149 Department of Veterinary Medical Sciences, Alma Mater Studiorum - University of Bologna, Bologna, Italy *Corresponding author at: Department of Veterinary Medical Sciences, Alma Mater Studiorum - University of Bologna, Bologna, Italy. Tel.: +39 333 448 5003, e-mail: mariana.roccaro2@unibo.it. Keywords Age determination, Dog, Forensic medicine, Puppy trade, Teeth examination, Veterinary legislation. Summary Age determination of puppies represents a significant issue of animal welfare and forensic medicine, particularly for what concerns trade and imports of dogs. Despite the movement of puppy dogs before the age of 15 weeks is forbidden by Regulation (EU) No 576/2013, the occurrence of illegal transport of younger puppies is not uncommon. The illegal trade of puppies increases instances of falsified documentation, the counterfeit of vaccine certificates and discrepancies between the declared age and the real age of the puppies. Consequently, determining the exact age of animals and evaluating their welfare become legally crucial. Dental examination currently represents the most common approach to estimate the age of a puppy in clinical practice and in forensic investigations. In this work we addressed the legal, health and welfare issues associated with dogs’ trade and import and we reviewed the existing literature referring to the assessment of age in dogs by dental examination. The imprecision and inaccuracy of this method make it poorly convincing in legal proceedings. The reasons for such vagueness are to be ascribed both to the lack of standardization and to many variability factors (size, breed, sex, diet, etc.) which influence dental eruption and development. Mariana Roccaro* and Angelo Peli Age determination in dog puppies by teeth examination: legal, health and welfare implications, review of the literature and practical considerations Veterinaria Italiana 2020, 56 (3), 149-162. doi: 10.12834/VetIt.1876.9968.2 Accepted: 26.06.2019 | Available on line: 31.12.2020 they are transported to the distribution countries, often when they are too young to be moved. Data collected within a project on the movement of pet animals funded by the Italian Ministry of Health and carried out by the Istituto Zooprofilattico Sperimentale dell’Abruzzo e del Molise revealed that, in 2014, 86% of imported puppies were under three months old (Arena et al. 2015). Consequently, determining the exact age of animals becomes legally crucial. It follows that the method used to assess age has to be reliable, precise and accurate. The methods available for assessing the age in dogs are different. Examination of teeth has long been used to age in man and domestic animals, it is non-invasive and requires no special equipment. Instrumental analyses such as the radiographic evaluation of limbs’ ossification centres (Hare 1959, Hare 1960, Seoudi 1947, Sumner‐Smith 1966, Thrall and Robertson 2015) and dental radiography to assess pulp cavity/tooth width ratio (Kershaw et  al. 2005, Mbizah et  al. 2016) have been developed. Introduction Age determination in dogs is of great importance both in clinical veterinary practice and in veterinary forensic medicine; it is called in question in the purchase of animals as well as for their movement for non-commercial or trading purposes and this is especially the case of young animals. In this regard, the movement of pet animals, including dogs, within the European Union (EU) has to comply with clearly defined rules that prevent them from being transported before they have reached a certain age. Despite the legislation currently in force, the growing demand of purebred puppies at low prices has been fostering the illegal puppy trade from Eastern European Countries to Western Europe. According to the FOUR PAWS International’s report on puppy trade in Europe, dog traffic in Italy and France is estimated at 43 million Euros (FOUR PAWS International 2013). Puppies are bred in extremely poor conditions in so-called ‘puppy mills’ in Eastern Europe, where low enforcement of transport, health and welfare legislation allows the cheap prices; later 150 Veterinaria Italiana 2020, 56 (3), 149-162. doi: 10.12834/VetIt.1876.9968.2 Legal and welfare implications of age determination by dental examination Roccaro & Peli For what concerns the non-commercial movement of dogs within the EU, the relevant legislation is Regulation (EU) No 576/20132, together with Commission Implementing Regulation (EU) No 577/20133. In order to be moved into an EU country from another EU country, the dog must be marked by the implantation of a transponder or by a clearly readable tattoo (applied before July 2011) and subsequently it must be vaccinated against rabies by an authorised veterinarian, no earlier than 12 weeks of age. The period of validity of the vaccination starts not less than 21 days from the completion of the vaccination protocol for the primary vaccination. Finally, the pet animal must be accompanied by a passport completed and issued by an authorised veterinarian documenting the alpha-numeric code displayed by the transponder, and the details of the vaccination against rabies. The intra-Union pet trade must comply with the requirements of the Directive 92/65/EEC4 (implemented in Italy by Legislative Decree No 633/19965), according to which the animals must come from registered establishments. Dogs must be marked, vaccinated against rabies, be accompanied by a passport and a health certificate issued by an official veterinarian, who notifies the movement to the competent authorities of destination through the Community Trade Control and Expert System (TRACES). Finally, dogs must undergo a clinical examination in order to verify that they show no signs of diseases and are fit to be transported for the intended journey, in accordance with Regulation (EC) No 1/20056 on the protection of animals during transport. This Regulation also states that puppies younger than 8 weeks of age are unfit for transport unless accompanied by their mother. According to the over mentioned laws, the movement of dogs, either for non-commercial or trading purposes, is strictly dependent on the completion of the rabies vaccination protocol; therefore, puppies cannot be moved before 15 weeks of age. However, EU countries have discretion whether or not they allow the introduction onto their territory of ‘young dogs’, i.e. dogs which are less than 12 weeks old However, radiographic investigations have not found wide application outside the research field due to the cost of the required equipment, the exposure of the practitioner and the animal to x-rays, the eventual need for sedation and the lack of a standardized protocol. Ocular lens examination, dental abrasion and tartar can be used in adult dogs, but are subject to a considerable individual variability (Gesierich et  al. 2015, Tobias et  al. 2000). Lastly, the histologic investigation of dental cementum deposition, widely used in wild animals, proved to be unreliable for age determination in dogs (Van Lancker et al. 2005). This work aims to address the issues associated with age determination in dog puppies by dental examination, which currently represents the most common approach to estimate the age of a puppy in clinical practice and in forensic investigations. The first part focuses on the regulatory framework that requires knowledge of the dog’s age and on the consequences of trade and movement of dogs that are too young; the second part starts with a brief summary of dental development, there follows an overview of the existing literature referring to the assessment of eruption of deciduous and permanent teeth; finally, the last part identifies limits and areas of improvement for the application of such methodology in practice. Legal, health and welfare implications The laws that imply the knowledge of the dogs’ age essentially concern the identification, the trade and the movement of animals for non-commercial and trading purposes. In Italy, Law No 281/19911 established an obligation to identify and register dogs in the national database; the definition of manner and timing was left to the individual Regions. The identification requirements were subsequently harmonized by the State-Regional Agreement of 24 January 2013, according to which dogs must be identified and registered within two months of age and the sale or transfer of unidentified dogs is forbidden. 1 L. 14 August 1991, No. 281. “Legge quadro in materia di animali di affezione e prevenzione del randagismo”. Off J, 203, 30/08/1991, 3-5. 2 Regulation (EU) No. 576/2013 of the European Parliament and of the Council of 12 June 2013 on the non-commercial movement of pet animals and repealing Regulation (EC) No. 998/2003. Off J, L 178, 28/06/2013, 1-26. 3 Commission Implementing Regulation (EU) No. 577/2013 of 28 June 2013 on the model identification documents for the non-commercial movement of dogs, cats and ferrets, the establishment of lists of territories and third countries and the format, layout and language requirements of the declarations attesting compliance with certain conditions provided for in Regulation (EU) No. 576/2013 of the European Parliament and of the Council. Off J, L 178, 28/06/2013, 109-148. 4 Council Directive 92/65/EEC of 13 July 1992 laying down animal health requirements governing trade in and imports into the Community of Animals, semen, ova and embryos not subject to animal health requirements laid down in specific Community rules referred to in Annex A (I) to Directive 90/425/ EEC. Off J, L 268, 14/09/1992, 54-72. 5 D.Lgs. 12 November 1996, No. 633. “Attuazione della direttiva 92/65/CEE che stabilisce norme sanitarie per gli scambi e le importazioni nella Comunità di animali, sperma, ovuli ed embrioni non soggetti, per quanto riguarda le condizioni di polizia sanitaria, alle normative comunitarie specifiche di cui all’Allegato A, sezione I, della direttiva 90/425, CEE”. Off J, 296, 18/12/1996, Ordinary Supplement No. 222. 6 Council Regulation (EC) No. 1/2005 of 22 December 2004 on the protection of animals during transport and related operations and amending Directives 64/432/EEC and 93/119/EC and Regulation (EC) No. 1255/97. Off J, L 3, 05/01/2005, 1-44. 151Veterinaria Italiana 2020, 56 (3), 149-162. doi: 10.12834/VetIt.1876.9968.2 Roccaro & Peli Legal and welfare implications of age determination by dental examination 12 weeks of age on and becomes effective 21 days after the completion of the vaccination protocol. The trade of puppies that are too young to be vaccinated effectively represents a great danger not only for the animals but also for humans, especially if they are born from a non-vaccinated mother and they come from a not rabies-free country. Other welfare issues include the inadequate socialization of puppies in their countries of origin before being sold and the early maternal separation, which may play a role in the development of behavioural disorders. Compared to puppies separated from their mother and littermates at two months of age, puppies separated at 30 to 40 days of age are more likely to develop a variety of behavioural problems as an adult, including excessive barking, destructiveness, attention-seeking, fearfulness on walks, noise reactivity and aversion to strangers (Pierantoni et  al. 2011). All of these factors lead to societal costs in terms of new owners needing to treat their sick animals and to manage potential behavioural problems, such as aggression, which also has a significant impact on public health (Sacks et al. 1996, Méndez Gallart et al. 2002, Langley 2009, Rosado et al. 2009). Besides, aggression is one of the main causes of abandon and euthanasia of dogs (Reisner et al. 1994, Overall and Love 2001). Dental development in dogs: general features and literature overview The assessment of age through the observation of deciduous and permanent eruption and succession of teeth has been used for a long time in veterinary practice, starting with production animals. This non-invasive method requires no special equipment and can be performed both in living and dead animals. Clinical examination of teeth includes the assessment of number, integrity, shape and colour of the teeth, as well as the potential presence of tartar, halitosis and bleeding. Therefore, it requires a thorough knowledge of the anatomy of the teeth and the physiology of their development. Dogs, like other mammals, are characterized by the possession of a diphyodont dentition. At birth, a puppy is toothless; within a few weeks, deciduous teeth, also known as ‘milk teeth’, develop, which are later replaced by permanent teeth. Teeth in the upper dental arcade normally erupt a few days earlier than the ones in the lower arcade (Girard 1845, Balasini 1995). The permanent set is deemed to be complete by the seventh month. The most accredited canine dental formulas are, for the deciduous dentition Id3/3, Cd1/1, Pd3/3 and for the permanent dentition I3/3, C1/1, P4/4, M2/3; where ‘I’ stands for incisor, ‘C’ for canine, ‘P’ for premolar, ‘M’ for molar and ‘d’ for deciduous (Nickel et  al. 1979, and have not received an anti-rabies vaccination, or between 12 or 16 weeks old and have received an anti-rabies vaccination but are not yet fully protected. This is possible for non-commercial movements from another EU country or a third country and for trade purposes within EU countries. It must be noted that countries like Italy, Germany, France, United Kingdom, Spain, The Netherlands do not admit this exception. On the contrary, imports from non-EU countries of young dogs not vaccinated against rabies are not allowed under any circumstances. As stated above, EU legislation and TRACES guidelines are often violated. Puppies are transported when they are still too young, with no identification, without or with incorrect vaccinations, dog-passports and transport papers, in unsanitary conditions and with little regard for their wellbeing (Benini 2008). In Italy, Law No 201/20107 establishes the punishment for the illegal trade and import of pet animals, with a harsher punishment if the animals have less than 12 weeks of age. The measure of punishment for illegal puppy trafficking is severe enough on paper, but rarely results in a conviction in court. Therefore, the deterring function of the punishment is close to nil. The difficulty in determining the exact age of the animals, in case the transport papers lack or in order to verify their regularity, is undoubtedly the most critical element that arises in this kind of judicial proceedings. The trade and movement of dogs that are too young have not only legal implications, but also impacts on animal health and welfare. It has been observed that removal from the litter prior to eight weeks of age may cause severe distress (Serpell and Jagoe 1995), and the separation of dog puppies from their mother at six weeks of age impairs their physical condition and weight gain, with an increase of disease susceptibility and mortality (Slabbert and Rasa 1993). The health risk is increased by transport and promiscuity, therefore puppies often become ill and die during the transport or soon after. An inquiry of the Italian Veterinary Councils Federation (Benini 2008) on control of pet import reveals that 52% of dog puppies were found to be sick: 34% were infested with endoparasites, 23% were infected with parvovirus, 17% had fungal infections, 16% had scab and 10% were carriers of distemper. The risk of infectious disease spread is therefore very high and becomes critical when zoonoses, such as rabies, are involved. Rabies vaccination of puppies can be regularly performed only from 7 L. 4 November 2001, No. 201. “Ratifica ed esecuzione della Convenzione europea per la protezione degli animali da compagnia, fatta a Strasburgo il 13 novembre 1987, nonché norme di adeguamento dell’ordinamento interno”. Off J, 283, 03/12/2010, 1-27. 152 Veterinaria Italiana 2020, 56 (3), 149-162. doi: 10.12834/VetIt.1876.9968.2 Legal and welfare implications of age determination by dental examination Roccaro & Peli as few are the observed animals. Mellanby (Mellanby 1929) obtained the eruption times for deciduous and permanent teeth through the observation of 17 and 4 puppies, respectively, of different types of dog. Deciduous teeth erupted 3 days earlier in larger dogs compared to the smaller ones, while permanent teeth erupted one week in advance. In a later study, carried out by Arnall (Arnall 1960), the sample included 5 Bull Terrier puppies. Eruption times for deciduous teeth fell between 20-35 days of age, while permanent dental eruption began at 105-125 days. However, as the author himself admitted, such a limited study can only allow to draw general principles of eruption and any detailed conclusions can only apply to the breed of dog observed. Regarding to the sample size, an exception is represented by the works of Shabestari and colleagues (Shabestari et  al. 1967) and Kremenak (Kremenak 1969). In order to study dental eruption chronology, Shabestari and colleagues used 106  closely related purebred Beagle puppies. Based on previously published data, the Authors conclude that in dog dental eruption times vary more between breeds than among individuals of the same breed. Kremenak’s study on deciduous dental eruption was based on the daily observation of 32 purebred (16 Beagles, 10 Labradors, 6 Pointers) and 48 mixed-breed puppies of known age, for a total of 40 male and 40 female puppies. On the average, all teeth erupted from 22 to 34 days of age, and this is quite in accordance with Arnall’s results (Arnall 1960). Males preceded females in eruption of 21 of the 28 teeth, but this sex difference was found not statistically significant. When comparing puppies of different breeds, results supported the view that deciduous teeth eruption in Beagles occurs later than in strains of larger dog breeds. Interestingly, additional data from the observation of eight female mixed-breed puppies from the same litter allowed the Author to notice a rather wide variability among individuals sharing the same sex and bloodline, with ranges of up to nine days. It is widely recognised that dental eruption is affected by several factors, such as general health state, diet, sex, breed and body size. Several Authors agree that teeth erupt earlier in the larger dog strains (Girard 1845, Mellanby 1929, Piérard 1967, Kremenak 1969, Barone 1981, Sisson and Daniels Grossman 1982, Evans and de Lahunta 2013, Dyce et al. 2018). Breed as well influences the timing of dental eruption, development and wear. There are in fact significant differences among dog breeds in terms of head size and shape: brachycephalic and dolichocephalic breeds represent the two extremes of such variability. For this reason, Nickel and colleagues (Nickel et  al. 1979) explicitly refers to the German Shepherd’s dentition as a prototype, as it is the closest to the Evans and de Lahunta 2013, Dyce et al. 2018). There are no deciduous precursors for the first premolar or the molar teeth in dogs. On the basis of the changes that take place in the evolution and alteration of the teeth, the life of an animal can be divided in four periods: eruption of the deciduous teeth, wearing of the deciduous teeth, eruption of the permanent teeth and wearing of the permanent teeth. This work consists of a historical overview of the existing literature pertaining to the timing of dental eruption in dogs. The information was searched through the Catalogue of National Library Service and the data-base available which included the collections of: Directory of Open Access Journals (DOAJ), Medline/Pubmed (NLM), ProQuest collection (Web of Science), Science Direct Journals (Elsevier), Wiley (CrossRef ), Wiley Online Library. The search key words were as follow: age determination and dog; age assessment and dog; age and dog; teeth and dog; dentistry and dog; dental eruption and dog. The literature search included 24 Anatomy, Dentistry, Paediatrics and Zoognostic textbooks and 8 papers. The literature search on the data-base gave unsatisfying results, therefore a scientometric approach was not possible and we opted to proceed with a retrospective analysis of the literature. Most of the bibliographic material on dental eruption and development is dated and sometimes hard to trace. The first documents on age assessment by teeth date back to the late 1800s (Girard 1845, Liautard 1885, Huidekoper 1891). However, we decided to exclude these sources from the present work because both of them state that, in puppies, the incisors are already present at birth. We have not been able to find any reasonable explanation for such a statement, other than a sloppiness in the description, as it seems unlikely that selection could have led to the creation of substantially different dogs in just two hundred years. The timing of dental eruption has been reported in numerous Anatomy, Dentistry, Paediatrics and Zoognostic textbooks (Cornevin and Lesbre 1894, Miller 1952, Bourdelle and Bressou 1953, Silver 1963, Ferrara 1965, Nickel et  al. 1979, Barone 1981, Harvey and Emily 1993, Balasini 1995, Bonetti 1995, Hoskins 2001, Vaissaire 2001, Squarzoni 2003, Reece 2009, Veggetti and Falaschini 2009, Van de Wetering 2011, Evans and de Lahunta 2013, Gorrel 2013, Veronesi et al. 2013, Dyce et al. 2018) on the basis of previous published data or presumably from direct observations on the animals, but often there is no remind to original data or references. Some journal articles merely cite data published in books (Barton 1939, Fulton et  al. 2014, Hale 2005, Piérard 1967), while research papers that explicitly declare the number of dogs used in the study are very few, as well 153Veterinaria Italiana 2020, 56 (3), 149-162. doi: 10.12834/VetIt.1876.9968.2 Roccaro & Peli Legal and welfare implications of age determination by dental examination all incisors, premolars, etc.). Very few Authors give separate timing for upper and lower teeth (Mellanby 1929, Arnall 1960, Shabestari et  al. 1967, Kremenak 1969, Bonetti 1995). More importantly, it became evident that there is a wide disagreement in the chronology of dental development among the Authors. In order to highlight this variability, we provide a diagram illustrating the timing of deciduous and permanent dental eruption according to the different Authors (Supplementary Table I and II). Data were derived from 8 manuscripts and 21 of the 24 consulted textbooks, which were selected for providing separate information for at least each tooth class. Diagrams summarizing the earliest and latest ages of deciduous and permanent teeth eruption (Figure 1) are given below. In this case, only sources indicating a time range for each single tooth were selected, otherwise the resulting ranges would have been wide to the point of losing usefulness. The first deciduous teeth to erupt are the canines. Some Authors placed their appearance at 15-20 days (Miller 1952, Ferrara 1965, Balasini 1995), while for the majority of the sources they do not erupt before 3 or 4 weeks of age. Incisors follow immediately after, usually starting from corner incisors (I3), then intermediate (I2) and lastly central incisors (I1). Their eruption window is quite variable, ranging from 3-5  days for some Authors (Ferrara 1965, Barone 1981, Balasini 1995) up to 15-20 days (Miller 1952, Arnall 1960, Kremenak 1969, Veronesi et  al. 2013). The deciduous premolars begin to erupt at the turn of the second and the third week of age, normally in this order: P3, P4, P2. For most Authors the eruption of the deciduous dentition is complete by 6 weeks of age, but for others it extends up to 8 (Miller 1952, original wild ancestor. Moreover, some breeds are known to be predisposed to dental anomalies, which can make age assessment by dental examination even more difficult. Hypodontia (missing teeth) is most common in small-breed dogs (Van de Wetering 2011, Lobprise 2012), but it also occurs in brachycephalic breeds (Akers and Denbow 2008, Dyce et  al. 2018) and in large breeds such as Dobermann, Rottweiler and German Shepherd (Van de Wetering 2011). The premolar teeth are the most commonly missing. Additional teeth are common in brachycephalic dogs (Boxer, Bulldog) and Mastiff (Van de Wetering 2011, Lobprise 2012). Delayed eruption has been observed in Tibetan Terrier, Irish Soft Coat Wheaten Terrier, Portuguese Water Dog and Chinese Crested Dog (Hoskins 2001, Lobprise 2012). For what concerns sex, contrarily to Kremenak's (Kremenak 1969) findings, according to Harvey and Emily (Harvey and Emily 1993) teeth of female dogs erupt earlier. Moreover, they state that season also affects the time of dental eruption, and teeth of dogs born in the summer erupt earlier. In addition to these factors, the dog’s diet and eating habits determine significant variations in tooth wear, therefore affecting the estimated age (Girard 1845, Piérard 1967, Barone 1981, Balasini 1995, Veggetti and Falaschini 2009, Liebich et al. 2014). The analysis of the available sources on dental eruption and development revealed terminological discrepancies in the described phenomena; for example, some Authors include the monophyodont first premolar among the deciduous teeth even if it does not shed, others number the deciduous premolars 1, 2 and 3, which can be confusing. Differences also exist in the degree of detail of the provided data, both in relation to the timing (days vs. weeks) and the tooth classes (time ranges available for each single tooth or, more generally, for 2 3 4 5 6 7 8 I1 I2 I3 Canine C P1 P2 P3 P4 Premolars Months Weeks 1 Incisors 2 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 I1 I2 I3 Canine C P1 P2 P3 P4 M1 M2 M3 72 3 4 5 6Months Weeks Incisors Premolars Molars A. Deciduous teeth B. Permanent teeth Figure 1. A. The interval between the earliest and the latest age of deciduous dental eruption (Mellanby 1929, Bourdelle and Bressou 1953, Ferrara 1965, Shabestari et al. 1967, Kremenak 1969, Balasini 1995, Vaissaire 2001, Veronesi et al. 2013.) B. The interval between the earliest and the latest age of permanent dental eruption (Mellanby 1929, Miller 1952, Bourdelle and Bressou 1953, Arnall 1960, Silver 1963, Nickel et al. 1979, Shabestari et al. 1967, Barone 1981, Balasini 1995, Vaissaire 2001, Reece 2009, Evans and de Lahunta 2013, Dyce et al. 2018). 154 Veterinaria Italiana 2020, 56 (3), 149-162. doi: 10.12834/VetIt.1876.9968.2 Legal and welfare implications of age determination by dental examination Roccaro & Peli Moreover, variability due to body size, breed and sex cannot be overlooked and shall be taken into consideration when assessing the dog’s age. Research studies on dental eruption have been performed on medium and large dog breeds (Mellanby 1929, Arnall 1960, Shabestari et al. 1967, Kremenak 1969) and in most cases they only provide pooled-sex data. No specific data are available for small-sized dog breeds, which are known to have a delayed dental eruption (Hoskins 2001, Lobprise 2012). This dearth could be addressed by carrying out studies on a consistent sample including different dog breeds, with the aim of defining a ‘standard eruption chronology’ for each one of them. Dental anomalies such as hypodontia or supernumerary teeth are relatively common, especially in purebred dogs, as a consequence of the genetic defect being perpetuated (Hoskins 2001, Akers and Denbow 2008, Van de Wetering 2011, Lobprise 2012). Furthermore, individual variations in subjects of the same sex and bloodline have been observed (Kremenak 1969). Finally, but no less importantly, we cannot omit the inevitable observer variability, related to the subjectivity of judgement which is inbuilt in any visual assessment. The assessor should therefore be adequately trained, have full knowledge of the anatomy and the physiology of dental development, as well as be fully aware of the wide range of physiological and pathological variability. On the basis of the available information, the assessment of a puppy’s age by dental examination is subject to either an overestimation or an underestimation of no less than 2 weeks during the first 2-3 months of age, which is mainly due to the wide genetic variability among breeds. This uncertainty increases hand in hand with the dog’s growth as a result to the intervention of other factors, such as the environment and the individual habits. Against this background, it is not surprising that dental examination cannot be considered a reliable method to determine the exact age of a dog, but at most to estimate it, as agreed by all the consulted Authors. The correspondence between the real age and its assessment by dental examination is at most 41% (Nickel et al. 1979). Conclusions The so-called Pet Travel Scheme (PETS), introduced by Regulation (EC) No 998/20038 (subsequently repealed by Regulation (EU) No 576/2013), is a Silver 1963, Evans and de Lahunta 2013), 10 (Van de Wetering 2011) or even 12 weeks (Harvey and Emily 1993, Squarzoni 2003, Hale 2005, Fulton et al. 2014). Time ranges for permanent dental eruption are even wider. The substitution process starts with incisors, this time in the opposite order (I1, I2, I3). Data on incisor eruption times are quite divergent. According to Miller (Miller 1952) and Evans and de Lahunta (Evans and de Lahunta 2013) incisors begin to erupt at 2 months of age, for other Authors the eruption window starts at 3 months (Silver 1963, Nickel et al. 1979, Harvey and Emily 1993, Hale 2005, Reece 2009, Fulton et al. 2014), and the remaining sources place their appearance at 4 months. For most Authors, canines erupt at 5-6 months, while for some of them they can appear one or two months earlier (Arnall 1960, Harvey and Emily 1993, Squarzoni 2003, Gorrel 2013, Fulton et  al. 2014). Premolars erupt between 4-6 months of age, starting from P1, according to almost all the Authors. Only Balasini (Balasini 1995) and Vaissaire (Vaissaire 2001) placed the eruption of the first premolar at 3 months. According to half of the consulted sources, the first molar erupts at 4 months, while for the remaining half one month later. The other molars normally follow with a gap of one month between each one. At 7 months, the permanent set is deemed to be complete. Practical considerations The analysis of the literature revealed that teeth eruption and development in dogs is far from being a uniform process. Eruption of deciduous teeth should be completed by the sixth week of age, but for some Authors it can last up to 10 or even 12 weeks (Harvey and Emily 1993, Hoskins 2001, Squarzoni 2003, Hale 2005, Van de Wetering 2011). At 15-16 weeks, age at which a puppy can be legally moved within EU countries, according to some Authors the eruption of I1 (Miller 1952, Silver 1963, Nickel et al. 1979, Harvey and Emily 1993, Hale 2005, Reece 2009, Evans and de Lahunta 2013, Dyce et al. 2018), C (Squarzoni 2003, Gorrel 2013, Fulton et  al. 2014) and P1 (Balasini 1995, Vaissaire 2001) should be already in place, while for others it is just about to start (Mellanby 1929, Barton 1939, Bourdelle and Bressou 1953, Arnall 1960, Ferrara 1965, Piérard 1967, Shabestari et  al. 1967, Barone 1981, Veggetti and Falaschini 2009, Veronesi et  al. 2013). The eruption window extends up to 5-6 months for incisors, premolars and the first molar, up to 7 months for canines and the remaining molars. Information provided by the consulted sources are often quite general, with wide time ranges frequently referred to the entire teeth class rather than the single tooth. Therefore, they are unlikely to be useful in forensic investigations. 8 Regulation (EC) No. 998/2003 of the European Parliament and of the Council of 26 May 2003 on the animal health requirements applicable to the non-commercial movement of pet animals and amending Council Directive 92/65/EEC. Off J, L 146, 13/06/2003, 1-9. 155Veterinaria Italiana 2020, 56 (3), 149-162. doi: 10.12834/VetIt.1876.9968.2 Roccaro & Peli Legal and welfare implications of age determination by dental examination disagreement between assessments performed by different experts, makes it arduous to stand up in court. A method for age estimation in dog puppies which is simple, fast, non-invasive, reproducible and, over all, accurate, that can be systematically used in different academic and forensic scenarios, is currently lacking. Our comprehensive analysis of the available information on the timing of tooth eruption in dogs has highlighted a wide disagreement in the chronology of dental development among the Authors. Age assessment by tooth examination in dogs is affected by a degree of uncertainty of no less than 4 weeks, attributable to the biological variability due to general health state, nutrition, sex and, above all, breed and body size. Of all animal species, in fact, the canine species displays the widest range of phenotypic diversity. As a consequence, individuals of the same chronological age may show a range of different biological ages. This inevitable uncertainty clashes with the certainties demanded by Legal Sciences. system which allows animals to travel easily between member countries without undergoing quarantine. According to this regulation, puppy dogs cannot be moved within the Member Countries before 15  weeks of age, they must be identified and vaccinated against rabies. Structural controls and law enforcement on the inner borders of the EU appear to be poorly implemented and the occurrence of illegal transport of younger puppies is not uncommon. The illegal trade of puppies increases instances of falsified documentation, the counterfeit of vaccine certificates and discrepancies between the declared age and the real age of the puppies. Invariably, it also supports puppy farms in countries where welfare standards for animal breeding and husbandry may be of dubious quality. The difficulty in determining the exact age of the animals in case the transport papers lack or in order to verify their regularity, which often results in a wide Akers R.M. & Denbow D.M. 2008. Teeth. In Anatomy and physiology of domestic animals. Blackwell Publishing,  443-445. Arena L., Messori S., Ferri N. & Ruggieri E. 2015. Introduzione di cuccioli dall’estero. 30 Giorni, 5, 25-28. Arnall L. 1960. Some Aspects of dental development in the dog - II. Eruption and Extrusion. 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In Functional anatomy and 157Veterinaria Italiana 2020, 56 (3), 149-162. doi: 10.12834/VetIt.1876.9968.2 Roccaro & Peli Legal and welfare implications of age determination by dental examination Annex 1 Publisher: Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise ‘G. Caporale’ | Journal: Veterinaria Italiana | Article Type: Reasearch Article | Volume: 56; Issue: 3; Year: 2020; doi: 10.12834/VetIt.1876.9968.2 Supplementary Table I. Timing of deciduous dental eruption according to the different Authors. —cont’d continued 1 2 3 2 3 4 5 6 7 8 9 10 11 12 Incisors I Canine C P2 P3 P4 I1 19-31d I2 20-27d I3 19-28d Canine C 20-28d P2 28-39d P3 21-35d P4 24-37d Incisors I Canine C I1 I2 I3 Canine C Premolars P I1 30d I2 28d I3 25d Canine C 21d P2 P3 P4 Incisors I 20-35d Canine C 20-35d Premolars P 20-35d Incisors I Canine C Premolars P I1 30-33d I2 28-29d I3 24-28d Canine C 15-20d Premolars Incisors Premolars Incisors Incisors Premolars Incisors AGE Months Weeks Mellanby 1929 Cornevin and Lesbre 1894 AUTHOR Barton 1939 Miller 1952 Bourdelle and Bressou 1953 Vaissaire 2001 Arnall 1960 Silver 1963 Ferrara 1965 158 Veterinaria Italiana 2020, 56 (3), 149-162. doi: 10.12834/VetIt.1876.9968.2 Legal and welfare implications of age determination by dental examination Roccaro & Peli Annex 1 Publisher: Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise ‘G. Caporale’ | Journal: Veterinaria Italiana | Article Type: Reasearch Article | Volume: 56; Issue: 3; Year: 2020; doi: 10.12834/VetIt.1876.9968.2 Supplementary Table I. Timing of deciduous dental eruption according to the different Authors. —cont’d continued I1 21-41d I2 19-35d I3 20-37d Canine C 18-28d P2 27-40d P3 20-34d P4 25-41d Incisors I Canine C Premolars P I1 30d I2 28d I3 25d Canine C 21d Premolars P I1 I2 I3 Canine C P2 P3 P4 Incisors I Canine C Premolars P Incisors Premolars Incisors Incisors Premolars Sisson and Grossman 1982 Harvey and Emily 1993 Squarzoni 2003 Hale 2005 Fulton 2014 Nickel et al. 1979 Gorrel 2013 Dyce et al. 2018 Barone 1981 Kremenak 1969 Incisors I Canine C I1 21-31d I2 21-30d I3 20-28d Canine C 19-25d P2 27-39d P3 22-33d P4 24-38 d Incisors Premolars Shabestari et al. 1967 Piérard 1967 1 2 3 2 3 4 5 6 7 8 9 10 11 12 AGE Months Weeks AUTHOR 159Veterinaria Italiana 2020, 56 (3), 149-162. doi: 10.12834/VetIt.1876.9968.2 Roccaro & Peli Legal and welfare implications of age determination by dental examination Annex 1 Publisher: Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise ‘G. Caporale’ | Journal: Veterinaria Italiana | Article Type: Reasearch Article | Volume: 56; Issue: 3; Year: 2020; doi: 10.12834/VetIt.1876.9968.2 Supplementary Table I. Timing of deciduous dental eruption according to the different Authors. —cont’d I1 30-33d I2 25-30d I3 21-25d Canine C 15-20d P2 28-34d P3 20-28d P4 20-28d Incisors I 20-30d Canine C 20-30d Veggetti et al. 2009 Incisors I Incisors I Canine C Premolars P I1 I2 I3 Canine C Premolars P I1 I2 I3 Canine C P2 P3 P4 Incisors I Canine C Premolars P Incisors Premolars Incisors Incisors Premolars Liebich et al. 2014 Evans and de Lahunta 2013 Veronesi et al . 2013 Balasini 1995 Bonetti 1995 Peterson and Kutzler 2011 1 2 3 2 3 4 5 6 7 8 9 10 11 12 AGE Months Weeks AUTHOR 160 Veterinaria Italiana 2020, 56 (3), 149-162. doi: 10.12834/VetIt.1876.9968.2 Legal and welfare implications of age determination by dental examination Roccaro & Peli Annex 1 Publisher: Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise ‘G. Caporale’ | Journal: Veterinaria Italiana | Article Type: Reasearch Article | Volume: 56; Issue: 3; Year: 2020; doi: 10.12834/VetIt.1876.9968.2 Supplementary Table II. Timing of permanent dental eruption according to the different Authors. —cont’d continued I1 I2 I3 Canine C P1 P2 P3 P4 M1 M2 M3 Incisors I Canine C P1 P2 P3 P4 M1 M2 M3 Incisors I Canine C Premolars P Molars M I1 I2 I3 Canine C P1 P2 P3 P4 M1 M2 M3 Incisors I Canine C Incisors I Canine C Premolars P Molars M Incisors Premolars Molars Premolars Molars Incisors Premolars Molars Squarzoni 2003 Barone 1981 Harvey and Emily 1993 Balasini 1995 Bonetti 1995 Sisson and Grossman 1982 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 AGE Months Weeks AUTHOR 161Veterinaria Italiana 2020, 56 (3), 149-162. doi: 10.12834/VetIt.1876.9968.2 Roccaro & Peli Legal and welfare implications of age determination by dental examination Annex 1 Publisher: Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise ‘G. Caporale’ | Journal: Veterinaria Italiana | Article Type: Reasearch Article | Volume: 56; Issue: 3; Year: 2020; doi: 10.12834/VetIt.1876.9968.2 Supplementary Table II. Timing of permanent dental eruption according to the different Authors. —cont’d continued I1 I2 I3 Canine C P1 P2 P3 P4 M1 M2 M3 Incisors I Canine C Premolars P Molars M I1 I2 I3 Canine C P1 P2 P3 P4 M1 M2 M3 Incisors I Molars M I1 I2 I3 Canine C P1 P2 P3 P4 M1 M2 M3 Incisors I Canine C Premolars P Molars M Hale 2005 Vaissaire 2001 Incisors Premolars Molars Incisors Premolars Molars Incisors Premolars Molars Reece 2009 Veggetti et al. 2009 Evans and de Lahunta 2013 Gorrel 2013 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 AGE Months Weeks AUTHOR 162 Veterinaria Italiana 2020, 56 (3), 149-162. doi: 10.12834/VetIt.1876.9968.2 Legal and welfare implications of age determination by dental examination Roccaro & Peli Annex 1 Publisher: Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise ‘G. Caporale’ | Journal: Veterinaria Italiana | Article Type: Reasearch Article | Volume: 56; Issue: 3; Year: 2020; doi: 10.12834/VetIt.1876.9968.2 Supplementary Table II. Timing of permanent dental eruption according to the different Authors. —cont’d Incisors I Canine C Premolars P Molars M Incisors I Canine C Premolars P Molars M I1 I2 I3 Canine C P1 P2 P3 P4 M1 M2 M3 Incisors Premolars Molars Fulton 2014 Dyce et al. 2018 Liebich et al. 2014 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 AGE Months Weeks AUTHOR