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A modified amphibian metamorphosis assay was performed in which Nieuwkoop and Faber (NF) stage 47 Xenopus laevis larvae were exposed to different concentrations of either perchlorate (ClO 4 - ) or nitrate (NO 3 - ) for 32 days. Larvae were exposed to 0.0 (control), 5, 25, 125, 625, and 3125 μg/L ClO 4 - , or 0 (control), 23, 71, 217, 660, and 2000 mg/L NO 3 - . The primary endpoints were survival, hind limb length (HLL), forelimb emergence and development, developmental stage (including time to NF stage 62 [MT 62 ]), thyroid histopathology, wet weight, and snout-vent length (SVL). Developmental delay as evidenced by altered stage distribution and increased MT 62 , a higher degree of thyroid follicular cell hypertrophy, and an increase in the prevalence of follicular cell hyperplasia was observed at concentrations ≥125 μg/L ClO 4 - . The no observed effect concentration (NOEC) for developmental endpoints was 25.0 μg/L ClO 4 - and the NOEC for growth endpoints was 3125 μg/L ClO 4 - . Exposure to nitrate did not adversely affect MT 62 , but a decreasing trend in stage distribution and median developmental stage at ≥217 mg/L NO 3 - was observed. No histopathologic effects associated with nitrate exposure were observed. An increasing trend in SVL-normalized HLL was observed at 2000 mg/L NO 3 - . Nitrate did not alter larval growth. The NOEC for developmental endpoints was 71 mg/L NO 3 - , and 2000 mg/L NO 3 - for growth endpoints. The present study provided additional evidence that the effects and potency of nitrate and perchlorate on metamorphosis and growth in X. laevis are considerably different., (© 2024 John Wiley & Sons Ltd.)

A larval amphibian growth and development assay was performed to evaluate the potential effects of environmentally-relevant concentrations of triclosan (TCS) on amphibian development and growth. Xenopus laevis were exposed to TCS 0.0 (control), 6.3, 12.5 and 25.0 μg l -1 (estimated maximum tolerable concentration) until 10 weeks post-metamorphosis. At median metamorphosis time (Nieuwkoop and Faber stage 62), five larvae per replicate were collected for snout-vent length, hind limb length and body weight measurements, and histopathological examination of thyroid glands. Endpoints evaluated at test termination were based on draft guidance (USEPA, ) and included: survival; snout-vent length; body weight; gender; nuptial pad development (males); and liver, kidney, gonad and gonadal ducts histopathology. Exposure to TCS did not decrease survival, induce general signs of toxicity, affect median metamorphosis time or alter sex ratios. Exposure to TCS 12.5 and 25 μg l -1 increased growth during the metamorphic stages relative to the control, but did not influence growth during the post-metamorphic phase. Overall, several statistically significant findings were found in larvae exposed to TCS, such as a decrease in the prevalence of stage 3 Müllerian ducts in the anterior trunk sections of TCS 25.0 μg l -1 dose group females as compared to controls; most were not considered toxicologically relevant. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

The potential impact of atrazine (ATZ) on gonadal malformations in larval Xenopus laevis has been controversially discussed, and a hypothesis has been generated that ATZ might induce the estrogen-synthesizing enzyme aromatase, leading to feminization or demasculinization. Recently, extensive long-term studies clearly indicate that no adverse effect of ATZ on larval development and sexual differentiation could be found. Therefore, to determine potential transient impacts of ATZ on sexual differentiation processes, short-term exposures were conducted using tadpoles treated for 4 days with ATZ at 25 microg/L. The expression levels of the key players for sexual differentiation in amphibians were determined in the brain, assessing aromatase, 5alpha-reductase type 1 (S1) and type 2 (S2), and the gonadotropins luteinizing hormone and follicle-stimulating hormone, and in the gonads, measuring aromatase, S1, and S2, by means of quantitative RT-PCR. No significant changes in any of these parameters have been found, implicating, in accordance with recent long-term exposures, that no aromatase induction by ATZ could be observed, and it seems likely that no further endocrine mechanism of ATZ affecting sexual differentiation in X. laevis exists.

Traditionally, the Amphibian Metamorphosis Assay (AMA; OECD TG 231) is performed by exposing Xenopus laevis tadpoles to test substances dissolved in laboratory water. Recently, the use of dietary administration has been proposed to combat poorly soluble test substances in ecotoxicologically‐based regulatory endocrine disruption (ED) studies, specifically the AMA warranting an investigation into the efficacy of dietary administration. An efficacy study comprised of two phases: 1) evaluation of the physical influence of the loading process via solvent and 10, 1, and 0.1 mg/l test substance or surrogate (sunflower oil, SFO) on the Sera® Micron Nature (SMN) diet, and 2) performance of a modified AMA in which Nieuwkoop and Faber (NF) stage 51 X. laevis larvae were exposed to dechlorinated tap water using one concentration of the SFO in the diet for 21 days, was performed. In phase 1, the addition of acetone or acetone with bis(2‐propylheptyl) phthalate (DPHP) or SFO to SMN with subsequent solvent purge altered the diet reducing the density of the liquified diet and dietary pellet size following centrifugation indicative of alteration of the physical properties of the diet. Treatments used in the modified AMA were acetone alone and 0.1 mg/l SFO dissolved in acetone. These treatments were evaluated against an SMN benchmark using standard AMA endpoints. Both the acetone‐treated SMN and 0.1 mg/l SFO‐treated diets significantly reduced survival rates, 67 and 70% relative to the SMN benchmark (100%), decreased developmental stage distribution and snout‐vent length‐normalized hind limb length relative to the SMN benchmark, and slightly increased the prevalence and severity of thyroid follicular cell hypertrophy. Although the acetone‐treated diets may have impacted the hypothalamo‐pituitary‐thyroid axis, clinical signs of gastrointestinal impaction and tail flexure were also observed in the acetone‐treated diets, but not the SMN diet alone. Ultimately, test substance exposure via the diet in an AMA study can produce results that may confound data interpretation, which suggests that the traditional aqueous exposure route is generally more appropriate. Dietary administration has been proposed to combat poorly soluble test substances in ecotoxicologically based regulatory endocrine disruption (ED) studies warranting an investigation into the efficacy of dietary administration. Acetone‐treated diets significantly reduced survival rates, decreased developmental stage distribution and snout‐vent length‐normalized hind limb length, and slightly increased the prevalence and severity of thyroid follicular cell hypertrophy. Test substance exposure via the diet in an AMA study can confound data interpretation, and traditional aqueous exposure route is generally more appropriate. [ABSTRACT FROM AUTHOR]

The amphibian metamorphosis assay (AMA) is a key in vivo endocrine screen to investigate chemicals with potential thyroid activity. The test guidelines and associated guidance consider that treatment‐related effects on thyroid gland histomorphology automatically result in the assay being considered positive for thyroid activity, independent of the direction of change or conflicting results in the other biological endpoints. An AMA study was conducted with five different feeding rations equivalent to 50%, 30%, 20%, 10%, and 5% of the recommended feeding rate. Biological endpoints relating to growth and development, including thyroid gland histopathology, were evaluated, and the specificity of these endpoints for the determination of thyroid activity was assessed. There was no effect on survival or clinical signs of toxicity. Effects related to feed reduction generally occurred in a feeding ration–response manner and included reduced development stage; reduced body weight and body length metrics; decreased prevalence of thyroid follicular cell hyperplasia and hypertrophy, and the occurrence of thyroid atrophy; reduced liver vacuolation; and the occurrence of liver atrophy. The results indicate that treatment‐related histopathological changes in the AMA can be induced by Non‐chemical factors; therefore histopathological results are not necessarily diagnostically specific for chemically induced thyroid endocrine activity. Consequently, the interpretation of data from AMA studies should be adjusted accordingly. We recommend that the decision logic presented in the test guidelines and associated guidance be changed to reflect a requirement for directional agreement between the thyroid histopathology findings and the growth and developmental endpoints before it is concluded that a test substance has thyroid endocrine activity. Environ Toxicol Chem 2023;42:1061–1074. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC. [ABSTRACT FROM AUTHOR]

The primary objective of the present study was to examine the influence of early systemic toxicity resulting from copper (Cu) exposure on metamorphic processes in Xenopus laevis. A 28‐day exposure study with copper, initiated at developmental stage 10, was performed using test concentrations of 3.0, 9.0, 27.2, 82.5, and 250 μg Cu/L. The primary endpoints included mortality, developmental stage, embryo‐larval malformation, behavioral effects, hindlimb length (HLL), growth (snout‐vent length [SVL] and wet body weight), and histopathology. The 28‐day LC50 value with 95% confidence intervals was 61.2 (51.4–72.9) μg Cu/L with 250 μg Cu/L resulting in complete lethality. Developmental arrest in the 82.5 and delay in the 27.2 μg Cu/L treatments was observed as early as study day 10 continuing throughout the remainder of exposure. SVL‐normalized HLL, body weight, and SVL in the 27.2 and 82.5 μg Cu/L treatments were significantly decreased relative to control. At 82.5 μg Cu/L, and thyroid gland size was markedly reduced when compared with controls consistent with the stage of developmental and growth arrest. Concentration‐dependent findings in the intestine, liver, gills, eyes, and pharyngeal mucosa were consistent with non‐endocrine systemic toxicity. These were prevalent in the 9.0 and 27.2 μg Cu/L treatment groups but were minimally evident or absent in the 82.5 μg/L group, which was attributed to developmental arrest. In conclusion, developmental delay in larvae exposed to 27.2 and 82.5 μg Cu/L was the result of systemic toxicity occurring in early development prior hypothalomo‐pituitary‐thyroid axis (HPT)‐driven metamorphosis and was not indicative of endocrine disruption. The primary objective of the present study was to examine the influence of early systemic toxicity resulting from copper (Cu) exposure on metamorphic processes in Xenopus laevis. A 28‐day exposure study with copper, initiated at developmental stage 10, was performed using test concentrations of 3.0, 9.0, 27.2, 82.5, and 250 μg Cu/L. Developmental delay in larvae exposed to 27.2 and 82.5 μg Cu/L was the result of systemic toxicity occurring in early development prior hypothalomo‐pituitary‐thyroid axis (HPT)‐driven metamorphosis and was not indicative of endocrine disruption. [ABSTRACT FROM AUTHOR]

The present study evaluated the hypothesis that dietary quality used in historical studies may impact the effects of chemical stressors on premetamorphic development and metamorphosis due to suboptimal nutritional quality. A modified Amphibian Metamorphosis Assay (AMA) was performed in which Nieuwkoop and Faber (NF) Stage 47 tadpoles of Xenopus laevis were exposed for 32 days to iodide (I−)‐deficient FETAX solution supplemented with <0.025, 0.17, 0.52, 1.58, and 4.80 μg I−/L (measured concentrations 0.061, 0.220, 0.614, 1.65, and 4.73 μg I−/L) and fed a pureed Frog Brittle (FB) diet. An AMA guideline benchmark group (four replicates) exposed to dechlorinated tap water and fed standard Sera Micron Nature® (SMN) diet was evaluated concurrently. Developmental delay, observed as changes in stage distribution or median developmental stage, occurred in FB treatments with 0.061, 0.220, and 0.614 μg/L I−, respectively. Developmental rates and hind limb length of the 1.65 and 4.73 μg/L I− groups were similar to each other, but both treatments fell short of the developmental rate achieved by the SMN benchmark. Iodide supplementation also had no impact on nonthyroidal growth endpoints, which were markedly reduced in FB‐fed frogs compared with their SMN‐fed counterparts. All larvae that received the FB diet had mildly to severely hypoplastic/atrophic thyroids, a condition for which iodine supplementation had little if any ameliorative effect. Collectively, these results suggested that nutritional deficiencies in the FB diet negatively affected both growth and metamorphic development, the latter of which was only compensated to a limited extent by iodine supplementation. The present study evaluated the hypothesis that dietary quality used in historical studies may impact the effects of chemical stressors on premetamorphic development and metamorphosis due to suboptimal nutritional quality. A modified AMA was performed in which NF Stage 47 tadpoles of Xenopus laevis were exposed for 32 days to iodide (I−) deficient FETAX solution supplemented with I− at five different concentrations and fed a pureed FB diet. The results suggest that nutritional deficiencies (beyond iodine deficiency) in the FB diet negatively affected both growth and metamorphic development, the latter of which was only compensated to a limited extent by iodine supplementation. [ABSTRACT FROM AUTHOR]

BACKGROUND: The herbicide atrazine has been proposed as a potential endocrine disrupting compound (EDC) for amphibians. Using atrazine concentrations below or at those typically found in surface waters (0.5, 5.0, 50 μg/L), we exposed Acris blanchardi (Blanchard's cricket frog) larvae throughout development until metamorphosis (i.e. Gosner stages 26–45). An additional 50 μg/L treatment (50s μg/L) was utilized where supplemented algae was added to control for indirect atrazine effects from reduced food sources. In addition to atrazine, experimental groups also included a negative control and two positive controls, 17β‐estradiol (E2) at 2.3 and 25 μg/L. At 60 days post‐metamorphosis, A. blanchardi metamorphs were euthanized for analysis of gross and histopathological development. RESULTS: Atrazine did not significantly influence mortality (mean recovery of 54% across treatments), sex ratio, body mass (BM), snout‐vent length (SVL), gonad size, nor gonad development of A. blanchardi. Females exposed to 50s μg/L atrazine had 29% less mass, were 10% shorter, and had a 29% lower mean ovary area (mm2) as compared to negative controls, suggesting algae enrichment had a significant negative effect. Males exposed to estradiol (25 μg/L) showed an increased level of oviduct development. Ovary area was also significantly influenced by estradiol treatment at 2.3 and 25 μg/L. CONCLUSION: Overall, estradiol had much less effect than predicted based on other model species (e.g. Xenopus laevis). Development of A. blanchardi, overall, was not affected by long‐term exposure to environmentally relevant concentrations of atrazine. However, this species also was largely insensitive to exogenous estradiol in this test system. © 2022 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

The Amphibian Metamorphosis Assay (AMA) is used to identify substances that potentially interfere with the normal function of the hypothalamic-pituitary-thyroid (HPT) axis. Although numerous AMA studies have been performed since the establishment of this assay a decade earlier, a comprehensive, large-scale examination of histopathology data obtained from control larvae has not been performed. The current investigation reviewed 51 AMA experiments conducted at 7 different laboratories in Europe and North America. Dilution water control and/or solvent control specimens from each study (1,335 animals total) had been evaluated microscopically by one of eight anatomic pathologists. In order of descending frequency, the most common findings in prometamorphic Xenopus laevis controls were the core criteria of follicular cell (FC) hypertrophy, FC hyperplasia, thyroid hypertrophy, and thyroid atrophy, respectively. Less frequently recorded were non-core and ad hoc diagnoses, the toxicological relevance and utility of which were in some cases uncertain. As anticipated, the prevalence of FC hypertrophy and FC hyperplasia diagnoses were at least partially dependent on the Nieuwkoop and Faber (NF) stage at sacrifice. The recorded frequencies of each of the four core diagnoses also differed according to pathologist, which suggests that pathologist diagnostic interpretation is a potential source of variability across AMA study outcomes. Based on the current examination of the AMA historical data, and further hands-on experience with this assay, diagnostic approaches to evaluating the histopathology endpoint are discussed, and several recommendations are proposed for the refinement of core diagnostic criteria assessment. [ABSTRACT FROM AUTHOR]

• The Amphibian Metamorphosis Assay (AMA) and Fish Short Term Reproduction Assay (FSTRA) are standardized regulatory tests used to determine if a chemical might affect the hypothalamic-pituitary-thyroid (HPT) axis or hypothalamic-pituitary-gonadal (HPG) axis, respectively. • Histopathology historical control data were assembled and evaluated from 55 AMA and 45 fathead minnow FSTRA studies. • Historical control data were found useful for investigating the existence of outlier results and aberrant control groups, identifying potential confounding variables, providing context for rare diagnoses, differentiating endocrine from non-endocrine effects, identifying background lesions, and helping to determine if specific improvements to established test guidance may be warranted. The Amphibian Metamorphosis Assay (AMA) is used to determine if a tested chemical has potential to impact the hypothalamic-pituitary-thyroid (HPT) axis of Xenopus laevis tadpoles, while the Fish Short Term Reproduction Assay (FSTRA) assesses potential effects on the hypothalamic-pituitary-gonadal (HPG) axis of fish such as the fathead minnow (Pimephales promelas). Several global regulatory programs routinely require these internationally validated tests be performed to determine the potential endocrine activity of chemicals. As such, they are conducted in accordance with standardized protocols and test criteria, which were originally developed more than a decade ago. Sizeable numbers of AMA and FSTRA studies have since been carried out, which allows for the mining of extensive historical control data (HCD). Such data are useful for investigating the existence of outlier results and aberrant control groups, identifying potential confounding variables, providing context for rare diagnoses, discriminating target from non-target effects, and for refining current testing paradigms. The present paper provides histopathology HCD from 55 AMA studies and 45 fathead minnow FSTRA studies, so that these data may become publicly available and thus aid in the interpretation of future study outcomes. Histopathology is a key endpoint in these assays, in which it is considered to be one of the most sensitive indicators of endocrine perturbation. In the current review, granular explorations of HCD data were used to identify background lesions, to assess the utility of particular diagnostic findings for distinguishing endocrine from non-endocrine effects, and to help determine if specific improvements to established regulatory guidance may be warranted. Knowledge gleaned from this investigation, supplemented by information from other recent studies, provided further context for the interpretation of AMA and FSTRA histopathology results. We recommend HCDs for the AMA and FSTRA be maintained to support the interpretation of study results. [ABSTRACT FROM AUTHOR]

The natural estrogen 17 β-estradiol (E2) is a potential environmental contaminant commonly employed as a positive control substance in bioassays involving estrogenic effects. The aquatic anuran Xenopus laevis is a frequent subject of reproductive endocrine disruptor research; however, histopathological investigations have tended to be less than comprehensive. Consequently, a study was designed to characterize gross and microscopic changes in the gonads of X. laevis as a result of E2 exposure. Additional goals of this study, which consisted of three separate experiments, included the standardization of diagnostic terminology and criteria, the validation of statistical methodology, and the establishment of a half maximal effective concentration (EC50) for E2 as defined by an approximately 50% conversion of presumptive genotypic males to phenotypic females. In the first experiment, frogs were exposed to nominal concentrations of 0, 0.2, 1.5, or 6.0 µg/L E2. From these experimental results and those of a subsequent range finding trial, the EC50 for E2 was determined to be approximately 0.2 µg/L. This E2 concentration was utilized in the other two experiments, which were performed at different facilities to confirm the reproducibility of results. Experiments were conducted according to Good Laboratory Practice guidelines, and the histopathologic evaluations were peer reviewed by an independent pathologist. Among the three trials, the histopathological findings that were strongly associated with E2-exposure (p < 0.001 to 0.0001) included an increase in the proportion of phenotypic females, mixed sex, dilated testis tubules, dividing gonocytes in the testis, and dilated ovarian cavities in phenotypic ovaries. A comparison of the gross and microscopic evaluations suggested that some morphologic changes in the gonads may potentially be missed if studies rely entirely on macroscopic assessment. Environ. Toxicol. Chem. 2010;29:1091–1105. © 2010 SETAC [ABSTRACT FROM AUTHOR]

2-Ethylhexyl 4-hydroxybenzoate (2-EHHB), 4-tert-octylphenol (4-OP), 4-nonylphenol-branched (4-NP), benzyl butyl phthalate (BBP) and dibutyl phthalate (DBP) were evaluated using a 21-day Amphibian Metamorphosis Assay (AMA). Xenopus laevis larvae were exposed nominally to each chemical at 3.6, 10.9, 33.0, and 100 μg/L, except 4-NP concentrations were 1.8, 5.5, 16.5 and 50 μg/L. Endpoints included mortality, developmental stage, hind limb length (HLL), snout-vent length (SVL), body weight (BW), and thyroid histopathology. BBP and 4-OP accelerated development compared to controls at the mean measured concentration of 3.5 and 39.8 μg/L, respectively. An increase in developmental stage frequency distribution was observed for 4-OP at 39.8 and 103 µg/L, BBP at all concentrations and DBP at 143 µg/L. Normalized HLL was increased on study day (SD) 21 for all tested substances except 4-NP. Histopathology revealed accelerated development and mild thyroid follicular cell hypertrophy at all BBP concentrations, but moderate severity at 105 μg/L. Increased BW occurred for all chemicals except 4-OP. Increased SVL was observed for 4-NP, BBP and DBP on SD 21. There was insufficient evidence that 4-NP and 2-EHHB affected the hypothalamic-pituitary thyroid axis, however, BBP, DBP and 4-OP showed potential effects on amphibian metamorphosis and thyroid activity, albeit through different lines of evidence. • BBP accelerated development, increased HLL, normalized HLL, SVL and BW compared to the control. • DBP increased developmental stage distribution, HLL, normalized HLL, SVL and BW compared to the control. • The prevalence of follicular cell hypertrophy was found to be increased relative to controls for both BBP and DBP. • The study displays the capability of AMA to distinguish potential adverse effects on the HPT axis from non-thyroid effects. [ABSTRACT FROM AUTHOR]