Multi-ancestry Genome-Wide Association Study of Early Childhood Caries.

Early childhood caries (ECC) is the most common non-communicable childhood disease. It is an important health problem with known environmental and social/behavioral influences that lacks evidence for specific associated genetic risk loci. To address this knowledge gap, we conducted a genome-wide association study of ECC in a multi-ancestry population of U.S. preschool-age children (n=6,103) participating in a community-based epidemiologic study of early childhood oral health. Calibrated examiners used ICDAS criteria to measure ECC with the primary trait using the dmfs index with decay classified as macroscopic enamel loss (ICDAS ≥3). We estimated heritability, concordance rates, and conducted genome-wide association analyses to estimate overall genetic effects; the effects stratified by sex, household water fluoride, and dietary sugar; and leveraged the combined gene/gene-environment effects using the 2-degree-of-freedom (2df) joint test. The common genetic variants explained 24% of the phenotypic variance (heritability) of the primary ECC trait and the concordance rate was higher with a higher degree of relatedness. We identified 21 novel non-overlapping genome-wide significant loci for ECC. Two loci, namely RP11-856F16 . 2 (rs74606067) and SLC41A3 (rs71327750) showed evidence of association with dental caries in external cohorts, namely the GLIDE consortium adult cohort (n=∼487,000) and the GLIDE pediatric cohort (n=19,000), respectively. The gene-based tests identified TAAR6 as a genome-wide significant gene. Implicated genes have relevant biological functions including roles in tooth development and taste. These novel associations expand the genomics knowledge base for this common childhood disease and underscore the importance of accounting for sex and pertinent environmental exposures in genetic investigations of oral health.

Metabolomics Insights in Early Childhood Caries.

Dental caries is characterized by a dysbiotic shift at the biofilm-tooth surface interface, yet comprehensive biochemical characterizations of the biofilm are scant. We used metabolomics to identify biochemical features of the supragingival biofilm associated with early childhood caries (ECC) prevalence and severity. The study's analytical sample comprised 289 children ages 3 to 5 (51% with ECC) who attended public preschools in North Carolina and were enrolled in a community-based cross-sectional study of early childhood oral health. Clinical examinations were conducted by calibrated examiners in community locations using International Caries Detection and Classification System (ICDAS) criteria. Supragingival plaque collected from the facial/buccal surfaces of all primary teeth in the upper-left quadrant was analyzed using ultra-performance liquid chromatography-tandem mass spectrometry. Associations between individual metabolites and 18 clinical traits (based on different ECC definitions and sets of tooth surfaces) were quantified using Brownian distance correlations (dCor) and linear regression modeling of log2-transformed values, applying a false discovery rate multiple testing correction. A tree-based pipeline optimization tool (TPOT)-machine learning process was used to identify the best-fitting ECC classification metabolite model. There were 503 named metabolites identified, including microbial, host, and exogenous biochemicals. Most significant ECC-metabolite associations were positive (i.e., upregulations/enrichments). The localized ECC case definition (ICDAS ≥1 caries experience within the surfaces from which plaque was collected) had the strongest correlation with the metabolome (dCor P = 8 × 10-3). Sixteen metabolites were significantly associated with ECC after multiple testing correction, including fucose (P = 3.0 × 10-6) and N-acetylneuraminate (p = 6.8 × 10-6) with higher ECC prevalence, as well as catechin (P = 4.7 × 10-6) and epicatechin (P = 2.9 × 10-6) with lower. Catechin, epicatechin, imidazole propionate, fucose, 9,10-DiHOME, and N-acetylneuraminate were among the top 15 metabolites in terms of ECC classification importance in the automated TPOT model. These supragingival biofilm metabolite findings provide novel insights in ECC biology and can serve as the basis for the development of measures of disease activity or risk assessment.

Flossing Is Associated with Improved Oral Health in Older Adults.

The effect of preventive oral habits is largely unexplored in older individuals. The purpose of this study was to evaluate the associations between home use of flossing and prevalence of periodontal disease and caries in older adults. Five-year incident tooth loss was also evaluated. Data on 686 individuals ≥65 y-old from the Piedmont 65+ Dental Study were examined including: 1) interproximal clinical attachment level (iCAL), 2) interproximal probing depth (iPD), 3) numbers of caries, and 4) missing teeth. Flossing behavior was evaluated according to the Periodontal Profile Class (PPC) system. Five-year follow-up data (n = 375) was evaluated for incident tooth loss. Dichotomous and categorical variables were analyzed using Pearson chi-square tests as well as covariate-adjusted Cochran-Mantel-Haenszel tests. Multiple linear regression compared clinical parameters based on flossing behavior. Elderly flossers had lower (mean, SE) %iCAL≥3 mm (38.2, 2.38 vs. 48.8, 1.56) and %iPD≥4 mm (8.70, 1.41 vs. 14.4, 0.93) compared to nonflossers (P ≤ 0.005). Flossers showed less coronal caries compared to nonflossers (P = 0.02). Baseline number of missing teeth (mean, SE) was 11.5 (0.35) in nonflossers compared to 8.6 (0.53) in flossers (P < 0.0001). Regular dental visitors had lower oral disease levels compared to episodic dental users. The majority of flossers classified into PPC-Stage I (health) whereas nonflossers classified as PPC-Stages V, VI, and VII (disease). At the 5-y follow-up visit, the average tooth loss for flossers was ~1 tooth compared to ~4 teeth lost for nonflossers (P < 0.0001). Among all teeth, molars showed the highest benefit (>40%) for flossing behavior (P = 0.0005). In conclusion, the extent of oral disease for older individuals was significantly less in flossers than in nonflossers. Flossers showed less periodontal disease, fewer dental caries, and loss of fewer teeth over a 5-y period. These findings further support flossing as an important oral hygiene behavior to prevent oral disease progression in older adults.

Interdental Cleaning Is Associated with Decreased Oral Disease Prevalence.

The purpose of this study was to evaluate the associations between interdental cleaning behavior and the prevalence of caries and periodontal disease and numbers of missing teeth, with data from the National Health and Nutrition Examination Survey (2011 to 2012 and 2013 to 2014). Analysis included the following parameters: interproximal clinical attachment level (iCAL) ≥3 mm, interproximal probing depth (iPD) ≥4 mm, number of coronal and interproximal caries, number of missing teeth, ≥1 surfaces with coronal caries, and periodontal profile classes (PPCs). Chi-square was used for bivariate associations. Associations of interdental cleaning with outcomes were assessed with multiple linear regression and generalized logit regression, adjusting for age, race, sex, diabetes, smoking, education, dental visits, and sugar consumption. Nonusers had a significantly higher percentage of sites with iCAL ≥3 mm and iPD ≥4 mm as compared with individuals who used interdental cleaning devices ( P < 0.0001). Individuals with a higher frequency of cleaning (4 to 7×/wk) had a significantly lower extent of sites with iCAL ≥3 mm as compared with lower-frequency cleaning (1 to 3×/wk; P ≤ 0.05). Interdental cleaning users showed lower numbers of coronal caries, interproximal coronal caries, and missing teeth as compared with nonusers ( P < 0.0001). Nonusers had 1.73-times (95% confidence interval, 1.53 to 1.94) higher odds for having ≥1 surfaces of coronal caries as compared with interdental cleaning users, regardless of the weekly frequency. Individuals were less likely to be in diseased PPCs if they were interdental cleaning users. Low-frequency cleaners (1 to 3×/wk) had significantly greater odds (1.43; 95% confidence interval, 1.08 to 1.88) to have severe disease (PPC-G) versus health (PPC-A) than were high-frequency cleaners (4 to 7×/wk). Interdental cleaning users showed lower levels of periodontal disease and caries and lower numbers of missing teeth. Higher frequency of interdental cleaning was correlated with increased periodontal health. Individuals with severe periodontal disease could show additional oral health benefits by increasing cleaning frequency. The data support the use of interdental cleaning devices as an oral hygiene behavior for promoting health.

Managing Carious Lesions: Consensus Recommendations on Terminology.

Variation in the terminology used to describe clinical management of carious lesions has contributed to a lack of clarity in the scientific literature and beyond. In this article, the International Caries Consensus Collaboration presents 1) issues around terminology, a scoping review of current words used in the literature for caries removal techniques, and 2) agreed terms and definitions, explaining how these were decided.Dental cariesis the name of the disease, and thecarious lesionis the consequence and manifestation of the disease-the signs or symptoms of the disease. The termdental caries managementshould be limited to situations involving control of the disease through preventive and noninvasive means at a patient level, whereascarious lesion managementcontrols the disease symptoms at the tooth level. While it is not possible to directly relate the visual appearance of carious lesions' clinical manifestations to the histopathology, we have based the terminology around the clinical consequences of disease (soft, leathery, firm, and hard dentine). Approaches to carious tissue removal are defined: 1)selective removal of carious tissue-includingselective removal to soft dentineandselective removal to firm dentine; 2)stepwise removal-including stage 1,selective removal to soft dentine, and stage 2,selective removal to firm dentine6 to 12 mo later; and 3)nonselective removal to hard dentine-formerly known ascomplete caries removal(technique no longer recommended). Adoption of these terms, around managing dental caries and its sequelae, will facilitate improved understanding and communication among researchers and within dental educators and the wider clinical dentistry community.

Managing Carious Lesions: Consensus Recommendations on Carious Tissue Removal.

The International Caries Consensus Collaboration undertook a consensus process and here presents clinical recommendations for carious tissue removal and managing cavitated carious lesions, including restoration, based on texture of demineralized dentine. Dentists should manage the disease dental caries and control activity of existing cavitated lesions to preserve hard tissues and retain teeth long-term. Entering the restorative cycle should be avoided as far as possible. Controlling the disease in cavitated carious lesions should be attempted using methods which are aimed at biofilm removal or control first. Only when cavitated carious lesions either are noncleansable or can no longer be sealed are restorative interventions indicated. When a restoration is indicated, the priorities are as follows: preserving healthy and remineralizable tissue, achieving a restorative seal, maintaining pulpal health, and maximizing restoration success. Carious tissue is removed purely to create conditions for long-lasting restorations. Bacterially contaminated or demineralized tissues close to the pulp do not need to be removed. In deeper lesions in teeth with sensible (vital) pulps, preserving pulpal health should be prioritized, while in shallow or moderately deep lesions, restoration longevity becomes more important. For teeth with shallow or moderately deep cavitated lesions, carious tissue removal is performed according toselective removal to firm dentine.In deep cavitated lesions in primary or permanent teeth,selective removal to soft dentineshould be performed, although in permanent teeth,stepwise removalis an option. The evidence and, therefore, these recommendations support less invasive carious lesion management, delaying entry to, and slowing down, the restorative cycle by preserving tooth tissue and retaining teeth long-term.

Detection of Caries Around Amalgam Restorations on Approximal Surfaces.

To evaluate the in vitro performance of the International Caries Detection and Assessment System (ICDAS) visual examination, bitewing radiography (BW), and the DIAGNOdent 2190, a pen-type laser fluorescence device (LFpen), in detecting caries around amalgam restorations on approximal surfaces. Approximal surfaces (N=136) of permanent posterior teeth (N=110) with Class II amalgam restorations were assessed twice by two experienced examiners using ICDAS, BW, and LFpen. The occurrence of proximal overhangs was also evaluated. The teeth were histologically prepared and assessed for caries extension. Different cutoff limits for the LFpen were used. Intraexaminer and interexaminer reproducibility showed moderate to good agreement for all the methods (weighted κ/intraclass correlation coefficient=0.40 to 0.87). The specificities at D1 (all visible lesions affecting enamel) and D3 (lesions extended into dentin) were, respectively, 0.41 and 0.82 for ICDAS, 0.70 and 0.82 for BW, and 0.77-0.89 and 0.88-0.94 for LFpen. The sensitivities were 0.80 and 0.52 for ICDAS, 0.56 and 0.51 for BW, and 0.04-0.23 and 0.01-0.02 for LFpen at D1 and D3, respectively. At the D1/D3 thresholds, the accuracy and the area under the receiver operating characteristic curve (Az) values were similar and statistically higher for ICDAS (0.65/0.68 and 0.633/0.688) and BW (0.64/0.68 and 0.655/0.719), respectively; whereas, LFpen presented lower accuracy (0.37-0.44/0.49-0.52) and Az (0.390-0.454/0.345-0.395) values. The occurrence of overhangs (26.8%) was shown to be irrelevant in determining the presence of secondary caries. The ICDAS and BW methods presented the best performance in detecting caries lesions affecting enamel and dentin on approximal surfaces of amalgam restorations.

In vitro progression of artificial white spot lesions sealed with an infiltrant resin.

This study assessed the ability of an infiltrant resin (Icon, DMG Chemisch-Pharmazeutische Fabrik GmbH, Hamburg, Germany) to prevent artificial lesion progression in vitro when used to impregnate white spot lesions and also assessed the effect of saliva contamination on resin infiltration. Enamel specimens (n=252) were prepared and covered with nail varnish, leaving a window of sound enamel. After demineralization (pH 5.0; four weeks), specimens were divided into six groups (n=42 per group): group 1, 2% fluoride gel (positive control); group 2, resin infiltrant; group 3, resin infiltrant + fluoride gel; group 4, no treatment (negative control); group 5, resin infiltrant application after saliva contamination; and group 6, resin infiltrant + fluoride gel after saliva contamination. Specimens from each group were cut perpendicular to the surface, and one-half of each specimen was exposed to a demineralizing solution for another four weeks. The other half was set aside as a record of initial lesion depth and was used later in the determination of lesion progression. Lesion progression and infiltrant penetration were measured using confocal laser scanning microscopy (CLSM) and transverse microradiography (TMR). For lesion depth, based on CLSM, groups 2 and 3 showed the least changes when submitted to demineralization challenge, followed by group 1, then groups 5 and 6, and finally group 4. There were no significant differences between groups 2 and 3 or groups 5 and 6 in their ability to inhibit further lesion progression (p<0.05). Based on TMR, groups 2 and 3 also showed the fewest changes when submitted to demineralization challenge, followed by group 5, then groups 1 and 6, and finally group 4. In terms of mineral loss as measured by TMR, all groups that contained fluoride (groups 1, 3, and 6) show less percentage change in mineral loss than the groups that did not contain fluoride (groups 2, 4, and 5). It can be concluded that infiltrant penetration into early enamel lesions inhibited further demineralization in vitro, especially in the presence of fluoride. Saliva contamination decreased the ability of the infiltrant to prevent further demineralization, but the presence of fluoride seemed to counteract this effect.

Longitudinal analyses of early lesions by fluorescence: an observational study.

Previous caries experience correlates to future caries risk; thus, early identification of lesions has importance for risk assessment and management. In this study, we aimed to determine if Quantitative Light-induced Fluorescence (QLF) parameters--area (A [mm(2)]), fluorescence loss (F [%]), and Q [% × mm(2)]--obtained by image analyses can predict lesion progression. We secured consent from 565 children (from 5-13 years old) and their parents/guardians and examined them at baseline and regular intervals over 48 months according to the International Caries Detection Assessment System (ICDAS), yearly radiographs, and QLF. QLF images from surfaces with ICDAS 0/1/2/3/4 at baseline that progressed (N = 2,191) to cavitation (ICDAS 5/6) or fillings and surfaces that did not progress to cavitation/fillings (N = 4,141) were analyzed independently for A, F, and Q. Linear mixed-effects models were used to compare means and slopes (changes over time) between surfaces that progressed and those that did not. QLF A, F, and Q increased at a faster rate for surfaces that progressed than for surfaces that did not progress (p = .0001), regardless of type of surface or baseline ICDAS score. AUC for ICDAS ranged from 0.65 to 0.80, but adding QLF information improved AUC (0.82-0.87, p < .0005). We concluded that faster changes in QLF variables can indicate lesion progression toward cavitation and be more clinically relevant than actual QLF values.

The natural history of dental caries lesions: a 4-year observational study.

Dental caries is a ubiquitous disease affecting all age groups and segments of the population. It is known that not all caries lesions progress to cavitation, but little is known regarding the progression pattern of caries lesions. This study's purpose was to evaluate the natural history of dental caries using a standardized, visually based system, the International Caries Detection and Assessment System (ICDAS). The study population consisted of 565 consenting children, who were enrolled and examined at baseline and at regular intervals over 48 months with ICDAS and yearly bitewing radiographs. Of these, 338 children completed all examinations. Not all lesions cavitated at the same rate, differing by surface type and baseline ICDAS severity score and activity status. With increasing severity, the percentage of lesions progressing to cavitation increased: 19%, 32%, 68%, and 66% for ICDAS scores 1, 2, 3, and 4, respectively. Lesions on occlusal surfaces were more likely to cavitate, followed by buccal pits, lingual grooves, proximal surfaces, and buccal and lingual surfaces. Cavitation was more likely on molars, followed by pre-molars and anterior teeth. Predictors of cavitation included age, gender, surfaces and tooth types, and ICDAS severity/activity at baseline. In conclusion, characterization of lesion severity with ICDAS can be a strong predictor of lesion progression to cavitation.

Risk factors of caries progression in a Hispanic school-aged population.

The purpose of this Institutional Review Board-approved study was to identify risk factors of caries lesion progression in children enrolled in rural schools in Puerto Rico. A convenience sample of 408 children (5-13 yrs old) was examined at baseline and at 12 and 24 mos with the International Caries Detection and Assessment System (ICDAS). A total of 395 caregivers completed a 25-item questionnaire including socio-demographic, dietary, protective factors, disease experience, and access to care. Caries progression was significant (89% and 91% at 12 and 24 mos, respectively). Multiple-variable models for predicting children with lesion progression and numbers of lesions progressing were calculated for 2 outcome variables (any-progression vs. progression-toward-cavitation). Models developed had areas under the receiver operating characteristic (ROC) curve ranging between 0.70 and 0.79 and were very similar regardless of the outcome (progression criteria), prediction time (12-24 mos), or inclusion (or not) of previous caries experience. Significant predictors of disease progression collected through a parent-completed questionnaire included questions related to caries experience in the child or caregiver, and the caregiver's rating of the child's oral health.

Identification of caries risk factors in toddlers.

The purpose of this study was to identify risk factors to predict caries progression in toddlers in primary-healthcare settings for the cost-effective targeting of preventive and referral strategies. We examined 329 children (26 ± 6 mos old) twice, one year apart, in Indiana, USA. A 107-item structured interview was used to collect information from the primary caregiver and child on factors/beliefs/perceptions/behaviors that could affect caries development, transmission of bacteria, medical-dental health, and access to care. Bacterial levels, gingivitis, dental plaque, and caries experience were assessed. Multiple-variable logistic regression models of caries progression toward cavitation included family caries experience, transmission-related behaviors, dietary factors, health beliefs, and lower income, but differed in selected predictors/predictive power by race/ethnicity. Addition of clinical variables did not significantly improve the prediction.

Use of ICDAS combined with quantitative light-induced fluorescence as a caries detection method.

The purpose of this study was to combine a standardized visually based system, the International Caries Detection and Assessment System (ICDAS), with a sensitive fluorescence-based system, quantitative light-induced fluorescence (QLF), to determine the ability to monitor caries lesion progression. This combination (QLF-I) has the potential to increase the sensitivity of the visual method without compromising specificity. A total of 460 children were enrolled and examined at baseline, 8 months and 12 months by ICDAS and QLF by a single examiner. The examiner repeatability for both methods was comparable, varying between weighted kappa of 0.70 and 0.79. The DMFT score was 6.0 (SD 5.8) at baseline and 6.4 (SD 6.3) at 12 months, and both methods were able to follow the increase in incidence. The QLF-I scored more surfaces at the early ICDAS scores (1 and 2) and score 4. Not all lesions progressed at the same rate, differing by score at baseline and surface type.

Dentition and lesion history.

Dental caries is a process that typically keeps recurring throughout life, and the consequences are too often seen as irreversible damage to the dentition. At various stages of life, different parts of the dentition are affected, and the effects continue to be seen in the dentition long after the events took place. They bear witness to previous occurrences of this process throughout the lifetime of an individual. This chapter reviews the linkage between the caries process and the dental caries lesion history of the human dentition. The prevalence and distribution of the caries burden are very variable and closely tied to cultural aspects. In the primary dentition, income and education have been found to be inversely associated with: (1) any early childhood caries and (2) the maxillary incisor caries pattern. A positive association between these caries patterns and minority ethnicity/race status was also identified. These patterns are different from those of the permanent dentition. Well-documented changes in caries prevalence have been observed throughout history, most closely tied to availability and amount of refined sugar consumed. Changes in caries rates are also well documented in the 20th century, mainly with the advent of fluoride in several forms, first as a steep decline and recently as being relatively unchanged. It is likely that there will be dramatic changes in the rates and distribution of dental caries in the future, due to changes in behavioural factors and therapeutic measures. The description drawn is based on the dental caries pattern experienced in modern western societies.

Dental caries diagnosis.

As with any disease process, the early initiation of therapy for dental caries is often the most effective means to ensure resolution. However, for any therapy to be effective, early diagnosis is paramount to success. Unfortunately, current tools used in dental caries detection are not sensitive enough to diagnose the disease process in its early stages and, frequently, once a diagnosis is made restoration is the only effective means of treatment. Quantitative light-induced fluorescence, electrical conductance measurements, direct digital radiography, and direct fiber optic transillumination are methods that may offer promise as effective tools in diagnosing early dental caries. This article describes some of the research that has been performed to make these methods a reality.

An in vitro comparison between laser fluorescence and visual examination for detection of demineralization in occlusal pits and fissures.

It has been demonstrated that when excited by laser light carious enamel appears dark compared to luminescent sound enamel. The aim of this study was to compare the sensitivity and specificity of visual exams (V), laser fluorescence (LF) and dye-enhanced LF (DELF) for detecting demineralization in occlusal pits and fissures. The actual presence of lesions was determined by subsequent confocal laser microscopy (CM), which was compared to histology (H). Independent clinical examiners visually graded three sites on occlusal surfaces of extracted, human premolars as sound or carious and also rated the color of each graded site as: 0 = same as surrounding enamel; 1 = white; 2 = light brown, or 3 = brown/dark brown. An argon laser was used to illuminate the teeth for LF and DELF; the images were captured with a CCD camera and then analyzed. DELF images were captured after the teeth had been exposed to 0.075% sodium fluorescein. Sections were then cut from each specimen and analyzed by CM and H for the presence or absence of caries. Results showed that DELF (0.72) was significantly more sensitive (p<0.05) than LF (0.49) and V (0.03) for detecting caries, but there were no significant differences among the methods in specificity (V 1.00; LF 0.67; DELF 0.60). When color was used as an indication of caries in V (VC, sensitivity 0.47; specificity 0.70), V exams were not different from LF. The area under the ROC curve, using H as the gold standard and CM as the test, was 0.78. Results indicated that DELF was the best diagnostic tool and that VC and LF were equally effective as diagnostic methods, when color of fissures was included as an indication of demineralization in the visual exam.

Laser fluorescence detection of demineralization in artificial occlusal fissures.

Laser fluorescence (LF) has been used previously to detect early smooth-surface lesions. Although its use for detection of occlusal demineralization has been implicated, it has not been demonstrated. The aim of this study was to determine whether LF could detect demineralization in the base of artificial fissures. To employ LF for detection of occlusal demineralization an apparatus was devised to direct laser light into fissures and simultaneously detect fluorescence from the base of the fissures. Three groups (n = 40/group) of differing fissure types were prepared (straight wall, converging and diverging wall) with either a sound or lesioned base. One half of each group was examined with LF and dye-enhanced LF (DELF); the other half was examined with LF, exposed to plaque, examined with LF and DELF, air-polished and examined with DELF. All images were scored twice as either (1) carious; (2) sound, or (3) undetermined, by a group of 3 examiners. For fissures without plaque, the average sensitivity was higher for DELF (0.76) than for LF (0.54) (p < 0.05). Likewise, the average specificity was higher for DELF (0.64) than for LF (0.29) (p < 0.05). In the presence of plaque, sensitivity was higher for DELF (0.91) compared to LF (0.43); however, specificity was lower for DELF (0.05) compared to LF (0.55). When the fissures were air-polished and then examined with DELF, sensitivity averaged 0.82 (p < 0.05), and specificity increased consistently (average 0.51, p < 0.05). It was concluded that, in the absence of plaque, DELF was a better diagnostic tool than LF for detection of demineralization in artificial fissures.

Effect of two fluorescent dyes on color of restorative materials.

PURPOSE: The objective of these two studies was to determine whether the use of dyes in vitro would affect the shade of tooth-colored restorative materials. MATERIALS AND METHODS: Two separate studies were performed. In Study 1, four groups (N = 36) of restorative materials; (1) resin composite (RSN), (2) resin-modified glass ionomer (RMGI), (3) conventional glass ionomer (CGI), and (4) porcelain (PCN), were subjected to a rinse in either tap water (control) or Pyrromethene 556 dye at a concentration of 0.01M for 30 seconds or 0.0015 M for 60 seconds. They were then rinsed with 25% ethanol as would be done in clinical usage. In Study 2, the same restorative materials (except PCN) were subjected to a rinse in either tap water (control) (N = 8) or 0.0018 M sodium fluorescein (N = 8) for 60 seconds followed by a 5-second rinse with tap water. For both studies the specimens were analyzed for overall color changes (delta E*) comparing baseline to immediately post dye exposure and then again after 1, 4, 24, and 48-hour storage under running tap water. In addition, an image of each specimen, illuminated under an argon laser light (HGM), was captured with a miniature charged-coupled device (CCD) color camera at the same time intervals when the color was measured. These images were analyzed for fluorescence using computer assisted methods. A software program then computed the mean and standard deviation of the fluorescence values for each image. The data were analyzed using repeated measures ANOVA. RESULTS: delta E* values from baseline on specimens treated with either Pyrromethene 556 or sodium fluorescein were not significantly different from water at any time. The only exceptions were CGI specimens exposed to 0.01 M Pyrromethene 556 and RMGI exposed to sodium fluorescein which had a higher delta E* immediately after treatment, however this effect was reversed after 1-hour rinse. Mean delta E* was less than 2.69 for specimens treated with Pyrromethene 556 and less than 3.20 for specimens treated with sodium fluorescein after 1-hour rinse in water. When the mean fluorescence level, as determined by the computer, was averaged there was no difference between the Pyrromethene 556 treatments and control for RMGI, RSN and PCN after 4-hour rinse in water and, as time in storage increased, the fluorescence level decreased. CGI treated with 0.01 M Pyrromethene 556 was significantly more fluorescent even after 48-hour rinse in water. All specimens treated with sodium fluorescein were also significantly more fluorescent than control even after 48-hour rinse in water. It was concluded that exposure of these restorative materials to Pyrromethene 556 at 0.01 M or 0.0015 M or to 0.0018 M sodium fluorescein will not lead to any prolonged visually detectable color changes.