Abstract
The authors performed a retrospective study to determine the incidences of spontaneous findings in the adrenal glands of control CD-1 mice. Data were collected from 2,163 mice from control dose groups in 104-week carcinogenicity studies carried out between 2000 and 2010. Adrenal gland nonproliferative lesions were more common in males than in females. In males, the most common nonproliferative lesions were cortical hypertrophy, cortical atrophy, pigment deposition/pigmentation, cysts, and extramedullary hematopoiesis. In females, the most common nonproliferative lesions were pigment deposition/pigmentation, extramedullary hematopoiesis, and cortical atrophy. Proliferative lesions were more common in females than in males. In both sexes, the most common proliferative lesions were subcapsular cell hyperplasia, focal cortical hyperplasia, and subcapsular cell tumor. Pheochromocytomas were uncommon in both sexes, with a slightly higher incidence in females, and the benign type was more frequent than the malignant type. Lymphoma was the most common metastatic tumor in both males and females, followed by histiocytic sarcoma and erythroid/myeloid leukemia. To the best knowledge of the authors, there are no recent reports on spontaneous pathological findings in the adrenal glands of CD-1 mice, and these results will facilitate the interpretation of background findings in carcinogenicity studies.
Introduction
The reporting and regular updating of background pathological findings from control animals used in nonclinical toxicology studies are required in order to properly interpret drug-induced lesions. Although CD-1 mice are the most common strain of mouse used for chronic studies in Europe, specific information on the incidence of spontaneous histopathological findings in the adrenal glands is not available.
The adrenal gland is considered to be the most common target of all endocrine tissues in nonclinical toxicology studies (Harwey, Everett, and Springall 2007; Ribelin 1984). This may be explained by various factors. The adrenal gland can accumulate lipophilic compounds, is rich in xenobiotic matabolizing enzymes (e.g., cytochrome P450), has a rich blood supply, and is susceptible to lipid peroxidation due to the high content of unsaturated fatty acids in adrenocortical cell membranes (Chandra, Hoenerhoff, and Peterson 2013). It is also interesting to note that the highest incidence of spontaneous pathological findings in CD-1 mouse occurs in adrenal glands as compared to other tissues (Johnson, Spaet, and Potenta 2013). Despite this, they have not been as extensively studied as some other endocrine tissues.
The incidence of some spontaneous lesions of the adrenal gland in rodents used in toxicologic studies varies depending on the species, strain, and sex (Laroque, Duprat, and Hollander 1997) and can also vary over time depending on the genetic stability of the strain. The aim of this study is to summarize the incidence of adrenal gland background lesions in Crl:CD-1(ICR)BR mice over a period of 10 years (2000–2010) and also determine whether the incidences of the most common lesions were stable during this period.
Materials and Methods
Animals
Tissue samples from a total of 2,163 CD-1 mice (1,081 males and 1,082 females) were obtained from eleven 104-week nonclinical toxicity studies conducted at Charles River Edinburgh between 2000 and 2010. The animals were purpose-bred for laboratory use and supplied by Charles River UK Ltd. (Margate, Kent, U.K.). All control animals were dosed with a vehicle (Table 1). Pretermination deaths were included in the study.
Details of data sources.
Males were housed individually and females were housed 3 per cage unless reduced by mortality. The temperature and humidity were automatically controlled at 19°C to 23°C and 40% to 70%, respectively, with a minimum of 15 air changes per hour. An automatic 12-hr light cycle of 07:00 to 19:00 was maintained. Animals were fed an ad libitum commercial rodent diet (Rat and Mouse modified No. 1 Diet SQC Expanded; Special Diet Service Ltd., Witham, Essex, U.K.). Wooden chew sticks and play tunnels were also offered to all animals for environmental enrichment.
All studies were conducted in accordance with the U.K. Animals (Scientific Procedures) Act 1986, which conforms to the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Strasbourg, Council of Europe).
Histopathological Evaluation
Animals were humanely euthanized by a rising concentration of carbon dioxide and exsanguinated via femoral veins. A detailed necropsy was performed by a trained technician, and a veterinary pathologist was either present or available for consultation throughout the necropsy. Tissues were preserved in 10% neutral-buffered formalin, embedded in paraffin wax, sectioned to a 4- to 5-μm thickness, and stained with hematoxylin and eosin. Histopathological evaluation was performed by a board-certified veterinary pathologist or a veterinary pathologist with training and experience in laboratory animal pathology. The findings were entered directly into a computerized database (PLACES 2000 Instem; Apoloco Limited Systems, Plymouth, PA). Generally accepted terms were used in the diagnosis of proliferative and nonproliferative lesions (STP/ARP/AFIP SSNDC Guides for Toxicologic Pathology; Patterson et al. 1995).
Study Design and Statistical Analysis
Data were collected retrospectively from control groups of CD-1 mouse 104-week carcinogenicity studies evaluated over a period of 10 years (2000–2010). From this pool of information, studies to be incorporated into the present investigation were selected based on the following criteria: At least 1 vehicle control group, Good laboratory practice–compliant toxicological studies, Evaluation of a full tissue list.
Study material including histological incidence tables and individual animal data listings was analyzed for pathology findings under each body and organ system. A few selected glass slides were retrieved from the archives for imaging purposes and to permit more detailed description of lesions. Data were available for a total of 2,163 control animals, and the lesions identified in unscheduled deaths animals were combined with those at scheduled termination.
Statistical analysis of lesions incidence was performed at 1% significance level (p < .01) using Fischer’s exact test comparing males and females within the same time points (2000–2005, 2006–2010, and 2000–2010). Fischer’s exact test was also used to evaluate differences in the incidence of each type of lesion at different time points analyzing males and females separately.
Results
The incidences of spontaneous adrenal gland findings encountered in CD-1 mice are presented in Tables 2 and 3. There was minor between-study variability in the incidence of some of the common lesions (both proliferative and nonproliferative) and also between the 2 time periods (2000–2005 and 2006–2010). However, there were no clear trends to suggest a major drift in the incidence of these lesions.
Histopathological findings of adrenal gland in CD-1 mice: nonproliferative lesions from 104-week carcinogenicity studies.
Note. Significantly different (p < .01) incidences of lesions comparing males versus females: a = at the time point 2000 to 2005; b = at the time point 2006 to 2010; and c = all time points together (2000–2010). Significantly different (p < .01) incidences of lesions comparing period of times: d = females 2006 to 2010 versus females 2000 to 2005 and e = males 2006 to 2010 versus males 2000 to 2005.
Histopathological findings of adrenal gland in CD-1 mice: proliferative lesions from 104-week carcinogenicity studies.
Note. Significantly different (p < .01) incidences of lesions comparing males versus females: a = at the time point 2000 to 2005; b = at the time point 2006 to 2010; and c = all time points together (2000–2010). Significantly different (p < .01) incidences of lesions comparing period of times: d = females 2006 to 2010 versus females 2000 to 2005.
Nonproliferative Lesions from 104-week Carcinogenicity Studies in CD-1 Mice
Across all studies, nonproliferative adrenal gland lesions were more common in males than in females (Table 2). However, the difference in the incidence was statistically significant (p < .01), at all the time points considered, only for cortical hypertrophy and cortical atrophy. Extramedullary hematopoiesis was the only nonproliferative lesion, which was significantly (p < .01) higher in females than in males but exclusively in the studies from 2006 and 2010. In the 5-year period from 2000 to 2005, pigment deposition/pigmentation was higher at a significant level (p < .01) in both males and females when compared to the incidence observed in the following 5 years (2006–2010). Similarly, extramedullary hematopoiesis was significantly higher comparing the 2 periods (2006–2010 vs. 2000–2005) but in female animals only.
Over the 10-year period (2000–2010), the most common nonproliferative lesions in males were, in decreasing order, cortical hypertrophy, cortical atrophy, pigment deposition/pigmentation, cysts, and extramedullary hematopoiesis. Other lesions, with an incidence below 1%, were inflammatory cell infiltration, accessory cortical tissue, cortical vacuolation, angiectasis, capsular fibrosis, cortical necrosis, cortical vacuolation, medullary vacuolation, and osseous metaplasia (Table 2).
Over the 10-year period (2000–2010), the most common nonproliferative lesions in females were, in decreasing order, pigment deposition/pigmentation, extramedullary hematopoiesis, and cortical atrophy. Lesions with an incidence lower than 1% included amyloidosis, accessory cortical tissue, angiectasis, cysts, capsular fibrosis, cortical cell hypertrophy, cortical vacuolation, arteritis–periarteritis, and persistence of X-zone (Table 2).
Cortical hypertrophy was most frequently observed in the zona fasciculata and occurred as foci of cells with increased amounts of eosinophilic or clear cytoplasm and large vesicular nuclei (Figure 1A and B). In cortical atrophy, the adrenal cortex is reduced to a variable extent making the medulla appear more prominent with the capsule making up the bulk of the remaining gland. Pigmentation occurs as deposition of ceroid or lipogenic pigment, which appears as granular to amorphous, yellowish-brown material at the corticomedullary junction, and in the degenerating X-zone. Cysts consisted of clear spaces lined by ciliated columnar epithelium or a nonciliated cuboidal epithelium (Figure 1C and D). Extramedullary hematopoiesis was present in the cortex as foci of hematopoietic cells. Amyloid was observed only in females and manifested as intercellular translucent, amorphous eosinophilic deposits.
Representative photomicrographs of nonproliferative spontaneous lesions in adrenal gland of CD-1 mice from control groups (hematoxylin and eosin). (A and B) Focal cortical hypertrophy at low and high magnification. (C and D) Adrenal gland cyst. The lesion consists of a blood-filled space surrounded by a thin rim of remaining adrenal gland tissue (C). The cyst is lined by ciliated epithelium (D).
Proliferative Lesions from 104-week Carcinogenicity Studies in CD-1 Mice
The incidence of proliferative lesions was higher in females than in males (Table 3) but with statistical significance (p < .01) in all the time points considered only for subcapsular cell hyperplasia. In contrast, focal cortical hyperplasia and subcapsular cell tumor were significantly (p < .01) more frequent in male than in female animals. In females only, subcapsular cell hyperplasia was significantly higher comparing the 2 periods (2006–2010 vs. 2000–2005).
In males, the most common lesions affecting the cortex were subcapsular cell hyperplasia, focal cortical hyperplasia, and subcapsular cell tumor, and the most common lesion affecting the medulla was medullary cell hyperplasia (Table 3). In females, the most common lesions in the cortex were in descending order subcapsular cell hyperplasia, subcapsular cell tumor, and focal cortical hyperplasia, and the most common lesion in the medulla was medullary diffuse hyperplasia (Table 3).
Subcapsular cell hyperplasia (Figure 2A and B) is composed of 1 of the 2 different cell types (type A and type B cells) or a mixture of the 2. Type A cells predominate in the early stages and the cells are spindle to fusiform, have flat to oval hyperchromatic nuclei, and scant basophilic cytoplasm. Type B cells are polygonal and have round vesicular nuclei, abundant clear, or eosinophilic cytoplasm, which may contain lipid-laden vacuoles, and they may occur in spherical nests or glandular structures. Subcapsular cell hyperplasia can begin as subcapsular scattered aggregates of type A cells arranged parallel to the capsule. As the lesion progresses, hyperplastic cells proliferate and expand horizontally between the cords of the zona fasciculata forming wedge-shaped foci (Nyska and Maronpot 1999).
Representative photomicrographs of cortical proliferative spontaneous lesions in adrenal gland of CD-1 mice from control groups (hematoxylin and eosin). (A and B) Diffuse subcapsular cell hyperplasia, low, and high magnification. (C and D) Benign subcapsular cell tumor with a pseudoinvasive growth pattern effacing the cortex, low, and high magnification. On the left-hand side of (C), a focal area of cortical hypertrophy is also present. (E) Cortical adenoma compressing the zona fasciculata and medulla.
Benign subcapsular cell tumor (Figure 2C and D) was the most common tumor of the adrenal gland, and the incidence of this tumor in females was twice that in males. In mice, subcapsular cell tumor can be classified into 2 categories depending on the predominance of type A or type B cells. Type A tumors develop from the subcapsular spindle cells, they are usually small and well demarcated and often compress the surrounding tissue. The neoplastic proliferation is composed of ovoid to spindle cells with ovoid nuclei, minimal cellular atypia, and there may be infiltration by mast cells. Type B tumors are composed of irregularly sized polygonal cells, which may also have cytoplasmic vacuolation.
Cortical adenoma and carcinoma were uncommon in both males and females (Table 3). Cortical adenomas are usually located in the zona fasciculata (Figure 2E), are well circumscribed, and compress the surrounding tissue. Cortical carcinomas are composed of large cells with variable features of atypia and tend to invade the adjacent tissue, the capsule, or have distant metastasis. Mitotic figures can be frequent.
Medullary hyperplasia can be diffuse (Figure 3A) or can occur as a distinct focus of medullary cells in minimally disordered islands and have only slight atypia. These cells are polyhedral, with a central nucleus and finely stippled cytoplasm. The nests of cells may be limited by a delicate extracellular stroma and are classified as focal hyperplasia when they are no larger than 50% of the normal medulla and do not compress the surrounding tissue.
Representative photomicrographs of medullary proliferative spontaneous lesions in adrenal gland of CD-1 mice from control groups (hematoxylin and eosin). (A) Diffuse medullary hyperplasia. No compression of adjacent cortical tissue is present. (B and C) Benign pheochromocytoma at low and high magnification composed of chromaffin cells organized in nests. No invasion of the surrounding tissue is present. In (B), a focal area of vacuolar change and hypertrophy is also present in the ventral region of the adrenal gland cortex.
In our study, pheochromocytomas (Figure 3B and C) were rare in both sexes. This tumor is composed of medullary cells arranged in nests but may also have a trabecular pattern. Cells that have poorly basophilic cytoplasm, hyperchromatic nuclei, and mitotic figures are usually infrequent. Necrotic areas within the tumor may also be seen.
Lymphoma was the most common secondary tumor in both sexes (Table 3), but it was significantly higher (p < .01) in females than in males for all the time points considered. Other secondary tumors observed were histiocytic sarcoma and erythroid/myeloid leukemia.
Discussion
The main aim of this study was to determine the incidences of the most common background findings of adrenal glands in control Crl:CD-1 mice used in 104-week carcinogenicity studies at Charles River, Edinburgh, over a 10-year period. In the current study, we observed a different sex-based incidence for both nonproliferative and proliferative lesions, with the nonproliferative lesions more common in males, and conversely the proliferative lesions more common in females.
Among the nonproliferative lesions, we noted some differences in their incidence between the 2 review periods. The incidence of cortical hypertrophy, cortical cell atrophy, and pigment deposition/pigmentation in the period 2006 to 2010 was lower than in the period 2000 to 2005. These differences were most likely to be a result of differences in diagnostic thresholds between pathologists, with higher incidence of those lesions in 1 oral gavage study.
Cortical hypertrophy, which is a common finding in B6C3F1 mice, was the main nonproliferative lesion observed in males but was less common in females, consistent with other studies (Nyska and Maronpot 1999). In rats and mice, cortical hypertrophy may be focal, multifocal, or diffuse (Nyska and Maronpot 1999; Frith et al. 2000). In this study, most cases of cortical cell hypertrophy were focal. Cortical hypertrophy is not considered a preneoplastic change and may reflect an adaptation to stress (Nyska and Maronpot 1999), ACTH administration, or deficient glucocorticoid feedback regulation (Chandra, Hoenerhoff, and Peterson 2013). This change may regress with cessation of stress and/or administration of Adrenocortical hormone (ACTH) (Greaves 2012).
Cortical atrophy was the second most common lesion in adrenal glands from both males and females. In rats, bilateral atrophy can occur secondary to exogenous administration of glucocorticoid or from destructive lesions of the pituitary gland causing ACTH deficiency (Frith et al. 2000). Unilateral cortical atrophy is commonly seen in association with cortical neoplasms in the contralateral gland (Frith et al. 2000). Atrophy of zona fasciculata and reticularis can be present in association with steroid-producing tumors or secondary to overtreatment with corticosteroids (Greaves 2012). In mice, prednisone-induced atrophy was more severe in males than in females treated with prednisone for an 18-month period (Dillberger, Cronin, and Carr 1992). In the adrenal gland of mice, this finding can also be observed in the X-zone, which is sensitive to androgenic steroid (Greaves 2012). Atrophy of the zona fasciculata has been reported secondary to the administration of exemestane (an inhibitor of cytochrome P450 aromatase) and an antisclerotic agent (PD 138142-15; Chandra, Hoenerhoff, and Peterson 2013).
Pigment deposition was the most frequent finding in females and the third most common finding in males. Pigmentation (brown degeneration, ceroid pigment, lipofuscin, and lipogenic pigmentation) is a spontaneous finding mostly occurring in aged animals. However, its earliest appearance has been reported at 4 months of age in mice (Nyska and Maronpot 1999). It is more frequent in females than in males (Taylor 2012) and is a common finding in BALB/c mice (Dunn 1970). Increased severity has been associated with administration of estrogens, adrenocorticosteroids, and ketoconazole (Greaves 2012).
In this study, cortical cysts were observed in only 1.7% of males and 0.16% females. Cortical cysts have been reported to be rare in B6C3F1 mice (Nyska and Maronpot 1999). Cortical cysts are lined by a ciliated columnar or cuboidal epithelium and must be differentiated from angiectasis or cystic degeneration (Nyska and Maronpot 1999). Cystic degeneration, which is most commonly observed in aging female Sprague-Dawley rats (Chandra, Hoenerhoff, and Peterson 2013), occurs as cystic and/or blood-filled spaces, and it may occur in association with hyperplastic foci and cortical vacuolation (McInnes 2012).
In CD-1 mice, amyloidosis is typically of the primary type rather than secondary to chronic diseases (Frith and Chandra 1991), and in the current study was reported only in female animals at a low incidence. Amyloidosis is a common finding in adrenals from aged mice, especially A/J, BL, C3H, C57BL/6 CBA, C57L/J, NH, and Y strains (Chandra, Hoenerhoff, and Peterson 2013; Sass 1996), but it has been rarely reported in B6C3F1 mice (which is the inbred result of the male low amyloid inbred C3H strain and female C56BL/6 strain, which develop mild amyloidosis; Nyska and Maronpot 1999). Although adrenal gland amyloid deposition can be associated with similar deposits in multiple tissues, the incidence of this finding in the adrenal glands does not necessarily coincide with that of generalized amyloidosis (Sass 1996).
Extramedullary hematopoiesis is occasionally seen in the adrenal glands of rodents (Chandra, Hoenerhoff, and Peterson 2013; Taylor 2012), and it may be observed when a higher production of hematopoietic cells is required, for example, in association with a regenerative anemia. The same finding can be seen, concurrently, in other tissues such as spleen and liver.
Subcapsular cell hyperplasia is a common finding in the adrenal gland in mice of many strains (Goodman 1996). The proliferation may occur in a focal or diffuse pattern and it may progress to subcapsular tumor (Taylor 2012; Bernichtein, Peltoketo, and Huhtaniemi 2009). In IQI/Jic female mice, it has been suggested that based on their ultrastructural features (e.g., elongated mitochondria with lamellar cristae and round mitochondria with tubular cristae), hyperplastic cells in subcapsular hyperplasia might be adrenal pluripotent progenitor cells that are located in the zona glomerulosa or in the junction of the zona glomerulosa/fasciculata (Kim et al. 2005). In our study, it was the most common proliferative lesion in both sexes, with a much higher incidence in females. In a study by Yoshida, Maita, and Shirasu (1986), in Institute for Cancer Research (ICR) mice the incidence was 59% in males and 91% in females at 19 months of age, with no significant increase in both sexes thereafter. In mice, subcapsular cell hyperplasia is reportedly linked to decreasing levels or altered balance of sex hormones (Bernichtein, Peltoketo, and Huhtaniemi 2009). Higher incidences have been reported in gonadectomized rats (Taylor 2012; Bernichtein, Peltoketo, and Huhtaniemi 2009; Johnsen et al. 2006), in rats subjected to repeated breeding (Wexler 1964), castrated mice (Dunn 1970), and in animals treated with anabolic substances (e.g., clenbuterol; Illera et al. 1998). It may also be induced by other condition such as thymectomy of mice at birth (Greaves 2012) and diet-related factors (e.g., high-protein and high-fat diets; Greaves 2012). The animal cage density may also have some role in increasing the prevalence of subcapsular cell hyperplasia in Swiss mice, but the underlying mechanism still remains unclear (Chevédoff et al. 1980). Compounds such as the vasodilator pinacidil (Greaves 2012), lovastatin (Rebuffat, Mazzocchi, and Nussdorfer 1987), and the hypocholesterolemic agent 4-aminopyrazolo-pyridine (Mazzocchi et al. 1987) have also been associated with an increased incidence of subcapsular cell hyperplasia.
Subcapsular cell tumors are commonly seen as background lesions in aged mice in long-term studies. In our study, subcapsular cell tumors were more common in males than in females, confirming the results from previous studies (Heath 1996). The incidence depends on the strain with BALB/c highest, followed in decreasing order by B6C3F1, NH, CD-1, and C57BL/6 (Heath 1996). However, the incidence in each strain can vary by age and sex, with NH and BALB/c females more affected, probably due to ovarian function decreasing with age (Heath 1996). It is considered that subcapsular cell tumors may represent a continuum between hyperplasia and carcinoma (Heath 1996). In order to differentiate subcapsular cell hyperplasia from tumor, the presence or absence of compression of the surrounding tissue is usually adopted as general diagnostic criteria (Chandra, Hoenerhoff, and Peterson 2013) but a definitive diagnosis may be challenging (Capen et al. 2001).
Cortical adenoma and carcinoma were uncommon in our study. In 1 study on CD-1 mice, cortical adenomas were more common in males (8 of 891) than in females (4 of 890), and adrenal cortical adenocarcinomas were observed only in females (3 of 890; Maita et al. 1988). In B6C3F1 mice, cortical adenoma occurred with higher incidence in males (3.4%) than in females (0.7%), and cortical carcinoma was observed only in females at a very low incidence (0.1%; Haseman, Hailey, and Morris 1998). These types of proliferation lesions are more common in some strains of rat (e.g., Osborne, Mendel, WAG/Rij, BUF, and BN/Bi) and hamster (e.g., BIO 4.24 and BIO 45.5) and in ferrets (Rosol et al. 2013). The zona fasciculata and reticularis are more prone to develop tumors than the zona glomerulosa (Rosol et al. 2013).
The incidence of medullary cell hyperplasia was low in the present study, and it is also rare in other strains such as B6C3F1 mice (Nyska and Maronpot 1999). It is considered a diagnostic challenge because medullary hyperplasia forms a morphological continuum with pheochromocytoma (Greaves 2012). An increased incidence of diffuse medullary hyperplasia has been reported to occur in C57BL/KsJ db/db mice, and this finding was associated with increased enzyme activities (tyrosine hydroxylase, dopamine-β-hydroxylase, and phenyl-ethanolamine-N-methyltransferase; Carson, Hanker, and Kirschner 1982).
Pheochromocytomas are the most common tumors found in the adrenal medulla in rats. Although they are commonly seen in different strains of rats (e.g., F344, Long-Evans, Wistar, and New England Deaconess Hospital rats) and hamsters as spontaneous lesions. In mice pheochromocytomas are less commonly seen as spontaneous lesions (Rosol et al. 2013; Nyska and Maronpot 1999) and even less easily induced by xenobiotics (Greaves 2012; Haseman, Hailey, and Morris 1998; Hill et al. 2003; Mahler et al. 1996). In 1 report on tumor incidence in B6C3F1 mice, the overall incidence of benign pheochromocytoma was 0.43% in males and 1.37% in females, and that for the malignant counterpart was 0.21% in males and 0.53% in females (National Toxicology Program 2013). In mice, the overall evaluation of 4 cohort studies in CD-1 mice and 4 more studies in B6C3F1 mice suggested that in the latter strain the incidence of pheochromocytomas was higher (Langeart 1996). The difference among species, specifically between rats and mice, suggests that the latter is a more suitable animal model for humans with respect to adrenal medullary gland risk assessment (Nyska and Maronpot 1999; Hoyer and Flaws 2013).
In conclusion, the CD-1 mouse is commonly used in long-term and carcinogenicity studies, and the data presented will provide a valuable source of information of historical control data to support correspondence with clients and regulatory authorities, and for pathologists and toxicologists involved in the interpretation of pathology data from preclinical toxicology studies.
Footnotes
Acknowledgments
The authors are grateful to Heather Telfer and Nyree Cowe for their support in collecting and tabulating the data. We also thank Murray Wellwood for help with figures layout.
Author Contribution
Authors contributed to conception or design (CP, NS, SW, AB); drafting the manuscript (CP); and critically revising the manuscript (CP, AB). All authors gave final approval and agreed to be accountable for all aspects of work in ensuring that questions relating to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
The author(s) received no financial support for the research, authorship, and/or publication of this article.