafresh Kids Bubble Fresh toothpaste fulfils all testing requirements listed in the anti-caries monograph. The fluoride uptake results were consistent with those measured in the ph cycling studies. Abrasives are added to toothpastes to remove plaque and further to remove the stained pellicle on the tooth surface and thus it is important to ensure that the abrasive will not cause mechanical damage to the teeth 39,40. In vitro abrasivity tests are routinely performed to provide information on the abrasive potential of toothpastes; however, the protective nature of the pellicle towards toothpaste abrasion means that any extrapolation of in vitro data to levels of in vivo abrasive wear should be treated with caution 41. While no specific REA and RDA limits have been established for children s toothpaste, ISO 11609:2010 sets toothpaste abrasivity limits of 250 for RDA and 40 for REA. As RDA is not a predictor of REA and vice versa, both abrasivity tests were conducted. In consideration of the differences between primary and permanent enamel with regard to its abrasion resistance, all children s toothpastes have been designed to have low levels of abrasivity. The results show that all toothpastes were below the recommended abrasivity limits and are therefore considered safe for everyday use. CONCLUSION In conclusion, a range of toothpastes have been developed for use by children at different stages of their development and contain different concentrations of fluoride for different markets. These toothpastes were specifically formulated to maximise fluoride availability and have low abrasivity. The in vitro findings reported here demonstrate that the experimental toothpastes were effective at promoting fluoride uptake and remineralisation of artificial caries lesions. Toothpastes for the US market were shown to comply with the in vitro testing requirements of the US anti-caries monograph. Conflict of interest Author Churchley is employed by GlaxoSmithKline Consumer Healthcare. 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