The last resort requirement under REACH: From principle to practice.

REACH (Registration, Evaluation, Authorisation and Restriction of Chemicals) is a European Union regulation that aims to protect human health and the environment from the risks posed by chemicals. Article 25 clearly states that: "[i]n order to avoid animal testing, testing on vertebrate animals for the purposes of this Regulation shall be undertaken only as a last resort." In practice, however, the standard information requirements under REACH are still primarily filled using animal studies. This paper presents examples illustrating that animal testing is not always undertaken only as a last resort. Six over-arching issues have been identified which contribute to this: (1) non-acceptance of existing animal or non-animal data, (2) non-acceptance of read-across, (3) inflexible administrative processes, (4) redundancy of testing, (5) testing despite animal welfare concerns and (6) testing for cosmetic-only ingredients. We, members of the Animal-Free Safety Assessment (AFSA) Collaboration, who work together to accelerate the global adoption of non-animal approaches for chemical safety assessment, herein propose several recommendations intended to aid the European Commission, the European Chemicals Agency and registrants to protect human health and the environment while avoiding unnecessary animal tests - truly upholding the last resort requirement in REACH.

Early Childhood Caries in a Preschool-based Sample in Northeast Italy: Socioeconomic Status and Behavioral Risk Factors.

Aim: To assess the relationship among caries and age, gender, immigrant condition, socioeconomic status (SES), and a behavioral risk factor (toothbrushing) in a sample of 3-5-year-old children. Materials and methods: We performed a random cross-sectional survey from January to December 2017, carrying out clinical examinations to estimate the decayed, missing, and filled teeth (dmft) score. Parents filled in a questionnaire reporting their education level (SES) and the daily frequency of children's toothbrushing. The multivariate analysis assessed the association between caries occurrence and the independent variables. The dmft score was assessed by zero-inflated negative binomial regression (ZINBR). Results: Out of 1,441 children in the sample, 357 (26.0%) presented at least one caries-affected tooth. Caries risk significantly increased with age and irregular toothbrushing, and it was significantly higher in children with lower SES levels. We modeled caries risk by means of ZINBR. The degree of caries experience increased in children from lower SES positions, immigrant status, and of older age; regular toothbrushing (twice a day) is a predicting factor to belong to the "zero caries" group. Conclusion: Dental caries represent a significant burden in preschool children and can be regarded as an early marker of social disadvantage. Clinical significance: The confirmation of the earliest preventive approach as the only chance to grant a "caries free" dentition in all ages and the first target for a pediatric dentist. How to cite this article: Ferro R, Besostri A, Olivieri A, et al. Early Childhood Caries in a Preschool-based Sample in Northeast Italy: Socioeconomic Status and Behavioral Risk Factors. Int J Clin Pediatr Dent 2022;15(6):717-723.

A synbio approach for selection of highly expressed gene variants in Gram-positive bacteria.

BACKGROUND: The market for recombinant proteins is on the rise, and Gram-positive strains are widely exploited for this purpose. Bacillus subtilis is a profitable host for protein production thanks to its ability to secrete large amounts of proteins, and Lactococcus lactis is an attractive production organism with a long history in food fermentation. RESULTS: We have developed a synbio approach for increasing gene expression in two Gram-positive bacteria. First of all, the gene of interest was coupled to an antibiotic resistance gene to create a growth-based selection system. We then randomised the translation initiation region (TIR) preceding the gene of interest and selected clones that produced high protein titres, as judged by their ability to survive on high concentrations of antibiotic. Using this approach, we were able to significantly increase production of two industrially relevant proteins; sialidase in B. subtilis and tyrosine ammonia lyase in L. lactis. CONCLUSION: Gram-positive bacteria are widely used to produce industrial enzymes. High titres are necessary to make the production economically feasible. The synbio approach presented here is a simple and inexpensive way to increase protein titres, which can be carried out in any laboratory within a few days. It could also be implemented as a tool for applications beyond TIR libraries, such as screening of synthetic, homologous or domain-shuffled genes.

Survey of Caries Experience in 3- to 5-year-old Children in Northeast Italy in 2011 and Its Trend 1984-2011.

PURPOSE: To describe dental caries experience by age and gender among preschool children in a health district in northeast Italy and to plot a trend of primary-dentition dental caries prevalence and severity over a 27-year time span (1984-2011). MATERIALS AND METHODS: In a cross-sectional survey, 27 out of 88 kindergartens were randomly selected with a sample of 2603 preschoolers drawn from a population of 8328 3-, 4- and 5-year-old children. Dental caries (d3 t) experience according to the criteria of the British Association for the Study of Community Dentistry was evaluated by two calibrated examiners at schools in 1960 (75.3%) 3- to 5-year-old children from October 2010 to May 2011. Three previous surveys performed in the same area, applying the criteria of the World Health Organization, were used to plot a trend over a 27-year period. Comparisons between groups were made using Pearson's chi-squared test, and caries occurrence was established by logistic regression analysis to assess the influence of sex and age (independent variables) on caries experience (dependent variable). RESULTS: Prevalence (%) and severity (mean dmft ± SD) increased with age (17% and 0.5 ± 1.7 at age 3; 24% and 0.8 ± 2.2 at age 4; 35% and 1.3 ± 2.6 at age 5). The level of untreated caries was 85.8%. No statistically significant difference was found for gender. From 1984 to 2004, the prevalence and severity of caries declined at all examined ages, but were unchanged from 2004 to 2011. CONCLUSION: The current caries scores in preschoolers are low and similar to those reported in other western European countries. Nevertheless, as very early childhood is a key opportunity to intervene, an effort must be made to provide clear oral health guidance and increase the cooperation among all health professionals.

Adult high-grade B-cell lymphoma with Burkitt lymphoma signature: genomic features and potential therapeutic targets.

The adult high-grade B-cell lymphomas sharing molecular features with Burkitt lymphoma (BL) are highly aggressive lymphomas with poor clinical outcome. High-resolution structural and functional genomic analysis of adult Burkitt lymphoma (BL) and high-grade B-cell lymphoma with BL gene signature (adult-molecularly defined BL [mBL]) revealed the MYC-ARF-p53 axis as the primary deregulated pathway. Adult-mBL had either unique or more frequent genomic aberrations (del13q14, del17p, gain8q24, and gain18q21) compared with pediatric-mBL, but shared commonly mutated genes. Mutations in genes promoting the tonic B-cell receptor (BCR)→PI3K pathway (TCF3 and ID3) did not differ by age, whereas effectors of chronic BCR→NF-κB signaling were associated with adult-mBL. A subset of adult-mBL had BCL2 translocation and mutation and elevated BCL2 mRNA and protein expression, but had a mutation profile similar to mBL. These double-hit lymphomas may have arisen from a tumor precursor that acquired both BCL2 and MYC translocations and/or KMT2D (MLL2) mutation. Gain/amplification of MIR17HG and its paralogue loci was observed in 50% of adult-mBL. In vitro studies suggested miR-17∼92's role in constitutive activation of BCR signaling and sensitivity to ibrutinib. Overall integrative analysis identified an interrelated gene network affected by copy number and mutation, leading to disruption of the p53 pathway and the BCR→PI3K or NF-κB activation, which can be further exploited in vivo by small-molecule inhibitors for effective therapy in adult-mBL.

Central nervous system relapse in adults with acute lymphoblastic leukemia after allogeneic hematopoietic stem cell transplantation.

Central nervous system (CNS) relapse after allogeneic hematopoietic stem cell transplantation (HSCT) confers a poor prognosis in adult patients with acute lymphoblastic leukemia (ALL). Preventing CNS relapse after HSCT remains a therapeutic challenge, and criteria for post-HSCT CNS prophylaxis have not been addressed. In a 3-center retrospective analysis, we reviewed the data for 457 adult patients with ALL who received a first allogeneic HSCT in first or second complete remission (CR). All patients received CNS prophylaxis as part of their upfront therapy for ALL, but post-transplantation CNS prophylaxis practice varied by institution and was administered to 48% of the patients. Eighteen patients (4%) developed CNS relapse after HSCT (isolated CNS relapse, n = 8; combined bone marrow and CNS relapse, n = 10). Patients with a previous history of CNS involvement with leukemia had a significantly higher rate for CNS relapse (P = .002), and pretransplantation CNS involvement was the only risk factor for post-transplantation CNS relapse found in this study. We failed to find a significant effect of post-transplantation CNS prophylaxis to prevent relapse after transplantation. Furthermore, no benefit for post-transplantation CNS prophylaxis could be detected when a subgroup analysis of patients with (P = .10) and without previous CNS involvement (P = .52) was performed. Finally, we could not find any significant effect for intensity of the transplantation conditioning regimen on CNS relapse after HSCT. In conclusion, CNS relapse is an uncommon event after HSCT for patients with ALL in CR1 or CR2, but with higher risk among patients with CNS involvement before transplantation. Furthermore, neither the use of post-HSCT CNS prophylaxis nor the intensity of the HSCT conditioning regimen made a significant difference in the rate of post-HSCT CNS relapse.

Validity of Italian version of the Child Perceptions Questionnaire (CPQ11-14).

BACKGROUND: The Child Perceptions Questionnaire (CPQ11-14) is the most commonly used indicator of child oral health-related quality of life (OHRQoL), and its validity and reliability have been studied both in English and in other linguistic contexts. The aim of this study was to develop a CPQ11-14 for use in Italy and to test its validity in a random sample of fourteen year-old Italian adolescents. METHODS: Once the CPQ11-14was translated into Italian and adapted for an Italian public, five hundred sixty-one adolescents were recruited for testing. Parents rated their social status; the children/adolescents were administered the questionnaire and underwent a dental examination during which their dental status was taken and recorded. Cronbach's alpha was used to assess the questionnaire's internal consistency. Spearman's correlation coefficients were calculated to assess construct validity between the total and subscale scores and the respondents' global ratings on oral health and well-being. Discriminant validity was analysed using the Kruskal-Wallis or Mann-Whitney tests in groups defined by gender, social position, caries experience and previous or no orthodontic treatment. RESULTS: The mean score on the CPQ11-14 was 15.4 (SD=11.9), and the scores on all the domains were found to be highly skewed. Cronbach's alpha ranged from 0.85 to 0.90. The global ratings on oral health and well-being were correlated to the total score and to the sub-scores except for those regarding the functional limitations. There were significant differences in the two genders, in the groups that had already or had not yet undergone orthodontic treatment, and in the social classification groups, while the difference between those who had and those who did not have caries experience did not reach statistical significance. CONCLUSIONS: The Italian version of the CPQ11-14 appears to be a reliable, valid instrument for Italian children/adolescents.

In vitro comparison of the flexibility of different splint systems used in dental traumatology.

AIM: The aim of the study was to evaluate the flexibility of five different splint systems [polyethylene fibre-reinforced splint (Ribbond THM, Ribbond Inc., Seattle, WA, USA), resin splint (RS), wire-composite splint (WCS), button-bracket splint (BS) and titanium trauma splint (TTS)] commonly used in clinical practice for the treatment of dental traumatic injuries involving the periodontal supporting tissues. MATERIALS AND METHODS: For the experimental study, a resin cast of the upper arch was manufactured, where teeth 11, 12 and 21 (used for the stress analysis) were inserted in a non-rigid fashion so as to allow for replacement, whereas the other teeth were permanently fixed to the corresponding sockets. Two different test sessions were performed for each splint: (i) stress analysis with increasing intensity ranging between 0 and 50 N directed along the tooth's longitudinal axis; (ii) stress analysis with 45 degrees of oblique force of increasing intensity ranging between 0 and 30 N. For each loading direction, five recordings were conducted without a splint, followed by five with the splint applied. The energy required to modify the position of the teeth was calculated for both the splinted and un-splinted teeth and the difference between the two values was determined. Energy variation was assessed for the testing of both axial (DeltaE(a)) and oblique force (DeltaE(o)). DeltaE represents the rigidity index of the analysed contention devices: high DeltaE values correspond to high rigidity materials. RESULTS: The RS showed the highest DeltaE value for the axial stress analysis, whereas the highest DeltaE value at a 45 degrees was recorded for the WCS and RS. For both tests, the lowest DeltaE values were recorded for the TTS and Ribbond THM splints. CONCLUSIONS: The data show that the contention devices with the highest flexibility are the TTS and the Ribbond THM as they exhibit a lower energy variation needed for splint deformation compared with the other materials that were examined.

Familial and cultural perceptions and beliefs of oral hygiene and dietary practices among ethnically and socio-economicall diverse groups.

UNLABELLED: OBJECTIVE; The aim of this international study was to develop a valid and reliable psychometric measure to examine the extent to which parents' attitudes about engaging in twice-daily tooth brushing and controlling sugar snacking predict these respective behaviours in their children. A supplementary objective was to assess whether ethnic group, culture, level of deprivation or children's caries experience impact upon the relationships between oral health related behaviours, attitudes to these respective behaviours and to dental caries. CLINICAL SETTING: Nurseries, health centres and dental clinics in 17 countries. PARTICIPANTS: 2822 children aged 3 to 4 years and their parents. MAIN OUTCOME MEASURES: Dental examination of children and questionnaire to parents. RESULTS: Factor analysis identified 8 coherent attitudes towards toothbrushing, sugar snacking and childhood caries. Attitudes were significantly different in families from deprived and non-deprived backgrounds and in families of children with and without caries. Parents perception of their ability to control their children's toothbrushing and sugar snacking habits were the most significant predictor of whether or not favourable habits were reported. Some differences were found by site and ethnic group. CONCLUSIONS: This study supports the hypothesis that parental attitudes significantly impact on the establishment of habits favourable to oral health. An appreciation of the impact of cultural and ethnic diversity is important in understanding how parental attitudes to oral health vary. Further research should examine in a prospective intervention whether enhancing parenting skills is an effective route to preventing childhood caries.

International comparisons of health inequalities in childhood dental caries.

OBJECTIVE: To undertake formative studies investigating how the experience of dental caries in young children living in diverse settings relates to familial and cultural perceptions and beliefs, oral health-related behaviour and oral microflora. PARTICIPANTS: The scientific consortium came from 27 sites in 17 countries, each site followed a common protocol. Each aimed to recruit 100 families with children aged 3 or 4 years, half from deprived backgrounds, and within deprived and non-deprived groups, half to be "caries-free" and half to have at least 3 decayed teeth. OUTCOME MEASURES: Parents completed a questionnaire, developed using psychological models, on their beliefs, attitudes and behaviours related to their child's oral health. 10% of children had plaque sampled. RESULTS: 2,822 children and families were recruited. In multivariate analyses, reported toothbrushing behaviours that doubled the odds of being caries-free were a combination of brushing before age 1, brushing twice a day and adult involvement in brushing. Analyses combining beliefs, attitudes and behaviours found that parents' perceived ability to implement regular toothbrushing into their child's daily routine was the most important predictor of whether children had caries and this factor persisted in children from disadvantaged communities. 90% of children with lactobacillus had caries. CONCLUSIONS: Parental beliefs and attitudes play a key role in moderating oral health related behaviour in young children and in determining whether they develop caries. Further research is indicated to determine whether supporting the development of parenting skills would reduce dental caries in children from disadvantaged communities independent of ethnic origin.

Relationship between children's first dental visit and their dental anxiety in the Veneto Region of Italy.

The aim of this study was to explore the association between negative experiences during children's first dental visit and any subsequent dental anxiety and related factors in three dental clinics in the Veneto Region of Italy. For this purpose, parents of 378 children filled out a questionnaire. Factors related to child dental anxiety (none-some/fairly much-very much) were explored by means of logistic regression analysis. The independent variables were: problems with tht first dental visit (no/yes), parental dental anxiety (none-some/fairly much-very much), number of previous visits (0-3/4 < or =) site visited (public/private) and age of the child (< 10 years/10 < or = years). Parental anxiety was associated with child's anxiety (OR = 2.3, 95% CI = 1.1-4.9). A problematic first visit was a strong predictor of dental anxiety. However, this effect was modified by the number of subsequent visits. Children with 4 or more visits after the first visit were less likely to be anxious after a problematic first visit (OR = 4.6, 95% CI = 1.5-14.1) than children with 3 visits or less after the first visit (OR = 19.8, 95% CI = 7.2-54.5). Thus, the negative effect of a problematic first visit may fade during subsequent dental visits.