Ocular Toxicity Studies in Mammalian Nonrodent Species
While all ocular toxicity testing is specialized, ocular toxicity studies in nonrodent species require an even more “customized” level of planning and often different methods than those used in rodent (rat/mouse) studies.
The species emphasized below are mammals with eyes much larger than those of rats and mice: domestic rabbit, dog (laboratory Beagle), monkey (cynomolgus and rhesus macaques), and domestic pig (including minipigs) but many points are also applicable to other nonrodent species such as cats, ferrets, marmosets and other monkey species, ruminants (sheep, goat, cow), and horses.
Considerations for ocular toxicity testing in nonrodent species include:
1) The eye does not exist in anatomic or functional isolation.
For all species, ocular histopathology should include not just the eye but at a minimum, the optic nerve as well as several major ocular adnexa (ancillary tissues that support the eye and vision and are components of the ocular surface system; Cher, 2007; Gipson, 2012). Ocular adnexa include the Harderian gland, nictitating membrane (and its nictitans gland), eyelids and their glands including Meibomian glands, lacrimal gland, and lacrimal drainage apparatus (including nasolacrimal ducts), associated lymphoid tissue, and even the precorneal tear film (not all of these are present in all species). Which adnexa are appropriately included can vary with species and study goals, so this is best decided on a case-by-case basis.
In a real-life example of how ocular adnexal findings can provide highly relevant “context”, treated rats and mice in two carcinogenicity studies of the same test article had prominent microscopic corneal ulceration and inflammation, but also exhibited extensive lacrimal gland inflammation and atrophy. The corneal changes were interpreted as most likely secondary to probable precorneal tear film deficits (related to the lacrimal gland pathology) rather than being primary test-article toxic effects.
2) Nonrodent species differences in ocular/ocular adnexal anatomy and histology
Anatomic and histologic differences in eyes and ocular adnexa of nonrodent species are numerous (Bayraktaroglu and Ergün, 2010; Dellman and Brown, 1976; Hartmann and Strauss 1933; Hermanson and de Lahunta , 2019); Jester et al., 1980; Kuszak, 1995; May, 2008; Morrison et al., 1987; Ollivier et al., 1984; Prince et al., 1960; de Schaepdrijver et al., 1989; Schlegel et al., 2001; Ts’o and Friedman, 1967; Wilson and Hong, 2000). Some species also have adjacent non-ocular structures that can be mistaken for ocular adnexa.
In nonrodent studies, it is especially important to avoid the “fallacy of rat/mouse extrapolation” – that is, the blanket assumption that nonrodent anatomy will be exactly as in the more familiar rats/mice, and that all rat/mouse necropsy/histology methods can be used without modification in nonrodent species.
Not taking anatomic differences into account has led to many problems in ocular toxicity studies: protocol-required tissues not collected at necropsy and thus lost forever; time-consuming, fruitless wet-tissue recut attempts; delayed report submissions; and even incorrect or inadequate study results.
To cite a real-life example, the rabbit Harderian gland is bilobed but only the “white” lobe grossly resembles the rat/mouse Harderian gland while the “red” lobe has been mistaken for a “lacrimal gland”. Other unfortunately common real-life examples involve the lacrimal glands of dogs and macaques, which are so small and inconspicuous that they are frequently overlooked at necropsy (perhaps in some cases because of a fallacious expectation that the dog and macaque lacrimal glands are just like the relatively large lacrimal glands of rats/mice). This problem is sometimes compounded by a related issue: dogs (and rabbits) have large extraorbitally located zygomatic salivary glands, and these zygomatic glands are all too often mistakenly collected at necropsy as “lacrimal gland” (perhaps in some cases because of an erroneous assumption that rabbit and dog zygomatic glands are exact analogues of the extraorbital lacrimal glands of rats/mice). In all such cases, there was very poor to no tissue accountability for lacrimal gland (deficits that could be unfavorably regarded during regulatory audits). Even worse, such studies were de facto inadequate for determination of any possible treatment-related effects in the lacrimal glands.
3) Specialized necropsy/histology procedures
Necropsy and immediate post-necropsy considerations include how to dissect, handle and identify tissues at necropsy; whether or not adjacent orbital tissues are trimmed off or left attached to the globe at necropsy; placement of orientation markers on the globes; choice of tissue fixatives and fixation regimens; storage container size and shape; and packing and shipping requirements if wet tissues are to be shipped to EPL.
The left/right identity of eyes and ocular adnexa must be maintained at every step from necropsy to glass slide (and even during the pathologist’s microscopic evaluation) – which in turn, requires advance preparation of separate and appropriately labelled wet tissue containers, cassettes, glass slides, and histology records.
In “routine” toxicity studies, one section (glass slide) of the eye is standard and is generally acceptable for appropriate microscopic examination. In contrast, multiple eye sections are needed for the more specialized evaluation in ocular toxicity studies (especially for the large globes of many nonrodent species). The exact number of multiple eye sections and the locations on the globe where these sections are taken are highly critical issues which require careful customization on a study-by-study basis.
In most nonrodent ocular toxicity studies, it is also extremely important to specially orient the eye sections in the blocks such that superior-inferior and nasal-temporal regions will be identifiable on the glass slide sections. This orientation allows the pathologist to more precisely localize microscopic lesions (an important factor in the interpretation of ocular findings).
Regulatory agencies have recently begun to recommend that nonrodent-species retinal cone-rich areas (e.g., macaque macula lutea/fovea centralis, rabbit visual streak) be explicitly evaluated and accounted for by the pathologist and addressed in the pathology report. Thus, these areas should be present in eye sections on the glass slides, which can require considerable extra histology effort and time because some these areas (like the macaque fovea/macula) are very small and localized.
For some species with very large eyes (e.g., cow, horse, sheep, goat), “giant” blocks and glass slides may be needed to fit the entire globe sections (these preparations also require extra histology effort).
4) Histopathologic evaluation (including data entry and tabulation)
In “routine” toxicity studies, it is generally acceptable to record ocular tissue accountability and microscopic findings implicitly under the general topography of “eye”. However, in ocular toxicity studies irrespective of species, tissue accountability and microscopic findings in the eye should be recorded explicitly – that is, at the level of topographic eye subsites (e.g., “cornea”, “retina”, “lens”) rather than “lumped” together under a collective topography of “eye”(Bolon et al., 2013). Ocular adnexa can be recorded implicitly at the level of the organ topography (e.g., “lacrimal gland”, “eyelid, upper”, “nictitating membrane”).
In the majority of ocular toxicity studies, one eye of a pair is administered the test article or treatment, while the other is a control. For this reason, left- and right-side microscopic diagnoses for each eye/animal cannot be “combined” but rather must be separately diagnosed, recorded, tabulated, and statistically analyzed (The same applies to the left/right ocular adnexa).
In-life clinical observations (e.g., slit-lamp and indirect ophthalmoscopy) in the eye are common (Munger, 2002) but they rarely remain visible postmortem and thus cannot be recorded as typical “gross lesions” or “macroscopic observations” at necropsy. Nevertheless, the relationship between many in-life clinical observations and corresponding microscopic lesions is often highly toxicologically relevant, and can be a critical factor in interpretation of study results. Probably because of this, regulatory agencies have recently begun to specifically recommend that such ophthalmoscopic clinical observations be addressed during the pathologist’s microscopic evaluations. Thus, in ocular toxicity studies, individual correlations of clinical observations to microscopic findings should be made by the pathologist and recorded in itemized tables, with appropriate discussion in the pathology report as needed.
Given the above, it follows that communication between the pathologist and clinical ophthalmologist is extremely important and should not only be permitted but actively encouraged. To cite one real-life example of the benefits of such collaboration, in a recent intracameral-injection study in dogs, unusual microscopic cataracts (with correlation to in-life lens opacities) were diagnosed by the pathologist. Discussion by the pathologist and ophthalmologist resulted in a consensus that these microscopic cataracts were most likely related to injection-procedure trauma rather than being primary test-article toxic effects.
5) Pathology peer review
Pathology peer review is appropriate and recommended for many ocular toxicity studies, especially studies supporting submissions to regulatory agencies.
Although detailed discussion is beyond the scope of this article, in brief, pathology peer review is not a “second opinion” or a “re-read” of the study, but rather is a collaborative process in which another pathologist reviews the study pathologist’s initial histopathology findings with the end result being mutual consensus reflected in a single final pathology report signed by the study pathologist (Morton et al., 2010).
In summary, to ensure that nonrodent ocular toxicity studies proceed in an optimal manner, EPL encourages study directors and sponsors to solicit pathologist input during the protocol review, necropsy preparations, and pathology report and peer-review planning phases of these studies.
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