Validation of the Third Molar Maturation Index (I3M) to assess the legal adult age in the Portuguese population

Age estimation is a major step in forensic and legal procedures. Its relevance has been increasing due to growing society issues, such as identification of missing people, crimes against minors or lack of valid identification papers from locals or foreigners. Evaluation of the cut-off value of the Third Molar Maturation Index (I3M) = 0.08 for discriminating minors from adults in the Portuguese population. The left lower third molars were analysed by applying a specific cut-off value of 0.08 determined by Cameriere et al. in 2008. A sample of 778 digital panoramic radiographs of a representative Portuguese sample (442 females and 336 males), in the age range of 12–24 years (mean age 17.7 ± 2.98 years in females and 18.1 ± 3.0 years in males), was retrospectively evaluated. I3M decreased as the real age gradually increased in both sexes. The 0.08 cut-off score was valuable in discriminating adults from minors. According to the pooled results, the accuracy, by means of area under the curve, was 92.8% (95% confidence interval (CI) 91.0–94.6%). The proportion of correctly classified subjects (sensitivity) was 90.7% (95% CI 88.7–92.8%) and the specificity was 94.9% (95% CI 93.3–96.4%). The results show that I3M is a valuable method to differentiate minors from adults in the Portuguese population.


Scientific Reports
| (2020) 10:18466 | https://doi.org/10.1038/s41598-020-75324-x www.nature.com/scientificreports/ of the lower left third molar. The bidimensional widths of the apical and tooth lengths are measured and used to calculate the I 3M . Then, a cutoff value (0.08) is set to differentiate minors from adults 13 . Over the last years, I 3M was successfully validated worldwide, in Europe 2,13-23 , Africa 1,24-27 , Asia 5,28-31 , America 3,32-36 and Oceania 37 . Overall, I 3M had considerable reliability among all countries, however, to date, this method is not validated for a Portuguese sample. Therefore, investigating if the I 3M is suitable for the Portuguese population would be of great interest.
Given the evidence on high population movements in the Portuguese population, contributing to a higher level of diversity than some neighboring populations 38,39 , we aimed to test the validity of the I 3M in a Portuguese representative sample, by using panoramic radiographs (PRs).. Secondly, we looked for the influence of sex on its validity. Our null hypothesis was that the I 3M has no validity to discriminate adult age in this Portuguese sample.

Materials and methods
Source of data and sample size. This retrospective observational study has received approval from the Egas Moniz Ethics Committee (ID 887). Written informed consent was obtained for each participant, during the first appointment at the Egas Moniz Dental Clinic (EMDC), (Almada, Portugal). Regarding participants under 18, a parent and/or legal guardian and this study gave and signed the informed consent. This research was conducted in accordance with the Declaration of Helsinki, as revised in 2013.
The present study follows the Transparent Reporting of a multivariable prediction model for Individual Prognosis Or Diagnosis (TRIPOD) reporting guidelines 40 for validation of prediction models. This study was conducted on a triple-blind basis with respect to: (1) diagnosis and clinical outcome; (2) data collection; and (3) analysis.

Participants.
A consecutive sample of 1267 digital PRs, taken between September 2017 and January 2020, were considered for this study.
The inclusion criteria were: the presence of the lower third molar; known chronological age between 12 and 24 years old 5,13,36 ; and absence of evident bone pathologies or systemic diseases that may affect tooth development. Exclusion criteria included spatial orientation of the third molar that prevents correct measuring, endodontic treatment and/or coronary restoration on the third molar, extensive cavities or abnormal dental anatomy, congenital anomalies and poor-quality or distorted X-rays 41 . Outcome, predictors and measurement reproducibility. All digital PRs were converted to JPEG documents for the examination with ImageJ image processing software (Graphics Suite X7, Ottawa, Canada) by one trained and calibrated observer (JAN). The I 3M index of each evaluated third molar was performed according to the Cameriere et al. method 13 . If the root development of the third molar is complete, then I 3M = 0.0. If not, I 3M was calculated as the distance between the inner sides of the open apex (A and B) divided by the tooth length (C) (Fig. 1). In case of I 3M < 0.08, the individual is classified as 18 years old or older and if I 3M ≥ 0.08, the individual as considered a minor.
The Kappa correlation index was used to test the agreement of classification of individuals younger than 18 years, aged 18 years or older. The Intraclass Correlation Coefficient (ICC) was used to analyze the measurements of open apices. Previously, twelve PRs were randomly chosen from the total sample, measured and  (Table 1). Then, several performance indicators were determined and detailed in Table 1 43 . Performance measurement was assessed through binary and multiclass area under the curve (AUC), through receiver operating characteristics (ROC) analysis. The correspondent 95% confidence intervals (95% CI) were also determined. Bayes post-test probability (p) of being 18 years or older is computed to discriminate between those who are or are not aged 18 years or more. According to Bayes' theorem, p may be written as: In the post-test probability p, p0 defines the probability that a participant is 18 years or older given that he or she is aged between 12 and 24 years, in the target population. In this study, we calculated the probability p0 as the proportion of participants between 18 and 24 years of age who live in Portugal and those who are aged between 12 and 24 years. This value (p0) was considered to be 0.55 (global, males and females) according to the Portuguese National Statistics Institute -Instituto Nacional de Estatística (INE) (https ://www.ine.pt/xport al).

Participants.
A total of 778 digital PRs (336 males and 442 females) met the inclusion criteria, with 489 being excluded due to absence of third molars. The distribution of age and sex is depicted in Table 2. The mean ages of the males and females, aged between 12 and 24 years, were 18.1 ± 3.0 years old and 17.7 ± 3.0, respectively, without statistically significant difference (p = 0.078).
Model performance. The estimated age of majority was correlated with the chronological age (p < 0.001).
The I 3M values decreased as age increased across all age groups in both sexes, showing that the lower third molar mineralization occurred earlier in females than in males (Fig. 2). Then, pooled data of both sexes as well as separately was analysed (

Discussion
The present study is the first to test the validity of I 3M in a Portuguese sample. Overall, the null hypothesis was rejected, that is, the I 3M is a reliable tool to discriminate adult age in this Portuguese population. Further, I 3M was more accurate in male participants. These results have important implications because this tool has potential to be used in legal and criminal settings. Discriminating minors from adults is important to prevent legal wrongful procedures, specially in cases where valid identification documents are lacking, in order to prevent legal wrongful procedures 4,32 . Notwithstanding, age estimation is challenging, particularly in the differentiation teens from young adults (aged 15 years old to early 20 s), as the physical appearance and characterization are not clearly related of being an adult 1,4 . From 15 years of age, the third molars are only teeth not fully developed, as this clinical circumstance is useful in forensic sciences 44 .        46 have evaluated third molars, however the method and the age range of these  www.nature.com/scientificreports/ The sensibility indicates the I 3M ability to correctly identify individuals who are 18 years or older (I 3M < 0.08). On the other hand, specificity is the ability to discriminate individuals younger than 18 years old (I 3M ≥ 0.08). Santiago et al. (2018) 41 reported that I 3M outperforms in individuals younger than 18 years due to the higher specificity of this tool. This is key in forensic science because if a minor is wrongly processed as an adult it would violate its rights 23,41 .
When analysing our data regarding males and females (Fig. 4), we demonstrated alike maturation of third molars in both males and females, which is in line with similar studies [14][15][16][19][20][21][22][23]44,51 . Previous European studies present slightly better accuracy and sensibility in males than in females [14][15][16][19][20][21][22][23]44,51 . Contrary, females presented better specificity than males [14][15][16][19][20][21][22][23]44,51 . Therefore, I 3M seems to be slightly more accurate in males, although it is equivalent to the female participants in this Portuguese population. The effect of age on I 3M performance is proposed to rely on the maturation development according to sex as women tend to develop at a younger ages than men 52 . This maturation difference is suggested to explain the I 3M results discrepancies between both sexes, however more studies are needed to fully understand the biological reasons upon this subject 19 20 and Kelmendi et al. 21 reported results very close to 100% in females, and Gulsahi et al. 19 reported results of 100% in both sexes, although the sample size was one of the smallest in all European studies (n = 293), which may influence these results.
The likelihood ratio is a practical measure of diagnostic accuracy. The higher the value of LR + , more the test has the capability of establishing the tested condition; an LR + value greater than 10 defines it as a good diagnostic test. In our study, balanced values of LR + and LR − have been achieved. Although I 3M presented higher LR + in females (20.5; 95% CI 15-23.3) then males (14.6; 95% CI 9.9-17.1), I 3M is an excellent prediction of the probability of majority; and lower LR − (0.06; 95% CI 0.04-0.08) in males than (0.13; 95% CI 0.11-0.15) in females, prove that the test is also an excellent tool at classifying individuals younger than 18 years of age, despite the sex of the individuals 19,23 .
In conclusion, I 3M is a suitable method in legal and forensic purposes to identify minors from adults in the Portuguese population. Further, the cut-off value used (0.08) is predictable and useful to discriminating individuals younger than 18 years of age (high specificity in both sexes).
Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.