Use of Both Fluoro-Jade B and Hematoxylin and Eosin to Detect Cell Death in the Juvenile Rat Brain Exposed to NMDA-Receptor Antagonists or GABA-Receptor Agonists in Safety Assessment.

Administration of pediatric anesthetics with N-methyl D-aspartate (NMDA)-receptor antagonist and/or γ-aminobutyric acid (GABA) agonist activities may result in neuronal degeneration and/or neuronal cell death in neonatal rats. Evaluating pediatric drug candidates for this potential neurotoxicity is often part of overall preclinical new drug development strategy. This specialized assessment may require dosing neonatal rats at postnatal day 7 at the peak of the brain growth spurt and evaluating brain tissue 24 to 48 hours following dosing. The need to identify methods to aid in the accurate and reproducible detection of lesions associated with this type of neurotoxic profile is paramount for meeting the changing needs of neuropathology assessment and addressing emerging challenges in the neuroscience field. We document the use of Fluoro-Jade B (FJB) staining, to be used in conjunction with standard hematoxylin and eosin staining, to detect acute neurodegeneration and neuronal cell death that can be caused by some NMDA-receptor antagonists and/or GABA agonists in the neonatal rat brain. The FJB staining is simple, specific, and sensitive and can be performed on brain specimens from the same cohort of animals utilized for standard neurotoxicity assessment, thus satisfying animal welfare recommendations with no effect on achievement of scientific and regulatory goals.

Extended One-Generation Reproductive Toxicity (EOGRT) study of benzoic acid in Sprague Dawley rats.

Benzoic acid (BA) was administered in the diet to male and female Sprague Dawley Crl:CD(SD) rats in an OECD Test Guideline 443 Extended One-Generation Reproductive Toxicity (EOGRT) study to test for effects that may occur as a result of pre- and postnatal exposure. The study included cohorts of F1 offspring to evaluate potential effects of benzoic acid on reproduction, the developing immune system, and the developing neurological system with the inclusion of learning and memory assessments. Benzoic acid was incorporated in the diet at concentrations of 0, 7,500, 11,500, and 15,000 mg/kg diet (ppm). These concentrations were selected based on the results of preliminary studies, and, based on average food consumption, were intended to supply BA doses of approximately 0, 500, 750, and 1000 mg/kg bw/day. To avoid exceeding these target dose levels, the dietary concentrations were adjusted (based on historical control body weight and food consumption data) to maintain the target mg/kg bw/day dose levels during those life periods when food intake per unit of body weight was increased to support milk production by females (gestation and lactation) and rapid pup growth (post-weaning). In the parental (F0) generation, survival, clinical observations, organ weights, pathology, hematology, serum chemistry, urinalysis, and bile acids were unaffected by BA administration. Reproductive parameters were also unaffected by BA administration. In the F1 generation, survival, growth and developmental landmarks, organ weights, pathology, immunotoxicity assessment, and neurotoxicity and neurobehavioral parameters such as auditory startle response, locomotor activity, learning and memory assessments were unaffected by BA administration, as were clinical pathology (hematology, serum chemistry, urinalysis, bile acids and thyroid hormones) and reproductive performance. Similarly, no adverse effects or systemic toxicity were observed in the F2 generation. Overall, the highest dietary concentration (15,000 ppm), providing a dosage of approximately 1000 mg/kg bw/day, was the NOAEL for benzoic acid in this EOGRT study.

Phage constructs targeting gonadotropin-releasing hormone for fertility control: evaluation in cats.

OBJECTIVES: Phage-gonadotropin-releasing hormone (GnRH) constructs with potential contraceptive properties were generated in our previous study via selection from a phage display library using neutralizing GnRH antibodies as selection targets. In mice, these constructs invoked the production of antibodies against GnRH and suppressed serum testosterone. The goal of this study was to evaluate this vaccine against GnRH for its potential to suppress reproductive characteristics in cats. METHODS: Sexually mature male cats were injected with a phage-GnRH vaccine using the following treatment groups: (1) single phage-GnRH vaccine with adjuvant; (2) phage-GnRH vaccine without adjuvant and half-dose booster 1 month later; or (3) phage-GnRH vaccine with adjuvant and two half-dose boosters with adjuvant 3 and 6 months later. Anti-GnRH antibodies and serum testosterone, testicular volume and sperm characteristics were evaluated monthly for 7-9 months. RESULTS: All cats developed anti-GnRH antibodies following immunization. Serum antibody titers increased significantly after booster immunizations. In group 3, serum testosterone was suppressed 8 months after primary immunization. Total testicular volume decreased in group 1 by 24-42% and in group 3 by 15-36% at 7 months after immunization, indicating potential gonadal atrophy. Vacuolation of epididymides was observed histologically. Although all cats produced sperm at the conclusion of the study, normal morphology was decreased as much as 38%. Phage alone produced no local or systemic reactions. Immunization of phage with AdjuVac produced unacceptable injection site reactions. CONCLUSIONS AND RELEVANCE: Our phage-based vaccine against GnRH demonstrated a potential for fertility impairment in cats. Future research is required to optimize vaccine regimens and identify animal age groups most responsive to the vaccine. If permanent contraception (highly desirable in feral and shelter cats) cannot be achieved, the vaccine has a potential use in zoo animals or pets where multiple administrations are more practical and/or reversible infertility is desirable.

Correction to: Letter to the editor regarding "safety of safety evaluation of pesticides: developmental neurotoxicity of chlorpyrifos and chlorpyrifos-methyl" by Mie et al. (environmental health. 2018. 17:77).

Following publication of the original article.

Soy-deficient diet induces renal lesions in juvenile rats.

Certified LabDiet® 5K96 Advanced Protocol™ Verified Casein Diet 10 IF (5K96) is a commercial diet low in soy isoflavones developed for use in developmental and reproductive toxicity (DART) studies, especially those designed to detect endocrine disruptors. The objective of this study was to determine the incidences and severities of 5K96-associated renal lesions in control F0 and F1 cohorts of rats fed the 5K96 diet. Kidneys from control animals of four DART studies involving Sprague-Dawley rats fed the 5K96 diet, were evaluated microscopically. Mineralization and basophilic tubules were present in high incidence/severity in males and females compared to historical controls fed conventional diets. F1 cohorts were affected to a far greater degree than F0 cohorts, and females were affected more than males. Consideration of target tissue and mode of action should be given before automatically incorporating the 5K96 diet into DART study designs, and caution should be exercised when identifying and interpreting renal toxicity in the F1 cohorts of such studies.

An Evaluation of Neurotoxicity Following Fluoride Exposure from Gestational Through Adult Ages in Long-Evans Hooded Rats.

At elevated levels, fluoride (F-) exposure has been associated with adverse human health effects. In rodents, F- exposure has been reported to induce deficits in motor performance and learning and memory. In this study, we examined Long-Evans hooded male rats maintained on a standard diet (20.5 ppm F-) or a low F- diet (3.24 ppm F-) with drinking water exposure to 0, 10, or 20 ppm F- from gestational day 6 through adulthood. At postnatal day 25, brain F- levels were 0.048 or 0.081 µg/g and femur 235 or 379.8 µg/g for 10 and 20 ppm F-, respectively. Levels increase with age and in adults, levels for plasma were 0.036 or 0.025 µg/ml; for the brain 0.266 or 0.850 µg/g; and for the femur, 681.2 or 993.4 µg/g. At these exposure levels, we observed no exposure-related differences in motor, sensory, or learning and memory performance on running wheel, open-field activity, light/dark place preference, elevated plus maze, pre-pulse startle inhibition, passive avoidance, hot-plate latency, Morris water maze acquisition, probe test, reversal learning, and Y-maze. Serum triiodothyronine (T3), thyroxine (T4), and thyroid stimulating hormone (TSH) levels were not altered as a function of 10 or 20 ppm F- in the drinking water. No exposure-related pathology was observed in the heart, liver, kidney, testes, seminal vesicles, or epididymides. Mild inflammation in the prostate gland was observed at 20 ppm F-. No evidence of neuronal death or glial activation was observed in the hippocampus at 20 ppm F-.

Comparative Aspects of Pre- and Postnatal Development of the Male Reproductive System.

This review describes pre- and postnatal development of the male reproductive system in humans and laboratory animals, and highlights species differences in the timing and control of hormonal and morphologic events. Major differences are that the fetal testis is dependent on gonadotropins in humans, but is independent of such in rats; humans have an extended postnatal quiescent period, whereas rats exhibit no quiescence; and events such as secretion by the prostate and seminal vesicles, testicular descent, and the appearance of spermatogonia are all prenatal events in humans, but are postnatal events in rats. Major differences in the timing of the developmental sequence between rats and humans include: gonocyte transformation period (rat: postnatal day 0-9; human: includes gestational week 22 to 9 months of age); masculinization programming window (rat: gestational day 15.5-17.5; human: gestational week 9-14); and mini-puberty (rat: 0-6 hr after birth; human: 3-6 months of age). Endocrine disruptors can cause unique lesions in the prenatal and early postnatal testis; therefore, it is important to consider the differences in the timing of the developmental sequence when designing preclinical studies as identification of windows of sensitivity for endocrine disruption or toxicants will aid in interpretation of results and provide clues to a mode of action. Birth Defects Research 110:190-227, 2018. © 2017 Wiley Periodicals, Inc.

Postnatal Organ Development as a Complicating Factor in Juvenile Toxicity Studies in Rats.

Toxicologic pathologists must evaluate tissues of immature animals from a number of types of nonclinical toxicity studies. The pathologist who is familiar with normal postnatal organ development is in a better position to appropriately detect and differentiate between abnormal, delayed, or precocious development. Vacuolation and apoptosis in multiple tissue types are normal components of development that could influence the interpretation of some tissues. Unique postnatal features such as the germal matrix in the brain, gonocytes in the testes, and saccules in the lung may complicate the histopathological evaluation. With the knowledge of normal organ development and critical windows therein, it is possible to design targeted studies to identify xenobiotic toxicity.

Impact of Age on the Male Reproductive System from the Pathologist's Perspective.

Age, and in particular young age, can significantly impact the response to toxicants in animals and can greatly influence the interpretation of tissue changes by the toxicologic pathologist. Although this applies to multiple organ systems, the current review focuses on the male reproductive system. When performing microscopic evaluation of male reproductive organs, the toxicologic pathologist must be aware of the dynamic changes in histomorphology, predominantly driven by timed hormonal alterations, at various life stages. Specific challenges pathologists face are understanding the appearance of male reproductive tissues throughout the neonatal, infantile, and juvenile developmental periods, recognizing when normal looks abnormal during tissue development, defining sexual maturity, and working with high interanimal variability in maturation rate and histologic appearance in developing large laboratory animals, such as nonhuman primates, dogs, and pigs. This review describes postnatal development of the male reproductive system in the rat, demonstrates how assessing toxicity during a defined window of postnatal development in the rat may improve definition of toxicant timing and targets, and discusses challenges associated with the interpretation of toxicity in immature large animal species. The emphasis is on key age-related characteristics that influence the interpretation of tissue changes by the toxicologic pathologist.

Localisation of Lactate Transporters in Rat and Rabbit Placentae.

The distribution of monocarboxylate transporter (MCT) isoforms 1 and 4, which mediate the plasmalemmal transport of l-lactic and pyruvic acids, has been identified in the placentae of rats and rabbits at different ages of gestation. Groups of three pregnant Sprague-Dawley rats and New Zealand White rabbits were sacrificed on gestation days (GD) 11, 14, 18, or 20 and on GD 13, 18, or 28, respectively. Placentae were removed and processed for immunohistochemical detection of MCT1 and MCT4. In the rat, staining for MCT1 was associated with lakes and blood vessels containing enucleated red blood cells (maternal vessels) while staining for MCT4 was associated with vessels containing nucleated red blood cells (embryofoetal vessels). In the rabbit, staining for MCT1 was associated with blood vessels containing nucleated red blood cells while staining for MCT4 was associated with vessels containing enucleated red blood cells. Strength of staining for MCT1 decreased during gestation in both species, but that for MCT4 was stronger than that for MCT1 and was consistent between gestation days. The results imply an opposite polarity of MCT1 and MCT4 across the trophoblast between rat and rabbit.

Implementing the extended one-generation reproductive toxicity study (EOGRTS): important points to consider.

The hallmark of the extended one-generation reproductive toxicity study (EOGRTS) is that, based on certain criteria or triggers, selected offspring are assigned at weaning to different cohorts for further investigation of sexual maturation, reproductive organ integrity and function, neuropathological and behavioral endpoints, and/or immune function. The triggers allow for a more customizable design based directly on the data, while minimizing animal usage. Compared to the two-generation reproductive toxicity study, the EOGRTS design increases the number, extent, and duration of F1-offspring assessments resulting in more thorough and efficient utilization of the first generation while excluding the second generation of offspring unless triggered. Therefore, the EOGRTS has the potential to reduce the number of rats required by nearly 1200 animals per study. When performing the EOGRTS, the complexity of this study should not be underestimated and experienced flexible testing laboratories with sufficient resources and historical control data for all parameters are essential. The aim of this review is to discuss the important aspects of this challenging study design and to share our knowledge on the implementation of this study in our laboratories. In addition, we elaborate on the type of criteria for expansion of the study and logistical considerations. Altogether, this review can be used as guidance by other labs, study monitors, and registration officers.

A two-year dietary carcinogenicity study of (2R,4R)-monatin salt in mice.

Groups of Crl:CD-1 (ICR) mice (60/group/sex) were fed 0 (2 control groups), 5000, 20,000, or 40,000 ppm of enzymatically sourced (2R,4R)-monatin salt ("R,R-monatin") in the diet for up to two years. There were no adverse effects on survival, incidence of palpable masses and tumors, feed consumption, hematology or serum chemistry parameters, organ weights, or ophthalmic, macroscopic, and microscopic examinations. The only notable effect was statistically significantly lower mean body weights and body weight gains in all treated groups, which generally occurred throughout the study and were most likely a result of caloric dilution of the test diets and not considered adverse. There were no test article-related changes in the incidence or occurrence of neoplastic diseases in mice on this study. The no-observed-effect-level (NOEL) for carcinogenicity of R,R-monatin fed to mice for 24 months was 40,000 ppm, the highest dietary concentration tested, which was equivalent to approximately 6502 and 7996 mg/kg bw/day in males and females, respectively.

Scientific and Regulatory Policy Committee (SRPC) Points to Consider: Histopathology Evaluation of the Pubertal Development and Thyroid Function Assay (OPPTS 890.1450, OPPTS 890.1500) in Rats to Screen for Endocrine Disruptors.

The U.S. Environmental Protection Agency Endocrine Disruptor Screening Program (EDSP) is a multitiered approach to determine the potential for environmental chemicals to alter the endocrine system. The Pubertal Development and Thyroid Function in Intact Juvenile/Peripubertal Female and Male Rats (OPPTS 890.1450, 890.1500) are 2 of the 9 EDSP tier 1 test Guidelines, which assess upstream mechanistic pathways along with downstream morphological end points including histological evaluation of the kidneys, thyroid, and select male/female reproductive tissues (ovaries, uterus, testes, and epididymides). These assays are part of a battery of in vivo and in vitro screens used for initial detection of test article endocrine activity. In this Points to Consider article, we describe tissue processing, evaluation, and nomenclature to aid in standardization of assay results across laboratories. Pubertal assay end points addressed include organ weights, estrous cyclicity, clinical pathology, hormonal assays, and histological evaluation. Potential treatment-related findings that may indicate endocrine disruption are reviewed. Additional tissues that may be useful in assessment of endocrine disruption (vagina, mammary glands, and liver) are discussed. This Points to Consider article is intended to provide information for evaluating peripubertal tissues within the context of individual assay end points, the overall pubertal assay, and tier I assays of the EDSP program.

Histologic Features of Postnatal Development of Immune System Organs in the Sprague-Dawley Rat.

The immune system of the rat undergoes substantial functional and morphological development during the postnatal period. Some aspects of this development are genetically predetermined, while other aspects depend on environmental influences. Detailed information on postnatal development is important in the interpretation of histopathologic findings in juvenile toxicology and pubertal assay studies, as well as other studies conducted in juvenile rats. Studies were conducted to provide detailed characterization of histologic features of the major functional compartments of immune system organs in male and female Sprague-Dawley rats at weekly intervals from the day of birth through postnatal day (PND) 42. Maturation of the individual immune system organs occurred across a range of ages, with histologic maturation of T-cell-related compartments typically occurring prior to maturation of B-cell-related compartments. The sequence of histologic maturation was bone marrow and thymus on PND 14, mesenteric lymph node on PND 21, Peyer's patches and bronchus-associated lymphoid tissue on PND 28, mandibular lymph node, nasopharynx-associated lymphoid tissue, and diffuse mucosal mononuclear cell population of small intestine on PND 35, and spleen on PND 42. An estimation of functional maturation can be made based on the morphological indications of maturity of each compartment of immune system organs, but histologic indications of maturity do not confirm functional immunocompetence.

Postnatal ovary development in the rat: morphologic study and correlation of morphology to neuroendocrine parameters.

Histopathologic examination of the immature ovary is a required end point on juvenile toxicity studies and female pubertal and thyroid function assays. To aid in this evaluation and interpretation of the immature ovary, the characteristic histologic features of rat ovary through the developmental periods are described. These histologic features are correlated with published changes in neuroendocrine profiles as the hypothalamic-pituitary-gonadal axis matures. During the neonatal stage (postnatal day [PND] 0-7), ovarian follicle development is independent of pituitary gonadotropins (luteinizing hormone [LH] or follicle-stimulating hormone [FSH]), and follicles remain preantral. Antral development of "atypical" follicles occurs in the early infantile period (PND 8-14) when the ovary becomes responsive to pituitary gonadotropins. In the late infantile period (PND 15-20), the zona pellucida appears, the hilus forms, and antral follicles mature by losing their "atypical" appearance. The juvenile stage (PND 21-32) is the stage when atresia of medullary follicles occurs corresponding to a nadir in FSH levels. In the peripubertal period (PND 33-37), atresia subsides as FSH levels rebound, and LH begins its bimodal surge pattern leading to ovulation. This report will provide pathologists with baseline morphologic and endocrinologic information to aid in identification and interpretation of xenobiotic effects in the ovary of the prepubertal rat.

Postnatal development of the testis in the rat: morphologic study and correlation of morphology to neuroendocrine parameters.

Histopathologic examination of the testis from juvenile rats is often necessary to characterize the safety of new drugs for pediatric use and is a required end point in male pubertal development and thyroid function assays. To aid in evaluation and interpretation of the immature testis, the characteristic histologic features of the developing rat testis throughout postnatal development are described and correlated with published neuroendocrine parameter changes. During the neonatal period (postnatal day [PND] 3-7), seminiferous tubules contained gonocytes and mitotically active immature Sertoli cells. Profound proliferation of spermatogonia and continued Sertoli cell proliferation occurred in the early infantile period (PND 8-14). The spermatogonia reached maximum density forming double-layered rosettes with Sertoli cells in the late infantile period (PND 15-20). Leptotene/zygotene spermatocytes appeared centrally as tubular lumina developed, and individual tubules segregated into stages. The juvenile period (PND 21-32) featured a dramatic increase in number and size of pachytene spermatocytes with the formation of round spermatids and loss of "infantile" rosette architecture. In the peri-pubertal period (PND 32-55), stage VII tubules containing step 19 spermatids were visible by PND 46. The presented baseline morphologic and endocrinologic information will help pathologists distinguish delayed development from xenobiotic effects, determine pathogenesis when confronted with nonspecific findings, and identify sensitive time points for targeted study design.

Histopathological and Immunohistochemical Characterization of Methyl Eugenol-induced Nonneoplastic and Neoplastic Neuroendocrine Cell Lesions in Glandular Stomach of Rats.

Methyl eugenol induces neuroendocrine (NE) cell hyperplasia and tumors in F344/N rat stomach. Detailed histopathological and immunohistochemical (IHC) characterization of these tumors has not been previously reported. The objective of this study was to fill that data gap. Archived slides and paraffin blocks were retrieved from the National Toxicology Program Archives. NE hyperplasias and tumors were stained with chromogranin A, synaptophysin, amylase, gastrin, H(+)/K(+) adenosine triphosphatase (ATPase), pepsinogen, somatostatin, and cytokeratin 18 (CK18) antibodies. Many of the rats had gastric mucosal atrophy, due to loss of chief and parietal cells. The hyperplasias and tumors were confined to fundic stomach, and females were more affected than the males. Hyperplasia of NE cells was not observed in the pyloric region. Approximately one-third of the females with malignant NE tumors had areas of pancreatic acinar differentiation. The rate of metastasis was 21%, with liver being the most common site of metastasis. Immunohistochemically, the hyperplasias and tumors stained consistently with chromogranin A and synaptophysin. Neoplastic cells were also positive for amylase and CK18 and negative for gastrin, somatostatin, H(+)/K(+) ATPase, and pepsinogen. Metastatic neoplasms histologically similar to the primary neoplasm stained positively for chromogranin A and synaptophysin. Based on the histopathological and IHC features, the neoplasms appear to arise from enterochromaffin-like cells.

Histologic features of prepubertal and pubertal reproductive development in female Sprague-Dawley rats.

In response to growing concerns that environmental chemicals may have adverse effects on human health by altering the endocrine system, the Endocrine Disruptor Screening Program (EDSP), under the auspices of the United States Environmental Protection Agency (U.S. EPA), recently instituted a Tier I battery of tests including a female pubertal assay. This assay requires dosing of female rats from postnatal day (PND) 22 through PND 42 (or 43), the period of pubertal development in the rat, to identify test articles that may have estrogenic or antiestrogenic effects, or may alter hormones or neurotransmitters. While certain landmarks in female rat reproductive development are published, little is published on the microscopic appearance of the female reproductive tract during prepubertal and pubertal development. In this study, reproductive tissues from three female Sprague-Dawley rats were collected each day from PND 20 through PND 50, such that tissues from a total of 93 rats were collected throughout the prepubertal and pubertal period. Tissues were formalin-fixed, trimmed, paraffin-embedded, sectioned at 5-µm thickness, and examined microscopically. The major histologic features of the female reproductive tract throughout this critical period were described in detail. This information will help pathologists interpret findings observed in female pubertal assays.

Acute inhalation study of allyl alcohol for derivation of acute exposure guideline levels.

An acute, whole-body inhalation study for allyl alcohol in Sprague-Dawley rats was designed to support derivation of AEGL values, with emphasis on establishing NOAELs for irreversible effects of different exposure concentrations and durations. Groups of 10 rats were exposed for 1 hour (0, 50, 200, or 400 ppm), 4 hours (0, 20, 50, or 100 ppm), or 8 hours (0, 10, 20, or 50 ppm). Clinical evaluations were performed during exposure and in an open field within 22-71 minutes after termination of exposure. Clinical pathology, gross necropsy, and histopathology (nasal tissues, larynx, trachea, lungs/bronchi, liver, and kidneys) were evaluated 14 days after exposure. Mortality was limited to 1 male exposed for 8 hours to 50 ppm. Clinical findings of gasping, rales, increased respiration noted at higher exposure levels were rapidly reversed. No treatment-related findings were observed in the liver and kidneys, or in the lungs of surviving animals. Histopathology in the nasal cavity was noted at all exposure levels following 1, 4, or 8 hours of exposure. Mild nasal inflammation was found at the lowest exposure levels (50-ppm/1-hour, 20-ppm/4-hour, and 10-ppm/8-hour). These effects were considered reversible and were not associated with related clinical signs. Severe, irreversible nasal olfactory epithelial lesions were present in 50 ppm/8-hour males. The NOELs for irreversible effects were 400-ppm/1-hour, 100-ppm/4-hour, and 20-ppm/8-hour. The incidence of severe findings was positively dependent on both concentration and the exposure duration. In contrast, the incidence of mild reversible findings did not appear to be dependent on duration.