A comprehensive review of the literature on the biological effects from dental X-ray exposures

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1 International Journal of Radiation Biology ISSN: (Print) (Online) Journal homepage: A comprehensive review of the literature on the biological effects from dental X-ray exposures Vinita Chauhan & Ruth C. Wilkins To cite this article: Vinita Chauhan & Ruth C. Wilkins (2019) A comprehensive review of the literature on the biological effects from dental X-ray exposures, International Journal of Radiation Biology, 95:2, , DOI: / To link to this article: Copyright of the Crown in Canada. Accepted author version posted online: 29 Nov Published online: 24 Jan Submit your article to this journal Article views: 433 View Crossmark data Full Terms & Conditions of access and use can be found at

2 INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 2019, VOL. 95, NO. 2, REVIEW A comprehensive review of the literature on the biological effects from dental X-ray exposures Vinita Chauhan and Ruth C. Wilkins Consumer and Clinical Radiation Protection Bureau, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Canada ABSTRACT Purpose: Routine dental X-rays are among the most common sources of ionizing radiation exposure for healthy individuals globally, with 300 examinations/1000 individuals/year as documented by the United Nations Scientific Committee on the Effects of Atomic Radiation (UNSCEAR) global survey of medical radiation usage and exposure. Furthermore, in the United States of America, an increased use of dental is evident. However, with the shift from using film to digital image receptors, the dose of radiation per routine examination has decreased. Despite this, there remains public concern of dental X-rays. This continuing concern highlights the need to review the literature on biological and health effects related to exposure, from dental X-rays. Material and methods: This report presents studies investigating biological and health effects related to exposures using dental X-rays in patients and provides a critical evaluation. Relevant studies specific to dental X-rays are reviewed from 1970 and onward with the bulk data in this field resulting from epidemiological and biomonitoring studies. Results: While, most epidemiological studies suggest a possible correlation between head/neck related tumors and exposure to dental X-rays, evidence for causation is lacking. Biomonitoring studies suggest that exposure to low-level radiation such as that of dental may not be a factor in inducing long-term chromosomal damage, but may result in localized cytotoxicity in the irradiated region of the mouth, with no long-term harm. Conclusions: In general, the total number of studies is low and the majority of the data has been generated from poorly designed experiments. This review will highlight shortcomings that could influence outcomes and provide a more balanced interpretation that could impact the public perception and the level of public concern on the health effects resulting from dental X-rays. ARTICLE HISTORY Received 17 May 2018 Revised 9 August 2018 Accepted 22 October 2018 Introduction Medical imaging procedures are essential for diagnosing disease, identifying injuries, and managing patient conditions. Dental is among these procedures and is an effective means for imaging dental and maxillofacial structures to identify dental decay, infections in the bones, root pathologies and many other dental issues. There are two main types of dental X-ray equipment: Intraoral equipment produces an image by placing an X-ray film inside the mouth of the patient providing detailed information about the health of the teeth, jawbones, tooth root, and also confirming the presence of cavities. Extraoral equipment situates the X-ray image receptor exterior to the mouth, providing images of the teeth and information on the jaw and skull. There are several types of extraoral X-ray equipment in dentistry, including cephalometric, panoramic, and, more recently, cone beam computed tomography (CBCT). Each type of equipment can deliver a range of radiation doses, depending on the imaging technique. Cephalometric captures a single image of the jaw and/or head, while with panoramic the X-ray tube and receptor holder rotate in a half circle around the front of the head forming a composite image of the entire mouth in a single image. Cone beam computed tomography (CBCT) is a technique that consists of X-ray computed tomography where the X-rays are divergent, forming a cone that provides 3-dimensional imaging information. Typical doses for each technique (Table 1) vary widely both between and within modalities, partially due to differences in how each technique is administered. In general, individual intraoral radiographs deliver the lowest dose. CBCT delivers a higher effective dose than conventional radiographic techniques; however, they have a lower effective dose than those found in multi-detector CT for dental applications (Stratis et al. 2017). As digital technologies have evolved over the years, so have dental X-ray image receptors. New technologies are emerging that have the capability to provide improved image quality. For example, digital has become more popular in the past 10 years allowing the viewing of digital images on a CONTACT Vinita Chauhan Vinita.Chauahn@canada.ca; Ruth Wilkins Ruth.Wilkins@canada.ca Consumer and Clinical Radiation Protection Bureau Health Canada 775 Brookfield Road, PL 6303B Ottawa, Ontario K1A-1C1, Canada ß 2019 The Author(s). Published by Taylor & Francis Group LLC. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives License ( which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited, and is not altered, transformed, or built upon in any way.

3 108 R. WILKINS ET AL. Table 1. Typical doses from radiographic examinations. Examination Effective Dose (ls) Dose Area Product (mgycm 2 ) References Intraoral Bitewing (4 images) (Poppe et al., 2007b; Ludlow et al. 2008) Extraoral Panoramic (Poppe et al. 2007a; Ludlow et al. 2008; Granlund et al. 2016) Cephalometric (Ludlow et al. 2008; Holroyd 2011) Cone beam CT Large Field of View (Ludlow et al., 2015; Granlund et al. 2016) Medium Field of View (Ludlow et al. 2015) Small Field of View (Ludlow et al. 2015) computer screen and eliminating the need for traditional film and its associated processing. Under optimized settings, this can lead to shorter exposure times which can translate to lower dosestothepatient(okanoandsur2010). Dental X-rays are the most frequently used radiologic procedure in the US for healthy individuals, with the frequency increasing from 54 million in 1964 to 500 million in 2006 (BEIR VII 2006; Mettler et al. 2009). Worldwide, however, there was no increase in the number of examinations per 1000 population reported between 1970 and 2007 in health-care level I countries (those with at least 1 physician for every 1000 people) (UNSCEAR 2010). Despite the lower doses and the use of improved technologies, there still remains a public perception of increased health risks associated with exposure to dental X-rays. While there are relatively large uncertainties associated with the low dose exposures, some of these public s concerns of radiation risks may stem from how scientific information is communicated by the media. This review will focus on the studies pertaining to health and biological effects related specifically to dental X-ray exposures to patients since 1970, as exposures prior to this date were substantially higher and not representative of current dental practice. However, the information presented here is also broadly relevant to low dose medical exposures of the head and neck. A comprehensive literature search was conducted using various engines e.g. PubMed, Google Scholar, MEDLINE, Embase, and Scopus. Included in this report are epidemiological investigations and biomonitoringbased studies on human patients, however, no data is currently available from animal-derived studies specific to dental. In reviewing these studies, commonalities with respect to cohort recruitment, study design, and data management and analysis were considered and overarching conclusions were drawn. In all studies, shortcomings of the research and study designs were examined and are discussed in order to highlight drawbacks that may have influenced the outcomes. Studies on health effects Epidemiological studies All epidemiological studies considered in this review were population-based case controls, with data collected from individuals with cancers of the head and neck (case) compared to those who were cancer-free (controls). Patients were typically obtained from population-based cancer registries such as the Surveillance, Epidemiology, and End Results (SEER) Program (Hankey et al. 1999) or medical records from hospitals. The control populations were either matched patients or neighborhood residents with similar socioeconomic status, age, race, and sex. Pertinent data were collected from both cohorts including information on lifestyle, age, race, gender, frequency of dental and medical visits, and job history including tasks, occupational illness and/ or injury. Depending on the rigor of the interview process between studies, questions may have been posed about head trauma, head X-rays, family history of cancer (including brain, head and neck and thyroid), as well as intake of tobacco, alcohol, vitamin supplements, and certain foods (Preston-Martin et al. 1983; Preston-Martin et al. 1989). It should be noted that case-control studies have inherent issues that may skew results leading to a positive association. These may include issues of case and control misclassification, low participation rates, recall and information bias, and survival bias. The strength of such types of studies is only realized when large sampling sizes are used, the selection of cases and controls is unbiased and information is available on potential confounders, diagnostic and/or therapeutic X-rays of head, neck, and chest, radiotherapy (to any part of the body), and the number of exposures. In general, the majority of the studies were lacking in the above factors. Most importantly, the studies were not able to provide detailed information on the type of dental procedure, number of dental X-ray examinations and the total number of exposures, thereby challenging the ability to estimate dose. A summary of the results and detailed methodologies for all studies is provided in Table 2. The first case-control study relating to dental X-rays was conducted in the late seventies. In this study, male laryngeal cancer patients were recruited to assess the relationship between exposure to dental X-rays and the development of cancer (Hinds et al. 1979). The study was derived from a low sample size using a total of 47 laryngeal cancers, all in Caucasian males, identified through a cancer surveillance system. The method of data collection was inconsistent and not blinded to the interviewer. Some cases were interviewed by clinical personnel while controls were interviewed in their home by a different interviewer. It was also not known whether the cases and controls were interviewed by the same individual using a standardized format. Therefore possibilities of bias exist. Furthermore, the necessary details to estimate the dose of radiation received by an individual based on the number of dental X-ray examinations were not recorded. As a consequence of the low sample size and lack of information on the dosimetry, no conclusions on the

4 INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 109 Table 2. Summary of epidemiological studies. Reference Dental procedure Study population Instrument Dose Disease Outcome Hinds et al. (1979) No indication 47 males with laryngeal cancer from Pierce King or Kisap counties between compared to neighborhood controls Preston-Martin et al. (1980) Preston-Martin et al. (1983) Full mouth intraoral dental X-rays Five or more full month intra-oral dental X- ray series 101 (of the total 185) matched pairs of woman patients and neighborhood controls residents of Los Angeles county with intracranial meningioma s from matched pairs of men and neighborhood controls in Los Angeles county with meningioma from 1972 to 1979 Burch et al. (1987) No indication 215 cases of brain tumors in adults in Southern Ontario between 1979 and 1982 with individually matched hospital control series Preston-Martin et al. (1988) Preston-Martin et al. (1989) Neuberger et al. (1991) Details of lifetime history of dental care (full mouth or panoramic dental or medical to the head ) Full mouth intra oral dental X- rays (18 intraoral films) 408 patients (269 benign tumors, 139 malignant) from Los Angeles county with tumor of the parotid gland compared to neighborhood controls from men with primary brain tumor and matched neighbor controls from No indication 7 cases and matched with 25 controls of a brain cancer cluster in Missouri from No indication of exposure, however cases report a mean of 14.3 dental X-ray visits and controls a mean of 12.3 visits over subject lifetime No indication Intracranial meningiomas Potential to be exposed to 315 R during full mouth examinations Laryngeal cancer Possible relationship of laryngeal cancer with exposure to dental X-rays among heavy smokers Intracranial meningiomas Dental X-rays associated with increase in meningioma risk associated with early exposure to full-mouth X-ray series (16 20 intraoral films used) Dental X-rays associated with increase in intracranial meningioma development, especially early exposure (before age of 20) and multiple exposures in early years (5 full mouth X-ray series before 1945) No indication Brain cancer Non-significant minor elevation in the risk for glioma in those ever exposed to dental X-rays Total cumulative parotid dose varied from 0 to >50 rad Benign/malignant tumors of the parotid gland No indication Gliomas or meningiomas Cumulative exposure of the parotid gland from diagnostic radiograph associated with dose-related increase in risk of malignant tumours. Benign tumors showed a weaker positive association Glioma risk increased with an increase in the frequency (from never to yearly) of full-mouth examinations after age 25; a similar trend was seen for meningiomas. Both glioma and meningioma risk also increased with an increase in the frequency of exposure to dental X-rays of any type before age 25, although these trends did not reach statistical significance. No indication Brain cancer Positive correlation of brain cancer with reported exposure to dental X-rays Measure on which outcome is based Relative risk with p value p value p value Relative risk with Relative risk with 90% CI (continued)

5 110 R. WILKINS ET AL. Table 2. Continued. Reference Dental procedure Study population Instrument Dose Disease Outcome Wingren et al. (1995) Ryan et al. (1992) Ordinary dental X-rays and panoramic full mouth X-rays Rodvall et al. (1998) Dental and frequency Wingren et al. (1997) Longstreth et al. (2004) No indication 185 female papillary or mixed cancer cases 110 subjects with glioma and 60 with meningioma and 417 controls from cases of glioma, 99 cases of meningioma and 42 cases of acoustic neurinoma between with a control group of 343 subjects No indication 186 thyroid cancer cases in two Swedish case-control studies on determinants for thyroid cancer from compared to population controls Posterior bitewings, full mouth series, and lateral cephalometric and panoramic radiographs Hujoel et al. (2004) Full mouth series, (21 radiographs, 1.6 mgy to thyroid), periapical dental radiograph (0.08 mgy), 4 bitewings (0.22 mgy), cephalometric radiograph (0.46 mgy to thyroid, panoramic radiograph, 0.12 mgy 200 case-control study of residents of Western Washington State with intracranial meningioma between ; two control subject matched to each case-patient based on age and gender 1117 women with low-birthweight infants (<2500 g). Four control pregnancies resulting in normal birth weight infant (>2500 g) were used for each case (4468) between Ma et al. (2008) No indication 1742 population-based case patients and 441 control subjects from neighbourhood in Los Angeles County No indication Papillary thyroid cancer No indication Gliomas or meningiomas No indication Gliomas or meningiomas Average number of radiographs has been 1.8/person/year versus 1.65 in the US and 0.25 in the UK Female papillary thyroid cancer No indication Intracranial meningiomas Exposure of higher than 0.4 mgy during gestation occurred in 1.9% of the mother with low birth infants. 3% of the mothers with low birth infants received doses higher than 0.4 mgy Low birth weight infants Increased risks were seen for women who had worked as dentist/dental assistant No excess risk for glioma associated with exposure to diagnostic dental X-rays, however statistically significant increased risk associated with dental X-rays for meningioma in males and not in females No clear indication that dental is related to the development of tumours of the central nervous system, however there is risk of meningioma with no risk associated with gliomas High risk for papillary thyroid cancer was found in the occupation group of dentist/dental assistants The full mouth series (>6 over a lifetime) was associated with a significant risk of meningioma, no doseresponse relation was observed Dental during pregnancy is associated with low birth weight No indication Breast cancer Increased risk among women who received dental X-rays without lead apron protection before age of 20 Measure on which outcome is based Relative risk with

6 INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 111 Memon et al. (2010) No indication 313 patients with thyroid cancer and matched control subjects in Kuwait Claus et al. (2012) Self-reported bitewing, full mouth series, and panoramic dental X-rays Mortazavi et al. (2013) Lin et al. (2013) Periapical, bitewing Neta et al. (2013) Full mouth intra oral, panoramic, bitewing Zhang et al. (2015) Full mouth intra oral series, panoramic exams Al-Attas (2014) Restorative treatment, periodontal, endodontic, extraction, TMJ CI ¼ confidence interval case-control study of residents of North Carolina, San Francisco Bay and Texas from diagnosed with intracranial meningioma; control group of 1350 individuals matched on age, sex and geography No indication 1200 mothers with their firstterm labor (vaginal or cesarean) whose newborns history had been registered in neonates screening program in Shiraz were interviewed and surveyed 4123 benign brain tumor cases and controls without tumors (study 1) and 197 malignant brain tumor cases and 788 controls without malignant tumours (study 2) from Taiwan National Health Insurance claim data radiologic technologists who were followed up for thyroid cancer by the American Registry of radiologic technologists between histologically confirmed incident thyroid cancer cases and 498 populationbased controls 47 cases and 58 controls of women who gave single live birth in Jeddah city Not indicated, however, doseresponse data was based on number of dental X-rays categorized as 1 4, 5 9 and 10þ No indication Intracranial meningiomas Thyroid cancer Exposure to dental X-rays was associated with an increased risk of thyroid cancer with a dose-response pattern Exposure to some dental X-rays performed in the past, when radiation exposure was greater than in the current era, appears to be associated with an increased risk of intracranial meningioma No indication Infant birth weight No statistical significant no differences between the mean weight of newborns whose mothers had been exposed to dental vs non-exposed No indication, indicates frequency of X-rays Malignant or benign brain tumors No significant association was found between malignant brain tumors and dental diagnostic X-ray. Small increase risk of developing benign brain tumors mgy Thyroid cancer 13% increase in thyroid cancer risk for every 10 reported dental radiographs No indication Thyroid cancer Borderline increase risk for thyroid cancer associated with a having more than one dental X-ray per year No indication Infant birth weight No association between maternal dental health or dental care with low birth weight infants

7 112 R. WILKINS ET AL. relationship between dental X-ray exposure and the development of laryngeal cancer could be drawn from this study. In the late eighties and early nineties Preston-Martin et al. conducted a series of studies on the risks associated with dental X-rays (Preston-Martin et al. 1980; Preston- Martin et al. 1983; Preston-Martin, Bernstein et al. 1985; Preston-Martin, Henderson et al. 1985; Preston-Martin et al. 1988; Preston-Martin et al. 1989). These studies were the first epidemiological investigations to assess the correlation between dental X-rays and the development of tumors in the brain and parotid glands. Following this, several other groups also examined the effects of dental X-rays on meningioma development (Neuberger et al. 1991; Ryan et al. 1992; Rodvall et al. 1998; Longstreth et al. 2004; Claus et al. 2012; Lin et al. 2013). The results and details of these studies have been summarized in a recent meta-analysis which included seven case-control studies involving 6174 patients and 19,459 controls that reported development of meningioma. Overall, it was found that exposure to dental X-rays had no effect on brain cancer risk. When different types of examinations including full-mouth, bitewing, panoramic and lateral cephalometric were analyzed individually, there was a slight increase in risk relative to control groups with exposure to dental bitewing X-rays but with no other type of examination (Xu et al. 2015). However as only two of the seven studies reported risk with dental bitewing, statistical power is lacking to support the increased risk. It was noted that there was substantial heterogeneity in cohort selection, study design, information collection, and determination of exposure among the included studies. The results were also derived from studies with a number of shortcomings, the most notable of these being recall bias. Furthermore, information on the type of dental equipment, loading factors and film used or the frequency and the type of dental X-rays were not accurately documented in most studies. With respect to data analysis, odds ratios were presented without confidence intervals, which are meaningless in terms of significance and therefore voids any conclusions that may have been drawn from the data. A commentary by Tetradis et al. also proposed that the association between meningiomas and dental exposure observed by Clause et al. may have been due to increased numbers of dental X-rays taken for referred facial pain from meningiomas or the presence of head trauma (Tetradis et al. 2012). As the thyroid gland is highly susceptible to radiation induced carcinogenesis, particularly in children, a series of studies have investigated the effects of dental radiation on thyroid cancer risk (Ron et al. 1995; Ron et al. 2012). Memon et al. conducted a population-based case control interview study among 313 patients with thyroid cancer, matched controls and information on confounders such as nationality, gender, and age (Memon et al. 2010). The result of this study showed that exposure to dental X-rays was associated with an increased risk of thyroid cancer. However, the study was based on self-reporting by the participants as no dental X-ray records had been maintained at the dental clinics. More recently, employing data from the US Radiologic Technologists, Neta et al. found a 13% increase in thyroid cancer risk for every 10 reported dental radiographs using a prospective cohort study design (Neta et al. 2013). This study ran from 1983 to 2006 and included data from almost 150,000 patients who were radiological technologists who had undergone dental examinations in the past, with detailed information on confounding factors. However, information on the type of procedure (e.g. full mouth, panoramic, bitewing) was not available, with only the approximate number of times/year they had the procedure being reported. It should be noted, however, that these results were driven by dental X-rays received prior to 1970 when doses from dental X-ray procedures were much higher (Johnson and Goetz 1986). No detailed radiation exposure assessment was conducted to enable quantitative evaluation of risk. Furthermore, in 2015, Zhang et al. found a borderline increased risk for thyroid cancer associated with a having more than one dental X-ray per year (OR 2.20, : ) (Zhang 2015). This study was conducted in Connecticut in and included 462 histologically confirmed incident thyroid cancer cases and 498 population-based controls. The study examined all types of medical diagnostic procedures including dental. Participants were questioned on frequency and type of dental procedure they underwent and information on confounding factors was available. However, similar to earlier studies, recall bias must be considered as a shortcoming and information on the radiation doses to the thyroid gland was not available. The data analysis was based solely on the number of X-ray procedures received/participant, therefore, conclusions should be interpreted with caution. In 2004, Hujoel et al. conducted a study using enrollees of a dental insurance company to investigate the relationship between antepartum dental radiographs and infant low birth weight (Hujoel et al. 2004). Cases consisted of 117 women with low birth weight infant and controls comprised pregnancies resulting in normal birth weight infants (4468 participants). The authors hypothesized that pregnant woman exposed to dental X-rays may be at risk for delivering low birth weight infants (<2500 g). The study was performed with women aged between 1993 and 2000 of singleton births. For each woman, the date and type of each dental radiograph taken were abstracted from the dental utilization database. Doses were assigned to each type of radiographic exposure and summed. Doses were estimated based on a nationwide 1993 (U.S. N.E.X.T) dental survey evaluation of X-ray trends and published thyroid radiation doses. It was noted that thyroid shields were not used during the procedures for the participants recruited. For each of the enrollees, infant birth dates were recorded, however, no information was collected on critical items that could bias the study outcomes including information on smoking status, previous non-dental radiation exposures, and whether the participant had a normal functioning thyroid. Not all information was available from the participants on other confounders such as existing disease. It was shown that 10% of the cohorts had dental during pregnancy and birth weights of infants were reduced by up to 5%. The authors indicate that low dose radiation can cause thyroid

8 INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 113 dysfunction, which disrupts fetal development which may be the rationale for the observed low birth weight infants. This article was reviewed critically, highlighting the association was indirect and not causally related and the lack of information on critical confounding factors, such as overall oral health of the mother and recall of dental procedures, make it difficult to interpret the data (Boice et al. 2004; Brent 2005). Furthermore, studies that followed by others did not support the above findings. Al-Attas showed that, in a study with 47 cases and 58 control mothers, there was no association between the use of dental in any trimester of the pregnancy and birth weight (Al-Attas 2014). Mortazavi et al. examined using a large sample size of 1200 newborns, whose mothers had been either exposed or not exposed to dental in Iran. It was shown that the children of mothers who had received dental during their pregnancy showed no significant decrease in their birth weight (Mortazavi et al. 2013). specifications of the instrument used, settings, loading factors, number of visits, age of first visit etc. 6. Data interpretation: Standard statistical methods for the analysis of case-control studies are based on odds ratios, which calculate relative risk for each categorical risk factor (Breslow and Day 1980a). However, there are limitations of the odds ratio in detecting a diagnostic outcome which have been described and reviewed by Pepe et al. (2004). Odds ratios, especially without confidence intervals, are not the best statistical method for classification of outcomes. Other approaches such as multilogistic regression are more suitable. 7. Confounding factors: These are not often adjusted for, or insufficient data is available to draw accurate conclusions. These adjustments are normally achieved by unconditional multiple logistic regression which, with currently accessible analytical tools, should be applied to all epidemiological data (Breslow and Day 1980b). Summary of epidemiological findings In general, at such low doses, the detection of significant adverse health effects will always be a challenge, even with well-designed epidemiological studies with strong statistical power. However, when designing future studies the following criteria should be taken into consideration in order to ensure high confidence results: 1. Selection Bias: The selection and number of controls is an important determinant to the study design and if not appropriately, blinded and matched, may skew the outcomes. 2. Statistical power: One of the most important limitations of each of the evaluated studies is lack of statistical power. The studies have recruited a small number of participants (<40) to elucidate correlations. It is recommended for any type of epidemiological study the sample size and power of association be large in order to obtain reliable statistical inference. 3. Questionnaires: These should be well designed, clear and not open for misinterpretation by the respondent. It should also be designed to collect information on confounders along with adequate detail to accurately determine the dose of radiation exposure. 4. Interview bias: Interviewers should be blinded as to the status of the patient (i.e. case or control), thereby ensuring impartiality. 5. Accurate Dose estimates/recall bias: As there are currently limited databases available for diagnostic dental imaging procedures, it is difficult to obtain accurate dental exposure records. As such, researchers have had to rely on patient self-reporting, which has potential for recall bias leading to inaccurate data collection. Without accurate dental records and dose per procedure information, dose-response trends have to be approximated by the recall of the frequency of dental visits and type of X-ray procedures performed. It is recommended that future studies obtain complete information on the dental history of the patient, including type of procedure, Cell-based studies In light of the concerns regarding epidemiological studies, cell-based investigations become important as they are not hindered by many of the issues described above. Over the past decade, the majority of these studies have examined cytogenetic modulations (micronuclei formation) and cytotoxicity-induced effects (, pyknosis, and ) in the oral mucosa following dental X-ray exposure. Cytotoxicity assays normally assess short-term changes in the vicinity of the exposure that could eventually result in degenerative nuclear changes which might lead to cell death. Cell death is transient in the sense that, once the cell dies, the surrounding tissue will regenerate with no lasting effects. Concurrent with assessing these short-term changes, studies also detect long-term chromosomal damage using the buccal cell micronucleus (MN) assay (Thomas et al. 2009; Bonassi et al. 2011). The MN assay is widely used to identify irreversible chromosomal damage. MN formation occurs in dividing cells which are most likely to be found in the basal layer and move to the surface through turnover of the epithelial layer. As this turnover is rapid (7 16 days), MN formation can be detected in exfoliated cells within this timeframe subsequent to the exposure (Thomas et al. 2009). The majority of the cell-based studies harvest oral mucosa cells from the cheeks of their subjects before and 10 days following exposure to dental X-rays and then assess the cells for cytotoxic damage and MN formation. Specific details on type of dental procedure and specifications of the dental instrument used in each study are provided in Table 3. Over the course of the past 12 years, these methods have been used to asses cytogenetic changes associated with dental X-ray exposures at the cellular level after panoramic. MN and nuclear cytotoxic changes were examined in harvested oral mucosa cells obtained from the cheeks of healthy adults before and 10 days post-procedure. These studies have shown that the frequency of nuclear alterations, indicative of apoptosis ( and condensed chromatin) are significantly higher following dental X-ray

9 114 R. WILKINS ET AL. Table 3. Summary of in vitro studies. Reference Dental Procedure Cell Type Instrument Instrument detailsa End Point Analysis Outcome Cerqueira et al. (2004) Panoramic dental da Silva et al. (2007) Panoramic dental Angelieri et al. (2007) Panoramic dental Ribeiro and Angelieri (2008) Panoramic dental Ribeiro et al. (2008) Panoramic dental Popova et al. (2007) Panoramic dental Cerqueira et al. (2008) Panoramic dental Carlin et al. (2010) Cone beam computed tomography Angelieri et al. (2010) Panoramic dental Oral mucosa cells from healthy adults (n¼ 31) Tongue epithelial cells from healthy adult males collected 10 days after exposure (n¼ 42) Exfoliated buccal mucosa cells from healthy children (n¼ 17) Exfoliated buccal mucosa cells from healthy adults (n¼ 39) Exfoliated buccal mucosa cells from healthy children and adults (n ¼ 34) Exfoliated buccal cells from healthy adults (n¼ 32) Epithelial gingival cells from healthy adults (n¼ 40) Buccal mucosa cells harvested from healthy adults (n¼ 19) Comparison between epithelial cells harvested Siemens Orthophos 71 kv, 15 ma, 14 s, 110 mgy/cm 2, effective dose 21.4 lsv Rotography Plus kv, 10 ma, s, dose on the lateral border of the tongue was msv Siemens Orthophos 71 kv, 15 ma, 14 s, 110 mgy/cm 2, entrance dose 0.08 R Siemens Orthophos 71 kv, 15 ma, 14 s, 110 mgy/cm 2, entrance dose 0.08 R Siemens Orthophos 71 kv, 15 ma, 14 s, 110 mgy/cm 2, entrance dose 0.08 R Micronucleus and cytotoxicity assays p>.05 in micronucleated oral mucosa cells before and after dental X-rays. p<.05 increase in nuclear alterations related to cytotoxicity, pyknosis and Increase in the number of nuclear anomalies (except micronuclei in exfoliated cells of the lateral border of the tongue, effect was pronounced when patients were exposed to repeat radiographs p>.05 in micronucleated oral mucosa cells before and after dental X-rays. p<.05 increase in nuclear alterations related to cytotoxicity, pyknosis and p>.05 in micronucleated oral mucosa cells before and after dental X-rays. p<.05 increase in nuclear alterations related to cytotoxicity, pyknosis and Both groups exhibited no effects in micronucleus formation after exposure. Increase in cytotoxicity endpoints was observed (, pyknosis and ) in both groups following exposure No indication No indication Micronucleus assay CBCT and conventional Siemens Orthophos kv, 15 ma, 14 s, 110 mgy/cm 2, effective dose 21.4 lsv i-cat CBCT scanner kv, 10 ma, s, entrance dose R radiographs p>.05 micronucleus frequency, p<.05, pyknosis and p<.05 increase in chromosomal damage and nuclear alterations p>.05 micronucleus frequency, p<.05, pyknosis and Siemens Orthophos Lateral border of the tongue is more sensitive

10 INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 115 Angelieri et al. (2010) Lateral and frontal cephalometric X-ray and panoramic X-ray from buccal mucosa and lateral border of the tongue in healthy adults (n¼ 32) Exfoliated oral mucosa cells from healthy adults (n¼ 18) El-Ashiry et al. (2010) Panoramic dental Exfoliated buccal mucosa cells from healthy children (n¼ 20) Ribeiro et al. (2011) Cephalometric Exfoliated oral mucosa cells from healthy adults (n¼ 30) Waingade and Medikeri (2012) Panoramic Buccal epithelial cells from healthy subjects (n¼ 60) Arora et al. (2014) Panoramic Nonkeratinized buccal cells and keratinized epithelial gingival cells from healthy adults (n¼ 53) Agarwal et al. (2015) Panoramic Exfoliated buccal mucosa cells (n¼ 20) pediatric patients, Lorenzoni et al. (2012) Lateral cephalograph, posteroanterior cephalograph, panoramic radiograph, full mouth intra-oral X-ray Exfoliated buccal cells from healthy children (n¼ 25) Lorenzoni et al.. (2013) Cone beam computed tomography, conventional radiograph Exfoliated oral mucosa cells from healthy children (n¼ 25) a as described in the publication. 71 kv, 15 ma, 14 s, 110 mgy/cm 2, entrance dose 0.08 R Siemens Orthophos 71 kv, 15 ma, 14 s, 110 mgy/cm 2, effective dose 21.4 lsv Orthopantomograph 70 kv, 24 ma, 16 s, 110 mgy/cm 2, entrance dose 0.08 R Rotography Plus kv, 10 ma, s, entrance dose R Gendex Orthoralix system kv, 10 ma, 12 s, output dose rate: m Gy/s Gendex Orthoralix system 74 kv, 10 ma, 12 s, output dose rate m Gy/s at 70 kv, 10 ma Kodak 8000 C system kv, 15 ma, 13 s, 110 mgy/cm 2, effective dose 21.4 msv Rotograph Plus equipment Spectro 70X selectronic equipment i-cat CBCT scanner/ Rotography plus Rotograph Plus equipment: LAT: 80 kv, 10 ma, 1.3 S, msv PA: 85 kv, 10 ma, 1.6 s, 0.03 msv, PAN: 70 kv, 10 ma, 17 s, 0.03 msv Spectro 70 selectronic equipment: anterior periapical: 70 kv, 8 ma, 0.4 s, msv, round collimation posterior periapical and bitewing: 70 kv, 8 ma, 0.45 s, msv, round collimation 120 kv, mas, 40 seconds/ KV, 10 ma, s, entrance dose R Micronucleus and cytotoxicity assays to cytotoxic insults combined with continuous cigarette smoke exposure p>.05 micronucleus frequency, p<.05, pyknosis and p>.05 micronucleus frequency, p<.05, pyknosis and p>.05 micronucleus frequency, p<.05, pyknosis and Micronucleus assay p<.047 micronucleated cell frequencies Micronucleus assay p<.05 of micronuclei frequency in buccal cells, No statistically significant increase was found of micronuclei frequency in gingiva cells Micronucleus assay,, pyknosis, and Micronucelus assay, cytotoxicity assay: pyknosis,, p>.001 no significant number of MN in buccal epithelial cells, p<.001 combined frequency of, pyknosis, and was observed p>.05 no statistically significant differences were observed in Mni formation after exposure, p<.05 increase in, pyknosis, and was observed following radiation exposure CBCT and conventional radiographs p>.05 micronucleus frequency, p<.05, pyknosis, and

11 116 R. WILKINS ET AL. procedures but, in most cases, no statistically significant increase in the number of MN was observed (Cerqueira et al. 2004; Popova et al. 2007; Ribeiro and Angelieri 2008; Angelieri et al. 2010). A follow-up study, in 2008, by Cerqueira et al. conducted in epithelial gingival cells showed significant cytogenetic effects following dental X-ray exposures (Cerqueira et al. 2008). In contrast to their previous study in 2004, the results indicate a higher MN induction in epithelial cells harvested from the gingiva. Significant cytotoxic nuclear alterations were also observed indicative of apoptosis similar to those found in oral mucosa cells. In 2011 Ribeiro et al. performed a study examining genetic damage in oral mucosa cells of patients after exposure to digital performed using panoramic radiographs and lateral and frontal cephalometric X-rays (Ribeiro et al. 2011). The results of this work were similar to previous studies in terms of evidence for cytotoxicity and a lack of MN formation. More recently, two new studies have emerged both using similar analysis of MN in buccal cells following panoramic radiographic exposures. The first study of 60 subjects found a small but significant increase in MN after exposure to dental X-rays (Al-Attas 2014; Waingade and Medikeri 2012) while another study found a significantly higher increase in both the frequency of MN and the percent of cells with MN after a similar exposure (Arora et al. 2014). Similarly, by using exfoliated cells from the lateral border of the tongue from men aged 18 40, da Silva et al. observed no genotoxicity at the chromosome level following one or two panoramic radiographs, however, the number of cells undergoing apoptosis was increased post-exposure (da Silva et al. 2007). A cytotoxic effect was more evident in patients who required a repeat radiograph exposure due to error in the first procedure, but no evidence of permanent cellular damage was observed. The authors do acknowledge that the sample size in this study was small, and any effects of alcohol, tobacco or other genotoxic agents, which could produce changes in the oral mucosa, were not accounted for. With the introduction of CBCT, research has shifted to examine biological markers of damage induced by this new technology. This technology, which offers three-dimensional (3D) imaging of dental structures, is administered at higher doses than bitewing, root end or panoramic X-rays. In 2010, Carlin et al. assessed DNA damage and cellular death in exfoliated buccal mucosa cells from adults following CBCT (Carlin et al. 2010). Exfoliated oral mucosa cells were collected immediately before and 10 days after CBCT in healthy adults and evaluated for DNA damage, using the MN test, as well as other indicators of cell death. Prior to CBCT exposure, the frequency of MN cells was small (0.04%) with no statistically significant increases observed following the CBCT procedure. However, nuclear alterations in the form of, pyknosis, and were shown to increase by more than two-fold. These results are in line with those shown previously, indicating the potential for ionizing radiation to induce localized temporary cellular damage. The above studies which have been conducted in adults have predominately shown no permanent cellular effects associated with dental X-ray exposures. However, children are thought to be more susceptible to adverse health effects from exposure to radiation, mainly because they are developing and have shown altered DNA repair capacity (Frush 2011). The following studies have examined DNA damage (MN formation) and cellular death in exfoliated buccal mucosa cells from healthy children following dental (Angelieri et al. 2007; Ribeiro et al. 2008; Ribeiro and Angelieri 2008; Carlin et al. 2010; El-Ashiry et al. 2010; Agarwal et al. 2015). Overall these studies indicate no increased susceptibility in these endpoints among children as compared to adults. In 2011, Lorenzoni et al. performed a comprehensive cytogenetic biomonitoring study in children following a complete set of radiographs for orthodontic planning. This included lateral and posteroanterior cephalographics, panoramic, full periapical exams and bitewing exams. The MN test indicated no increase in chromosomal damage, however, a significant increase in nuclear alterations related to cytotoxicity was observed similar to results observed in adult patients (Lorenzoni et al. 2012). The study was repeated with CBCT and similar findings were observed (Lorenzoni et al. 2013). Summary of cell-based studies In general, the results from the cell-based studies described above predominantly indicate that there are no cytogenetic changes associated with exposures from dental X-rays. However, there is some indication of a localized cytotoxic response in the tissue. The most recent studies that do show increased MN formation may be the result of improvements to and standardization of the MN assay for biomonitoring, thereby, increasing the ability to measure small changes with greater confidence (Holland et al. 2008). However, these minute cellular changes may not pose any long-term adverse health effects. Furthermore, as with the epidemiological studies, there are some possible concerns and limitations to these cellular assays as listed here: 1. Confounding factors: confounding factors such as age, smoking and alcohol consumption, are all known to be associated with increased frequencies of chromosome aberrations and therefore, MN (Fenech 1998; Bonassi et al. 2011; Nefic et al. 2013). This can lead to large inter-individual variation in background levels which are difficult to control for and can skew results and bias outcomes. Robust statistical methods must be employed to appropriately analyze this type of data. 2. Sensitivity: Due to large variations in the background levels of MN, the sensitivity of the assay may be insufficient to detect changes after very low doses of radiation (Ceppi et al. 2010). New technologies have emerged over the years that may have greater sensitivity to detect effects at lower doses. 3. Sample size: Results from many of the studies were derived from low sample numbers (<40) and in some cases, these were divided into multiple treatment

12 INTERNATIONAL JOURNAL OF RADIATION BIOLOGY 117 groups. For detecting small changes with statistical confidence, larger sample sizes would be required. 4. Scorer bias: It is not clear from the studies whether the scorers were blinded to the sample identification. Future studies should control for selection and information collection bias by randomizing data and ensuring evaluators are blinded to the process. 5. Dosimetry: Dose estimates, if available were based on equipment settings with no evidence of actual physical dose measurements to the area of sampling. In addition, the reporting of dosimetry outputs was inconsistent across studies, making it difficult to compare results. Future studies should measure the physical doses to the area of sampling, thereby ensuring proper dose estimates. Concluding remarks Dental X-rays account for most of the collective population dose to the head and neck from diagnostic in healthy individuals. The question remains whether low-dose exposure to ionizing radiation could lead to a detectable increase in the risk of developing cancer. Epidemiological investigations have attempted to address this question; however not successfully, as these studies are challenged by inherent limitations in design, dosimetry, lack of information on confounding factors and most importantly sampling and recall bias, which are of particular importance when trying to detect small changes after such a low dose. Genetic effects from dental have also been assessed by investigating MN formation and cytotoxicity markers, but these studies have shortcomings related to the use of biomonitoring assays that are not sensitive to detect changes at low doses and a statistical analysis approach that does not account for confounding factors. Overall, the scientific literature on the possible biological implications of dental X- rays is insufficient and, based on decades of research on low dose ionizing radiation (<10 mgy), there is no clear indication of health effects. Studies using higher doses of radiation (i.e skull radiographs, sinus radiograph or head computed tomography) and extensive work with Japanese atomic bomb survivors have evidence of increased cancer risk below 0.1 Gy. However, more mechanistic studies are needed and research efforts should be put forth to developing innovative approaches for interpolating dose-response between data from epidemiological investigations and incremental doses above background and the correlation to longterm health effects. At such low doses, the detection of significant adverse health effects will always be a challenge, even with well-designed epidemiological studies with strong statistical power, ultimately what is of relevance is the detection of early biological signatures that may be indicative of a long-term adverse health effect. As new technologies evolve these may prove valuable in detecting minute changes in biological tissue and future studies should be centered on identifying key events that are associated with an adverse outcome. Until this time, the public should rest assured that clinicians ensure that each radiographic examination is appropriately justified as beneficial, and any required exposures are optimized to ensure the lowest reasonable radiation dose is used without compromising diagnostic information (Annals of the ICRP 2007a; Annals of the ICRP 2007b; European Commission 2004). Acknowledgments The authors are grateful to Matthew Rodrigues, Richard Smith, Christian Lavoie and Narine Martel for critical review of the manuscript. Disclosure statement No potential conflict of interest was reported by the authors. Funding This study was supported by Health Canada A-Based funds #8R1055. Notes on contributors Vinita Chauhan and Ruth C. Wilkins are research scientists at Health Canada. ORCID Vinita Chauhan Ruth C. Wilkins References Agarwal P, Vinuth DP, Haranal S, Thippanna CK, Naresh N, Moger G Genotoxic and cytotoxic effects of X-ray on buccal epithelial cells following panoramic : a pediatric study. J Cytol. 32: Al-Attas S Maternal dental health and low birth weight among term deliveries. Source Life Sci J. 11: Angelieri F, Carlin V, Saez DM, Pozzi R, Ribeiro DA Mutagenicity and cytotoxicity assessment in patients undergoing orthodontic radiographs. 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