Toxicologic Pathology, 43: 733-736, 2015 Copyright # 2014 by The Author(s) ISSN: 0192-6233 print / 1533-1601 online DOI: 10.1177/0192623314558464

Artifactual Stratum Corneum Calcification of the Beagle Dog Tongue CHRISTIANA GLOVER1 1

AND

RICARDO OCHOA2

ITR Laboratories Canada Inc., Montreal, Quebec, Canada 2 Pre-Clinical Safety Inc., Niantic, Connecticut, USA ABSTRACT

Examination of H&E-stained tongue samples from a 26-week intravenous infusion study of Beagle dogs, utilizing a compound with no recognized effect on mineral metabolism, exhibited superficial stratum corneum calcification in both treated and control animals. This resulted in the search for possible causes of the finding to help clarify confounding issues. Retrospective examination of 11 studies performed before the signal case indicated that the problem existed in the testing facility but was not recognized. Prior to 2008, this finding was not observed, perhaps indicating the requirement for a change in procedures or suppliers. Based on the hypothesis that the calcium salts were deposited from bone during processing, a series of tests was performed by fixing tongue and femur along with different tissues, processed routinely to slide, and stained with H&E and von Kossa stains. We conclude that the presence of superficial stratum corneum calcification of the tongue in dogs demonstrated in toxicology studies is an artifactual change related to the processing of tissues, specifically the fixation of tongue in the same container as bone and stomach. This change should not be confused with compound-related effects, even when the incidence varies between controls and treated animals. Keywords:

toxicologic pathology; dog pathology; digestive system; calcification.

INTRODUCTION

TABLE 1.—Incidence of stratum corneum calcification in the tongue in a 26-week intravenous study in Beagle dogs.

Artifacts during processing of tissues are often confusing to the pathologist and difficult to interpret. In toxicology studies, where some artifacts may mimic known or new conditions, the discrimination between effect and artifact is critical. If an artifact is suspected, then a search for possible causes of the artifact is important. The publication of these artifacts is valuable to the discipline, as it helps clarify confounding issues. The present brief report stems from the conduct of a 26-week intravenous infusion study in Beagle dogs. Superficial stratum corneum calcification of the tongue was observed in both treated and control animals. We considered this change to be an artifact possibly related to fixation of tongue in 10% neutral-buffered formalin (NBF) in the presence of bone possibly in combination with other tissues, which has been the standard procedure of the facility for 20 years. To investigate this, we conducted a retrospective examination of tongue sections from control dogs from 11 studies over 14 years to assess the incidence of the finding. In addition, we prospectively determined the effects of fixation of tongue samples from control dogs in NBF in the presence of bone in combination with other selected

Sex Male Group numbers Number of animals examined Tongue: stratum corneum calcification

1 3 3

2 3 3

3 3 3

Female 4 3 2

1 3 3

2 3 3

3 3 3

4 3 3

tissues on the incidence of this finding (stratum corneum calcification of the tongue). To the best of the authors’ knowledge, this finding has not been previously reported in the literature. METHODS Examination of slides from tongue samples from a 26-week intravenous infusion study, utilizing a compound without known effect on mineral metabolism, presented superficial stratum corneum calcification as indicated by positive results on the von Kossa stain for calcium, in response to an initial observation of the unusual change on the H&E-stained slides. Table 1 presents the incidence of superficial stratum corneum calcification of the tongue in the different test groups. A retrospective analysis was made of tongues from control dogs in 11 studies encompassing 14 years at the same facility. Table 2 displays the different studies reviewed and the incidence of superficial stratum corneum tongue calcification in these studies. Based on the hypothesis that the calcium salts

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. Address correspondence to: Christiana Glover, ITR Laboratories— 19601 Clark, Graham Baie d’Urfe, Montreal, Quebec, Canada H9X 3T1; e-mail: [email protected]. Abbreviation: NBF, neutral-buffered formalin. 733

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TOXICOLOGIC PATHOLOGY

TABLE 2.—Incidence of calcification in the tongue of control Beagle dogs (1998–2012). Year 1998–2007 1

2008–2012

2

3

4

5

6

7

8

9

10

11

Study number Sex

M

F

M

F

M

F

M

F

M

F

M

F

M

F

M

F

M

F

M

F

M

F

Number of animals examined Tongue: calcification, stratum corneum

4 0

4 0

4 0

4 0

6 0

6 0

8 2

8 4

8 3

8 0

6 0

6 2

3 3

3 3

7 5

7 6

3 3

3 3

5 1

5 0

1 0

1 1

Note. M ¼ male; F ¼ female.

TABLE 3.—Fixation of tongue with other tissues for varying times to study the effects of fixation on calcium deposition on the tongue. Organs/tissues fixed in 10% formalin

Duration of fixation 1 Month

Tongue Tongue Tongue Tongue

þ þ þ þ

femur þ eyesa femur þ stomach femur þ testesb all tissues

3 Months

16 Months

Tongue: calcification, stratum corneum Absent Absent Absent Present Present Present N/A Absent Absent Present Present Present

Note. N/A ¼ not applicable. a Eyes were first fixed in Davidson’s and added to formalin 1 week later. b Testes were first fixed in Bouin’s and added to formalin 72 hr later.

were deposited from bone during processing, a prospective study was performed. Representative samples of major tissues/organs, including tongue, femur, stomach, eyes, testes, liver, kidney, pancreas, spleen, heart, small intestine, large intestine, adrenal, skeletal muscle, salivary gland, brain, esophagus, lung, trachea, and haired skin, were fixed in commercially prepared 10% NBF, 9 volumes of fixative for each volume of tissue fixed (fixative–tissue ratio ¼ 9:1) with pH 7.2 supplied by ACP Chemicals Inc. (Canada). Tissues were grouped as presented in Table 3 and fixed with an average tissue volume to average 10% NBF volume of 600 to 8,000 ml. The average duration of fixation for the facility is 24 to 48 hr; however, for the prospective study, tissues were fixed in 10% NBF for a duration of 1, 3, and 16 months. The femur was fixed sawed with a bone band saw, and 50% of the stomach was placed in fixative. The eyes were fixed in Davidson’s solution for 96 hr, transferred into 70% ethanol for 72 hr, and then finally transferred into 10% NBF. The testes were fixed in Bouin’s solution for 96 hr, rinsed under tap water for 2 hr, placed in denatured 50% ethanol for 7 days, and finally transferred into 10% NBF. The fixed tissues were processed using defined paraffin-embedding procedure with a Tissue-Tex VIP automated tissue processor (Tissue Tek E300 and Leica ASP300, suppliers Leica Biosystems and Rankin Biomedical Corporation). The paraffin-embedded tissue blocks were cut at 6-mm thick sections onto glass slides and were stained with H&E or von Kossa methods.

FIGURE 1.—Calcification of the stratum corneum in the Beagle dog tongue (H&E stain, 40) from the 26-week study. Magnification bar indicates 50 mm.

RESULTS The signal 26-week study consisted of groups of 3 dogs each. The incidence of superficial stratum corneum calcification of the tongue is presented in Table 1. The tongue calcifications were observed in all but 1 animal (Figures 1 and 2). Examination of tongue sections from control dogs from 11 previous studies performed at the facility demonstrated that the incidence of this finding (superficial stratum corneum calcification of the tongue) was variable across studies, as presented in Table 2. It is also noteworthy that prior to 2008, the artifact was not observed, perhaps indicating a change in procedures or suppliers, although evidence for such change could not be documented, or perhaps indicating variability on the chemical characteristics of the fixative. As presented in Table 3, fixation of tongue in combination with femur and eyes or with femur and testes did not show superficial stratum corneum calcification. The finding was noted when the tongue was fixed with the femur and stomach as well as when fixed with all tissues (Figures 3 and 4). The presence of calcification on tongue samples in the presence of stomach and bone with or without other tissues

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Vol. 43, No. 5, 2015

ARTIFACTUAL TONGUE CALCIFICATION

FIGURE 2.—Calcification of the stratum corneum in the Beagle dog tongue (von Kossa stain, 40) from the 26-week study. Magnification bar indicates 50 mm.

FIGURE 3.—Calcification of the stratum corneum in the Beagle dog tongue (H&E stain, 40) from the experimental study. Magnification bar indicates 50 mm.

and the absence of calcification in the samples from tongues fixed in combination with only either eyes or testes, both of which are fixed in Davidson’s solution or Bouin’s solution, indicate that the presence of the stomach in the fixation jar made a difference in the presentation of the superficial stratum corneum calcification of the tongue of these dogs. DISCUSSION Based on the absence of reports of this finding, the timeline of presentation, the presentation in controls and treated animals, and the reproduction of the change when tongues are fixed together with stomach seem to imply that the acidification of the medium by the presence of the stomach

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FIGURE 4.—Calcification of the stratum corneum in the Beagle dog tongue (von Kossa stain, 40) from the experimental study. Magnification bar indicates 50 mm.

may be responsible for the presentation of this change. Since the combined fixation of tongue with bone and stomach is a common practice, it is reasonable to think that the effect may be limited to specific chemical characteristics of the processing solutions utilized at this facility. However, more work would have to be done to fully characterize the pH and concentrations necessary to produce this effect. Such work was beyond the scope of our investigation on this issue. No changes in the processes could be identified that would explain the onset of this finding (superficial stratum corneum calcification) beginning in 2008. The facility’s supplier (ACP Chemical Canada Inc.) has not changed over the years. Most of the reports of calcification of the tongue in dogs relate to calcification of muscle cells and connective tissue such as calcinosis circumscripta (Jeong et al. 2003) and are generally related to kidney failure or hyperparathyroidism. A study of calcinosis circumscripta in dogs revealed 18 spontaneous cases of calcinosis, and in these, only 3 animals had involvement of the tongue, and the calcification was present in the connective tissue at the base of the tongue (Lojszczykszczepaniak, Orzelski, and Smiech 2008). In these reported series, only 1 animal had involvement of just 1 site, while all others involved multiple sites.

CONCLUSION The presence of calcification of the stratum corneum in dogs during toxicology studies is an artifactual change related to the processing of tissues, specifically the fixation of tongue in the same container as bone and stomach and perhaps other tissues. This change should not be confused with compound-related effects, even when the incidence varies between controls and treated animals.

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AUTHOR CONTRIBUTIONS Both the authors contributed to conception or design; data acquisition, analysis, or interpretation; drafting the manuscript; and critically revising the manuscript. Both the 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.

TOXICOLOGIC PATHOLOGY

REFERENCES Jeong, W., Noh, D., Kwon, O.–D., Williams, B. H., Park, S.-C., Lee, M., Do, S., Chung, J., Lee, G., Yun, H., and Jeong, K.-S. (2003). Calcinosis circumscripta on lingual Muscles and dermis in a dog. J Vet Med Sci 66, 433–35. Lojszczyk-szczepaniak, A., Orzelski, M., and Smiech, A. (2008). Canine calcinosis circumscripta-retrospective studies. Medycyna Wet 64, 1397–400.

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Artifactual Stratum Corneum Calcification of the Beagle Dog Tongue.

Examination of H&E-stained tongue samples from a 26-week intravenous infusion study of Beagle dogs, utilizing a compound with no recognized effect on ...
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