2.1

Study design

This randomized clinical trial was approved by the IRB of Araçatuba Dental School (Protocol 2006-014112). The study population comprised children between 18 and 60 months old, attending public kindergartens of 2 cities of the State of São Paulo, Brazil. The aims were detailed explained to parents/caregivers and signed informed consent was obtained prior to the beginning of the study. Children were randomly assigned into 3 treatment groups, according to the dentifrice to be used, following a double-blind protocol. Parents/caregivers and children were instructed on oral hygiene procedures, brushing frequency and amount of dentifrice to be used. Caries status (dmfs) was determined at the beginning of the study and 18 months after using the dentifrices.

2.2

Study areas

The study was conducted in two cities of the State of São Paulo (Southeast macro region of Brazil): (1) Araçatuba, latitude 21°12′32” south and longitude 50°25′58” west, mean annual temperature 27.0 °C, Human Development Index 0.848 and mean fluoride concentration in the public water supply of 0.7 mg/L; (2) Fernandópolis, latitude 20°17′02′′ south and longitude 50°14′47′′ west, mean annual temperature 25.5 °C, Human Development Index 0.823 and mean fluoride concentration in the public water supply of 0.7 mg/L.

2.3

Sample size determination and selection of volunteers

Based on a pilot study assessing dmfs scores in 50 children from 18 to 60 months old (mean = 0.1, SD = 0.48), a sample size of 125 children for each treatment group was determined (α = 0.05 e β = 0.2), considering a dropout rate of 20% at the end of the clinical trial.

The inclusion criteria were: (1) being a resident of Araçatuba or Fernandópolis; (2) age between 18 and 60 months old; (3) parents should sign the informed consent document; (4) children should present good general health status and not taking medicines likely to interfere with salivary flow and/or biofilm formation; and (5) not having participated in another clinical trial at least 3 months prior to the beginning of the study. The exclusion criteria were: (1) voluntary withdrawal; (2) use of mouthrinses or dentifrices different to those provided by the researchers; and (3) not following up the experimental protocol (assessed by periodic visits to kindergartens). Children were selected from public kindergartens from urban areas of the 2 above-mentioned cities, totaling 747 children from 15 kindergartens which were randomly selected from a list provided by the Education Department of each city. Among those, informed consent was obtained from 600 children who fulfilled the inclusion criteria. For random allocation to the groups, classrooms were considered as units of draw (conglomerate sampling), by simple drawing using the Microsoft Excel software, so that only 1 type of dentifrice was distributed in each classroom. Allocation was done by one investigator not involved in the clinical examinations (ACBD). This planning facilitated the controlled delivery and collection of the dentifrices by the classroom’s teacher, as well as daily supervised toothbrushing at school. The flowchart with the distribution of children at the beginning and at the end of the study is presented in Fig. 1 .

Flowchart of the experimental design, showing the random allocation of the children into the groups according to the dentifrices used.

2.4

Dentifrice formulation

Experimental dentifrices contained the following components: titanium dioxide, carboxymethylcellulose, methyl- p -hydroxybenzoate, sodium saccharin, Tutti-frutti flavoring, glycerol, hydrated silica, sodium lauryl sulfate, and water. Sodium fluoride (NaF, Merck ^® , Darmstadt, Germany) was added to the formulations at 500 μg F/g (test dentifrices) and 1100 μg F/g (positive control). Sodium trimetaphosphate (TMP, Sigma™–Aldrich Co., USA) or Calcium glycerophosphate (CaGP, Sigma™–Aldrich Co., USA, dl, 50% α- and 50% β-isomer) were added to the 500 μg F/g formulations at concentrations of 1% and 0.25%, respectively. Prior to the beginning of the study, dentifrices were submitted to laboratory tests regarding their composition, pH, density, foaming ability, abrasiveness, consistency, rheological properties, concentration of fluoride, calcium and phosphate, as well as toxicological and microbiological tests, performed by the Brazilian Dental Association (Associação Brasileira de Odontologia – ABO) and by BITUFO Dental Products (Industry and commerce, Itupeva, SP, Brazil). All dentifrices were identical regarding their color, taste and appearance of the tube, and were coded by one author not involved in the clinical examinations (ACBD) so that the protocol was double-blind (children and examiners).

2.5

Study protocol

At the beginning of the study, school directors, teachers, and children’s parents were instructed about the study protocol. Parents were asked to brush their children’s teeth for 1 min at least twice a day at home (in the morning and before going to bed), with no restrictions regarding rinsing procedures. The amount of dentifrice to be used was standardized as the size of a rice grain (“smear”). For standardization of brushing procedures and the amount of dentifrice to be used, parents/caregivers were also instructed during a lecture.

Kits for oral hygiene contained 1 children’s toothbrush, 4 dentifrice tubes (50 g each) and 1 leaflet about oral hygiene care and compliance need, which were replaced every three months. Compliance was facilitated by supplying the dentifrices to all family members, in order to guarantee that children would use the experimental dentifrice. Moreover, extra kits were available at the schools if families needed them to be replaced prior to the regular quarterly supplying periods. Besides the kits for home use, toothbrushes and dentifrices were also available for use at kindergartens, for toothbrushing once/day after the main meal. Therefore, children were expected to brush their teeth at least twice/day.

2.6

Clinical examinations

Children were examined at schools by 2 calibrated dentists, at the beginning of the study and every 6 months, totaling 4 examinations. The dmfs index (decayed, missed, filled surfaces) was used, following the criteria and codes established by the World Health Organization , by visual inspection using a plane mirror and a ballpoint probe. Tooth surfaces were cleaned by toothbrushing performed by the professional and dried with gauze, to improve the visualization of tooth surfaces. The intra- and inter-examiner reliability was measured using kappa on a surface basis. The intra-examiner agreement was tested by duplication of 30% of the examinations for examiner 1 (κ = 0.97, p = 0.982, CI = 0.9694–0.9828) and 21% of the examinations for the examiner 2 (κ = 0.95, p = 0.969, CI = 0.9250–0.9861). The inter-examiner reliability was also evaluated; twenty children were examined twice by both dentists in each survey. The related kappa values were 0.85 and 0.87 for the first and second examinations, respectively. Initial and final dmfs scores were used for the calculation of caries increment (final dmfs – initial dmfs).

2.7

Statistical analysis

For the statistical analysis, SPSS program version 17 (SPSS Inc., Chicago) was used, with the significance level set at 5%. Kruskal-Wallis test was used to verify differences among the groups regarding mean age at baseline. Multivariate linear regression test was applied to verify the influence of socioeconomic characteristics (city and gender), caries status at baseline and type of dentifrice upon caries increment at the end of the trial.

2.1

Study design

This randomized clinical trial was approved by the IRB of Araçatuba Dental School (Protocol 2006-014112). The study population comprised children between 18 and 60 months old, attending public kindergartens of 2 cities of the State of São Paulo, Brazil. The aims were detailed explained to parents/caregivers and signed informed consent was obtained prior to the beginning of the study. Children were randomly assigned into 3 treatment groups, according to the dentifrice to be used, following a double-blind protocol. Parents/caregivers and children were instructed on oral hygiene procedures, brushing frequency and amount of dentifrice to be used. Caries status (dmfs) was determined at the beginning of the study and 18 months after using the dentifrices.

2.2

Study areas

The study was conducted in two cities of the State of São Paulo (Southeast macro region of Brazil): (1) Araçatuba, latitude 21°12′32” south and longitude 50°25′58” west, mean annual temperature 27.0 °C, Human Development Index 0.848 and mean fluoride concentration in the public water supply of 0.7 mg/L; (2) Fernandópolis, latitude 20°17′02′′ south and longitude 50°14′47′′ west, mean annual temperature 25.5 °C, Human Development Index 0.823 and mean fluoride concentration in the public water supply of 0.7 mg/L.

2.3

Sample size determination and selection of volunteers

Based on a pilot study assessing dmfs scores in 50 children from 18 to 60 months old (mean = 0.1, SD = 0.48), a sample size of 125 children for each treatment group was determined (α = 0.05 e β = 0.2), considering a dropout rate of 20% at the end of the clinical trial.

The inclusion criteria were: (1) being a resident of Araçatuba or Fernandópolis; (2) age between 18 and 60 months old; (3) parents should sign the informed consent document; (4) children should present good general health status and not taking medicines likely to interfere with salivary flow and/or biofilm formation; and (5) not having participated in another clinical trial at least 3 months prior to the beginning of the study. The exclusion criteria were: (1) voluntary withdrawal; (2) use of mouthrinses or dentifrices different to those provided by the researchers; and (3) not following up the experimental protocol (assessed by periodic visits to kindergartens). Children were selected from public kindergartens from urban areas of the 2 above-mentioned cities, totaling 747 children from 15 kindergartens which were randomly selected from a list provided by the Education Department of each city. Among those, informed consent was obtained from 600 children who fulfilled the inclusion criteria. For random allocation to the groups, classrooms were considered as units of draw (conglomerate sampling), by simple drawing using the Microsoft Excel software, so that only 1 type of dentifrice was distributed in each classroom. Allocation was done by one investigator not involved in the clinical examinations (ACBD). This planning facilitated the controlled delivery and collection of the dentifrices by the classroom’s teacher, as well as daily supervised toothbrushing at school. The flowchart with the distribution of children at the beginning and at the end of the study is presented in Fig. 1 .

Flowchart of the experimental design, showing the random allocation of the children into the groups according to the dentifrices used.

2.4

Dentifrice formulation

Experimental dentifrices contained the following components: titanium dioxide, carboxymethylcellulose, methyl- p -hydroxybenzoate, sodium saccharin, Tutti-frutti flavoring, glycerol, hydrated silica, sodium lauryl sulfate, and water. Sodium fluoride (NaF, Merck ^® , Darmstadt, Germany) was added to the formulations at 500 μg F/g (test dentifrices) and 1100 μg F/g (positive control). Sodium trimetaphosphate (TMP, Sigma™–Aldrich Co., USA) or Calcium glycerophosphate (CaGP, Sigma™–Aldrich Co., USA, dl, 50% α- and 50% β-isomer) were added to the 500 μg F/g formulations at concentrations of 1% and 0.25%, respectively. Prior to the beginning of the study, dentifrices were submitted to laboratory tests regarding their composition, pH, density, foaming ability, abrasiveness, consistency, rheological properties, concentration of fluoride, calcium and phosphate, as well as toxicological and microbiological tests, performed by the Brazilian Dental Association (Associação Brasileira de Odontologia – ABO) and by BITUFO Dental Products (Industry and commerce, Itupeva, SP, Brazil). All dentifrices were identical regarding their color, taste and appearance of the tube, and were coded by one author not involved in the clinical examinations (ACBD) so that the protocol was double-blind (children and examiners).

2.5

Study protocol

At the beginning of the study, school directors, teachers, and children’s parents were instructed about the study protocol. Parents were asked to brush their children’s teeth for 1 min at least twice a day at home (in the morning and before going to bed), with no restrictions regarding rinsing procedures. The amount of dentifrice to be used was standardized as the size of a rice grain (“smear”). For standardization of brushing procedures and the amount of dentifrice to be used, parents/caregivers were also instructed during a lecture.

Kits for oral hygiene contained 1 children’s toothbrush, 4 dentifrice tubes (50 g each) and 1 leaflet about oral hygiene care and compliance need, which were replaced every three months. Compliance was facilitated by supplying the dentifrices to all family members, in order to guarantee that children would use the experimental dentifrice. Moreover, extra kits were available at the schools if families needed them to be replaced prior to the regular quarterly supplying periods. Besides the kits for home use, toothbrushes and dentifrices were also available for use at kindergartens, for toothbrushing once/day after the main meal. Therefore, children were expected to brush their teeth at least twice/day.

2.6

Clinical examinations

Children were examined at schools by 2 calibrated dentists, at the beginning of the study and every 6 months, totaling 4 examinations. The dmfs index (decayed, missed, filled surfaces) was used, following the criteria and codes established by the World Health Organization , by visual inspection using a plane mirror and a ballpoint probe. Tooth surfaces were cleaned by toothbrushing performed by the professional and dried with gauze, to improve the visualization of tooth surfaces. The intra- and inter-examiner reliability was measured using kappa on a surface basis. The intra-examiner agreement was tested by duplication of 30% of the examinations for examiner 1 (κ = 0.97, p = 0.982, CI = 0.9694–0.9828) and 21% of the examinations for the examiner 2 (κ = 0.95, p = 0.969, CI = 0.9250–0.9861). The inter-examiner reliability was also evaluated; twenty children were examined twice by both dentists in each survey. The related kappa values were 0.85 and 0.87 for the first and second examinations, respectively. Initial and final dmfs scores were used for the calculation of caries increment (final dmfs – initial dmfs).

2.7

Statistical analysis

For the statistical analysis, SPSS program version 17 (SPSS Inc., Chicago) was used, with the significance level set at 5%. Kruskal-Wallis test was used to verify differences among the groups regarding mean age at baseline. Multivariate linear regression test was applied to verify the influence of socioeconomic characteristics (city and gender), caries status at baseline and type of dentifrice upon caries increment at the end of the trial.