Abstract
We propose a modified Comparative Thyroid Assay (CTA, USEPA) utilizing a smaller number of Sprague-Dawley rats (N=10/group) that assesses brain thyroid hormone (TH) concentrations and periventricular heterotopia while maintaining assay sensitivity. Our recent findings demonstrated that a prenatal test cohort of the modified CTA detected a dose-dependent decrease in maternal serum T3 (up to -26%) and T4 (up to -44%) with sodium phenobarbital (NaPB) exposure at 1000 ppm and 1500 ppm, equivalent to intakes of 60 and 84 mg/kg/day, respectively. On gestation day (GD) 20, fetuses exhibited reduced serum (-26%) and brain (-29%) TH concentrations, although these reductions were not dose dependent. The present study expanded the treatment in a postnatal test cohort, with maternal exposure to NaPB (81-93 mg/kg/day) from GD6 to lactation day (LD) 21. We assessed serum and brain TH concentrations, and periventricular heterotopia in pups on postnatal days (PND) 4, 21, and 28. While LD21 dams showed significant reductions in serum T3 (up to -34%) and T4 (up to -54%), the pups did not exhibit significant TH suppression or periventricular heterotopia at any test point. Instead, a compensatory increase in T4 was observed in serum and brain of PND21 pups. The present study confirmed that perinatal maternal exposure to high doses of NaPB leads to a moderate decrease in maternal TH concentrations; however, the exposure of maternal rats to a similar dose of NaPB did not significantly reduce serum or brain TH concentrations in their postnatal offspring.
INTRODUCTION
Thyroid hormone (TH) is essential for normal human brain development, both before and after birth (Bernal, 2017; Zoeller and Crofton, 2005). Therefore, environmental chemicals that interfere with thyroid function have the potential to cause TH insufficiency to such an extent that adverse effects on the offspring may result. Considering the large number of environmental chemicals known to interfere with thyroid function, it is important to establish an approach to evaluate the potential of these chemicals to produce neurotoxic effects through the reduction of circulating levels of TH (Crofton, 2008; Miller et al., 2009; Murk et al., 2013; Noyes et al., 2019). A developmental insult in TH systems of fetuses and/or neonatal pups can initiate a cascade of alterations which may not be detected structurally or functionally until later in life (Howdeshell, 2002; Zoeller and Rovet, 2004). Therefore, the detection of fetal and/or neonatal TH disruption is of great importance in assessing TH-mediated developmental neurotoxicity (DNT) of chemicals. Standardized studies designed to identify DNT are costly, time-consuming, and require the use of large numbers of animals. As such, they are not practical for screening a wide variety of chemicals (Coecke et al., 2007; Crofton et al., 2012, 2022; Lein et al., 2007). The comparative thyroid assay (CTA) is designed to determine whether the potentially sensitive life stages for TH disruption are adequately protected by the points-of-departure used for hazard assessments, and not for further elucidation of endocrine and/or neurodevelopmental hazard (Marty et al., 2021; USEPA, 2005).
The CTA consists of two cohorts: a prenatal test cohort treated from gestation day (GD)6 to GD20, and a postnatal test cohort treated from GD6 to lactation day (LD)21 in maternal rats (USEPA, 2005). Since rat central nervous system embryogenesis begins with formation of the neural tube around GD8–9 (Rice and Barone, 2000), the CTA encompasses this critical period. Development of the early central nervous system in utero is reliant on TH transported from the mother, as many thyroid-dependent processes in fetuses commence in late gestation (de Escobar et al., 1989; Gilbert et al., 2012; Howdeshell, 2002). Furthermore, since the development of thyroid-dependent processes continues into the first few weeks of postnatal life in rats, assessing TH status during neonatal period is also crucial. Historically, serum THs and thyroid weight/histopathology have been the usual endpoints used in the regulatory setting (Gilbert et al., 2020; USEPA, 2005). Although serum T4 is the primary source for brain THs (de Escobar et al., 1989; Landers and Richard, 2017), circulating concentrations may not be representative for the local TH changes in the brain (Reyns et al., 2003). Recently, reliance on peripheral THs alone has been suggested to be insufficient predictive markers of adverse neurodevelopment in the brain (Gilbert et al., 2020, 2021, 2024; O’Shaughnessy and Gilbert, 2020), and thus may under- or over-estimate adverse points of departure in hazard assessments. We recently proposed adding examination of brain TH concentration to the CTA to assess TH disruption at the target site (i.e., brain) (Minami et al., 2023, 2024).
Many contract research organizations (CROs) may have limited experience in measuring serum and in particular brain TH concentrations in fetuses and newborns, as such assessments have not been commonly requested in numerous toxicology studies (Li et al., 2019; Marty et al., 2021; OECD, 2018). This lack of experience could raise concerns regarding the ability to accurately evaluate such data. Therefore, to ensure precise evaluation of TH data, we suggest that CROs incorporate additional reliable measures. Specifically, we recommend the inclusion of an assessment for periventricular heterotopia in the CTA (Minami et al., 2023; Ogata et al., 2024), as this finding has been suggested as a direct brain-based readout of TH insufficiency, which could be applicable in regulatory contexts (European Commission, 2019; Gilbert et al., 2020, 2022, 2023; Hassan et al., 2017; Kortenkamp et al., 2020; O’Shaughnessy and Gilbert, 2020).
Periventricular heterotopia results from the inhibition of neuronal migration due to perinatal TH insufficiency (Gilbert et al., 2014, 2023; Goodman and Gilbert, 2007; Minami et al., 2023; O’Shaughnessy et al., 2018, 2019; Ogata et al., 2024). The critical time window for heterotopia formation in rats is known to be from GD19 to LD2 (O’Shaughnessy et al., 2019). The postnatal cohort of CTA encompasses this critical period, as the maternal dosing extends from GD 6 through PND21 (USEPA, 2005). Furthermore, periventricular heterotopia can be detected at a relatively early developmental stage after birth (i.e., from PND14 onwards) (Gilbert et al., 2014, 2023; Goodman and Gilbert, 2007; O’Shaughnessy et al., 2018, 2019). Therefore, periventricular heterotopia can be examined on PND21 without altering the standard CTA protocol (Minami et al., 2023; Ogata et al., 2024).
Recently, the hazard assessment of chemicals has been undergoing a paradigm shift with new approach methodologies (NAMs). According to the OECD definition, NAMs include animal tests that contribute to the reduction or refinement of other animal tests, encompassing any mechanistic and sophisticated approach (OECD, 2020). The original CTA requires a significant number of animals (20 pregnant rats per dose group per cohort, totaling 160 pregnant rats for a study with 4 dose groups) (USEPA, 2005). Therefore, we propose a modified CTA using a smaller number of rats (10 maternal rats per dose group per cohort) by incorporating parameters that are considered more reliable than blood TH concentrations and are closer to the manifestation of adverse outcome, namely the measurement of brain TH concentrations and brain histological evaluations (Minami et al., 2023). Consequently, this protocol has a statistical power of over 95% for detecting a 25% decrease in serum T4, and brain T3 and T4 concentrations in GD20 fetuses (Minami et al., 2024).
The adverse outcome pathway (AOP) network for chemically induced thyroid activity indicates the integration of multiple individual AOPs that are either under development or proposed. The network includes molecular initiation events such as hypothalamic-pituitary feedback, TH synthesis in the thyroid, xenobiotic receptor activation in the liver, serum TH transport, peripheral TH metabolism, cellular TH transport, TH receptor transactivation (reviewed by Noyes et al., 2019). The USEPA CTA guidance recommends 6-propylthiouracil (6-PTU), a potent inhibitor of TH synthesis, as a positive control (USEPA, 2005). However, it is unlikely that all environmental chemicals disrupt THs as effectively as 6-PTU. The impact of altered THs on neurodevelopment is often a secondary consequence of a chemical’s primary action, which occurs outside of the thyroid. Furthermore, many such chemicals may cause only mild to moderate disruption of TH (Crofton, 2008; Gilbert et al., 2012, 2020).
The xenobiotic hepatic receptor activator phenobarbital (PB or its salt, NaPB) has been shown to cause a dose-dependent increase in the activity of hepatic uridine diphosphate-glucuronosyltransferases (UDPGTs), which are specific to T4, through the activation of the constitutive androstane receptor (CAR). This, in turn, leads to a dose-dependent decrease in serum TH concentrations in rats or mice (Barter and Klaassen, 1994; Capen, 1997; Haines et al., 2019; Hood et al., 1999b; Liu et al., 1995; McClain et al., 1989; Noyes et al., 2019; O’Connor et al., 1999). Consequently, PB is recognized as a prototypical inducer of hepatic UDPGTs and is also known as a mild to moderate TH disruptor in adult rats. Similar to PB, numerous chemicals, including agrochemicals, have been reported to cause mild to moderate TH disruption in adult rats by upregulating hepatic UDPGT activity (Bartsch et al., 2018; Crofton, 2008; Dellarco et al., 2006; Finch et al., 2006; Foster et al., 2021). While it cannot be denied that these chemicals may reduce blood TH concentrations in humans, species-specific differences in the blood dynamics of TH and the reactivity of the hypothalamus-pituitary-thyroid axis have been clearly identified, and thus, regulatory authorities also consider that enzyme-induced thyroid carcinogenicity in rats is not relevant to humans (Bartsch et al., 2018; Crofton, 2008; Dellarco et al., 2006; Finch et al., 2006; Foster et al., 2021).
As depicted in Fig. 1, the AOP summarizes the predicted process by which maternal exposure (Key Event 1) to NaPB can lead to potential DNT in the rat offspring (Adverse Outcome). Through the activation of CAR (Key Event 2), NaPB causes an increase in the activity of hepatic UDPGTs (Key Event 3). As in all current proposed AOPs examining developmental TH disruption, a decrease in serum T3/T4 concentrations in dams and their offspring (Key Event 4) is a critical key event preceding reduced TH action in the offspring brain (Key Event 5). However, the degree of serum T3/T4 reduction (Key Event 4) that is harmful remains unclear, and there may be maternal exposures that do not significantly decrease serum TH levels (Key Event 4) yet still affect hormone concentrations in the offspring’s brain (Key Event 5). We propose that further evaluation of a downstream key event in the CTA, reflective of TH function in the offspring’s brain, could be utilized as a targeted testing strategy to support the regulatory assessment of chemical TH disruption (Minami et al., 2023, 2024; Ogata et al., 2024).
In our recent study, we investigated the effects of NaPB (1000 and 1500 ppm; corresponds to the maximum tolerated dose) to assess the sensitivity of the modified CTA’s prenatal test cohort to TH disruption caused by hepatic UDPGT induction (Minami et al., 2024). The modified CTA successfully detected reductions in maternal serum T3 (up to -26%) and T4 (up to -44%) at these NaPB concentrations on GD20. Similar suppressions in serum T3 and T4 (up to -26%), as well as decreases in brain T3 (up to -18%) and T4 (up to -29%), were observed in GD20 fetuses. The study confirmed that the modified CTA protocol is capable of detecting mild to moderate TH disruptions in pregnant dams and their GD20 fetuses (Minami et al., 2024).
In addition to the prenatal phase, the postnatal test cohort in the CTA is also crucial for determining whether continued chemical exposure in maternal rats after the birth of their pups leads to early postnatal (i.e., pre-weaning) TH disruption in pups with fully functioning thyroids (Howdeshell, 2002). In the current study, the postnatal test cohort of the modified CTA (dosed from GD6 to LD21) aimed to investigate potential postnatal TH disruption caused by NaPB in SD rats. Modifications to the CTA protocol included: 1) a 50% reduction in the group size (from N=20 to N=10 rats); and 2) the inclusion of additional endpoints, such as measurements of brain TH concentrations and qualitative assessments of brain histopathology in the offspring. Furthermore, the acceptability of 3) pooling blood samples per littermates and 4) assessing combined data from both male and female animals for heterotopia formation is also discussed in this paper.
MATERIALS AND METHODS
Test chemicals
Phenobarbital sodium salt (NaPB; CAS# 57-30-7, Lot No.KCP6847; purity >98%; FUJIFILM Wako Pure Chemical Co, Ltd., Osaka, Japan) was used.
Animals, husbandry, and mating
This study was conducted in the Institute of Environmental Toxicology (IET), which was the same facility as the previous studies (Minami et al., 2023, 2024; Ogata et al., 2024), fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International) in accordance with the Animal Care and Use Program of IET (IET IACUC Approval No. AC21019). Briefly, Specific Pathogen-Free (SPF) Crl:CD(SD) rats of both sexes were purchased from Atsugi Breeding Center, Charles River Laboratories Japan, Inc. (currently Jackson Laboratory Japan, Kanagawa) at 12 weeks of age. After quarantine and acclimatization for 10 to 14 days, males and females were paired at 13–14 weeks of age. Animals were housed in a barrier-sustained animal room with targeted controlled temperature (22 ± 2°C), humidity (50 ± 20%), ventilation (at least 10 times per hour; all fresh air system), and illumination (12 hr per day, lights on at 7:00 a.m. and off at 7:00 p.m.) throughout the study period. A commercially available solid or pulverized diet (MF or MF Mash diet, Oriental Yeast Co., Ltd., Tokyo) and local tap (chlorinated) water were provided ad libitum throughout the study. The animals were not fasted overnight prior to sacrifice. Copulated females were prepared by standard reproductive study methods as previously; the day on which vaginal plugs and/or sperm were observed was designated as GD0 (Minami et al., 2023; Ogata et al., 2024).
Study design
The present study was conducted with modifications of the CTA protocol designated by the USEPA guidance (USEPA, 2005). In the original CTA, both prenatal test cohort and postnatal test cohort are designated. Since another study reports the data from the prenatal test cohort with NaPB at 1000 and 1500 ppm (Minami et al., 2024), the present work utilized only the postnatal test cohort (Fig. 2). In a previous study using adult rats (Hood et al., 1999a), significant hepatic enzyme up-regulation and mild but significant TH reductions were reported at 1200 ppm PB (T3 and T4 concentrations decreased by 16˗38% and by 18˗28%, respectively). Based on these findings, the concentration of 1000 ppm NaPB (based on the salt in diet) was selected as a reference chemical for mild maternal TH disruption in our previous 1st CTA study (Minami et al., 2023). The modified CTA detected 1000 ppm NaPB (equivalent to intakes of 84 mg/kg/day)-induced suppression of serum THs in dams (T3 and T4 concentrations decreased by 24% and 16%, respectively), with less than -35% reduction of serum/brain TH levels in GD20 fetuses but not in pups (Minami et al., 2023). Since the magnitude of the reduction of THs in GD20 dams treated with 1000 ppm NaPB was less than our expectation, no suppression of brain TH concentrations in pups may be due to lower NaPB dosing in maternal rats. In the prenatal test cohort of the 2nd CTA study (Minami et al., 2024), to better assess the sensitivity of the modified CTA to mild or moderate TH disruption via liver enzyme induction, the effects of a somewhat higher dose of NaPB (1500 ppm) and the same dose of the previous study (1000 ppm, Minami et al., 2023) were investigated. Consequently, the study detected a dose-related decrease in maternal serum T3 (up to -26%) and T4 (up to -44%) induced by NaPB at dietary dosing of 1000 ppm and 1500 ppm in GD20 dams (equivalent to intakes of 60 and 84 mg/kg/day), and also decreases of serum T3 (up to -26%) and T4 (up to -26%), and brain T3 (up to -18%) and T4 (up to -29%), but without dose-dependency, in GD20 fetuses (Minami et al., 2024). These findings suggest that gestational treatment with 1000 ppm NaPB (equivalent to intakes of 60 mg/kg/day) can induce the maximum effect of NaPB on fetal TH status (Minami et al., 2024) .
In the present study, based on background information described above, NaPB was administered to pregnant rats, 10 females/dose group, at 0, 1000 or 1500 ppm based on the salt in diet, from GD6 through GD20 (Fig. 2). The day of parturition completion was designated as LD0 for dams (equivalent to PND0 for offspring). Since continued treatment with 1000 ppm NaPB at birth can induce many neonatal deaths, dosing at birth was required to be stopped (GD20-LD0; i.e., 2 or 3 days), as in the previous study (Minami et al., 2023). In the previous 1st CTA study, the dose level of 1000 ppm NaPB group was reduced to 500 ppm from LD13 to 21 to keep the test substance intake (mg/kg/day) constant during the entire study period and to avoid maternal excessive toxicity due to an excessive substance intake during later lactation (Minami et al., 2023). Thus, the dose level of the 1000 ppm group in the present study was reduced the same as in the previous 1st CTA study. In terms of the 1500 ppm group, the dose level was reduced to 1000 ppm after birth and continued to the end of treatment (LD21) to examine the possible effects as much as possible.
Furthermore, for determining persistence/transience of the brain abnormalities of TH concentrations and/or morphologies on PND28, which was one week after the termination of the treatment on LD21, some selected weanlings were housed in each group with the same conditions described above without NaPB treatment, and serum/brain TH concentrations and brain histopathology of these weanlings were assessed on PND28.
On PND4, the size of each litter was standardized by random pup selection to 8 pups, four males and four females, if possible, with attention to avoid eliminating runts only. Mortality, clinical signs, body weights and food consumption of dams–which may include data from pups during the later periods–were periodically monitored throughout the study. Pup mortality, clinical signs, and body weights were also periodically monitored. Cage-side observations were made of rats once daily for clinical signs and mortality. Each rat was also observed in more detail for the presence of abnormalities such as excitement, convulsion, sedation, and abnormal gait when it was weighed instead of cage-side observation. Maternal rats were examined for the status of pregnancy. All abnormalities observed were recorded for individual rats. Data from common control animals were shared with a concurrent 6-PTU study to determine the optimal timing for assessing heterotopia in the modified CTA (Ogata et al., 2024).
Tissue collection
Regarding the examination items in the present study, the implications in the AOP and the timing of testing are summarized in Figs. 1 and 2. Tissue collection from dams was conducted as with the prenatal test cohort (Minami et al., 2024). Briefly, to minimize the impact of circadian changes in hormone, samples were collected from the animals in all three groups at similar times of day and in an order of a control dam, a 1000 ppm dam, a 1500 ppm dam, followed by a control dam, a 1000 ppm dam, a 1500 ppm dam and so on, as in the previous prenatal test cohort study. Maternal rats were bled from the posterior vena cava using plain syringes (19G needle) under isoflurane anesthesia and euthanized on LD21. The blood was allowed to clot and centrifuged at 2,000 × g for 10 min. at 4°C to separate sera for hormone analyses. Blood sampling was generally performed in the afternoon (13:00-16:00) to ensure practical flexibility in conducting studies as in previous studies (Minami et al., 2023, 2024; Ogata et al., 2024). Dams were held in a curtained-off holding area adjacent to the necropsy room to avoid effects of any stress on hormone levels. In dams of the NaPB-groups, plasma for measurement of blood PB concentrations was additionally prepared from approximately 2 mL of collected blood from the posterior vena cava. The sera and plasma were stored in a freezer (-70°C or below) until use. Dams were subjected to necropsy and gross pathology after blood collection. The thyroid and liver of the dams were weighed and fixed in 10% neutral-buffered formalin for histopathology. Portions of the left lateral lobe of the liver were also collected for UDPGT activity measurements and mRNA analyses prior to fixation.
Extra pups culled on PND4 were anesthetized by an intraperitoneal injection of thiamylal sodium solution. Blood was then collected from 1 or 2 pups/sex/litter when available, in the same manner as for the fetuses in the prenatal test cohort, and pooled by sex and litter (Minami et al., 2024), as suggested by USEPA CTA guidance (USEPA, 2005). The brains were also sampled from the same pups used for blood sampling, snap frozen and stored for brain hormone analyses. In addition, the brain, thyroid, and liver were collected from extra pups, at least one male and one female in each litter when available, for histopathology. The thyroid of PND4 pups was dissected in the same manner as the fetuses (Minami et al., 2024). Liver samples (left lateral lobe) were also collected from pups on PND4 for mRNA analyses.
For pups on PND21 and PND28, blood samples were collected from a set of pups, 1/sex/litter, by the same method as that for dams. The brain and liver (left lateral lobe) were also collected from the same animals, snap frozen and stored for brain hormone analyses and UDPGT activity measurements, respectively. In addition, a piece of liver was excised from the left lateral lobe of the selected pups for mRNA analyses. Another set of pups, 1/sex/litter, was sacrificed by exsanguination under isoflurane anesthesia for the collection of histopathology samples: brain and liver (fixed using the same methods as for the fetal tissues in the prenatal cohort) (Minami et al., 2024).
Thyroid hormone analyses
Concentrations of T3 and T4 in serum and brain were analyzed as previously (Minami et al., 2024; Ogata et al., 2024). Briefly, the frozen brains of pups (1/sex/litter) were individually weighed and homogenized with the same weight of water for injection as the brain sample. Serum samples collected from dams and pups and the homogenized brain samples of pups were analyzed for the concentrations of T3 and T4 by liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS) analysis. An aliquot (50 μL) of serum or brain homogenate was placed into a test tube, added with 50 μL of water for injection and 300 μL of internal standard solution (methanol solution containing [13C6]T3 and [13C6]T4). After mixing, the mixture solution was centrifuged (16,000×g, 5 min, 10°C). Then, 200 μL of 0.1% formic acid was added to the solution, and the mixture solution was centrifuged (16,000×g, 5 min, 10°C). An aliquot of the obtained supernatant was purified by a MonoSpin (MonoSpin Phospholipid, GL Sciences Inc., Tokyo). The eluate was then injected into the On-Line SPE LC-MS/MS (LC: 1290 HPLC, Agilent Technologies, Santa Clara, California; MS/MS: 6470 Triple Quad LC/MS, Agilent Technologies; SPE column: Shim-pack MAYI-ODS (G), 2.0 mm × 10 mm, Shimadzu Corporation, Japan; LC column: ZORBAX Eclipse C18, 1.8 μm × 2.1 mm × 50 mm, Agilent Technologies). The limits of quantification (LOQ) for serum T3 and T4 were 0.010 and 0.2 ng/mL, respectively, and the limits of detection (LOD) for serum T3 and T4 were 0.005 and 0.1 ng/mL respectively. The LOQ for brain T3 and T4 were 0.02 and 0.20 ng/g brain weight, respectively, and the LOD for brain T3 and T4 were 0.01 and 0.10 ng/g brain weight, respectively. Serum T3 concentrations in one male and female fetus in the NaPB 1500 ppm group were below the LOQ value (0.010 ng/mL), and thus group mean values for the NaPB 1500 ppm group were calculated as 0.010 ng/mL as individual data for these animals.
Serum thyroid stimulating hormone (TSH) analyses
Serum TSH concentration was measured by Immuno-beads assay using Milliplex Map Rat Thyroid Hormone TSH Panel (EMD Millipore, Burlington, Massachusetts) as previously (Minami et al., 2024; Ogata et al., 2024). Each serum sample was incubated with beads and detection antibody according to the kit manual. The fluorescence intensity of each sample was analyzed after gating of immune-bead population on Forward Scatter Area and Side Scatter Area using a flow cytometer (FACSVerse, BD, Tokyo) with the FACSuite program. The LOQ for TSH was 62.5 pg/mL and there were no cases below the LOQ value in this study. Regarding the intra-assay precision of the TSH immuno-beads assay in the analytical validation, coefficients of variation (CVs) ranged from 3.0–13.6% and from 4.3–9.1% for 8 samples each for males and females, respectively.
Measurement of liver UDPGT activity, liver Ugt2b1 and Cyp2b1/2 mRNA expression levels, and plasma phenobarbital concentrations
Increased inductions of both Ugt2b1 mRNA and UDPGT (one of the enzymes responsible for UDPGT activity is translated from Ugt2b1 mRNA) were previously observed following NaPB treatment (Minami et al., 2023, 2024). These measurements were conducted by standardized methods as previously described. For each microsomal suspension derived from the frozen liver samples of the maternal rats, microsomal protein content was determined by DC Protein Assay Kit (Nippon Bio-Rad Laboratories, Tokyo) according to the augmented method of Lowry. Total RNA in the liver was extracted from liver from dams and pups using RNeasy Mini kit (Qiagen, Hilden, Germany) in accordance with manufacturer’s instructions. The forward and reverse primer and probe sets for quantitative real-time PCR are shown in Supplemental Table 1. The mRNA measurement was conducted by Sumika Technoservice Corp. (Takarazuka, Japan). Concentrations of phenobarbital were determined in maternal plasma by LC-MS/MS analysis.
Histopathology
Liver and thyroid glands from the dams, and brain and liver from the offspring were processed and subjected to histopathological examination. For offspring brain, the cerebrum was cut coronally at the levels anterior and caudal to the infundibulum, as vertically as possible, to make two paraffin blocks. Cerebellum and brainstem were cut in the mid-sagittal plane as vertically as possible to make another paraffin block. After trimming, the brains, liver, and thyroids were embedded in paraffin, sectioned at 3 μm, stained with hematoxylin and eosin (H&E), and examined by light microscopy.
The three blocks of the brain prepared as described above were used and carefully thinned to obtain the three levels of planes (the first level included periventricular cortex and caudate/putamen, the second level included periventricular cortex, hippocampus, thalamus, and hypothalamus, and the third level included cerebellum and brain stem along the mid-sagittal plane) homogeneously throughout all animals (Garman et al., 2016) (Supplemental Fig. 1). Histopathological examination of the brain for offspring was conducted at the three levels to evaluate for any qualitative changes. In addition to these three levels of examination, for PND4, PND21, and PND28 pups, “step sections” of 3 µm thickness were made every third section in the mid-region of the brain including the hippocampus using the posterior block of the cerebrum. The method for analyzing heterotopia in the present study was identical to that used in a concurrent 10 ppm 6-PTU study, which successfully detected significant heterotopia formation in the modified CTA (Ogata et al., 2024).
Although there is a theoretical concern that NaPB may have direct effects on brain tissue as a barbiturate antiepileptic drug (Dingemanse et al., 1989; Lumley et al., 2021), our study aimed to examine whether maternal NaPB exposure reduces TH concentrations in the offspring’s brain. As shown in the prenatal test cohort study (Minami et al., 2024), the fetal TH reductions were similarly observed at 1000 and 1500 ppm (approximately -18% for T3 and -29% for T4). In addition, there were no significant reductions of THs in serum and brain of pups, as described in the Results Section of the present study. Thus, histopathological examination of the brain was conducted in the 1000 ppm group to eliminate the possible NaPB’ pharmacological effects as much as possible. Left lateral lobe and right medial lobes of the liver (dams and offspring) and both lobes of the thyroid (dams) were processed and subjected to light microscopic examination. The effects of NaPB on these organs are already well-known (Capen, 1997; Hood et al., 1999b), and given that pathological examinations of these organs served as supplementary data to understand the impact on TH, they were conducted only in the 1000 ppm group that also underwent brain pathology assessments, in an effort to conserve resources.
Statistical analyses
The following statistical tests were used to evaluate significance of differences between the control and each NaPB group. The data sets, including body weight, food consumption, organ weight, UDPGT activity and mRNA expression in maternal females; body weight and UDPGT activity and mRNA expression in offspring, were first evaluated for homogeneity of variance by Bartlett’s test (α=0.05). When group variances were homogenous, a parametric one-way ANOVA in one-way classifications (α=0.05) was used to determine if there was a main effect of treatment. When the ANOVA was significant, Dunnett’s multiple comparison test (α=0.05 or 0.01) was performed to detect a statistically significant difference between each treated group and their corresponding controls. When Bartlett’s test indicated that the variances were not homogeneous, the Kruskal-Wallis test (α=0.05 or 0.01) was used for test for a main effect of treatment, and when significant, Dunnett-type nonparametric multiple comparison (α=0.05 or 0.01) was performed to detect statistical differences between each treated group and their corresponding controls. The litter was the statistical unit for the above analyses.
Serum hormone data in dams were also analyzed in the same manner as the other parametric data (e.g., body weight) with Dunnett’s multiple comparison test. Regarding serum and brain hormone data in fetuses, a two-way ANOVA was used to analyze for main effects of treatment and sex, and for a treatment-by-sex interaction. When there were significant effect of treatment and no treatment-by-sex interaction, Dunnett’s multiple comparison test (α=0.05 or 0.01) was performed to detect a statistically significant difference between each treated group and their corresponding controls in the combined data of males and females. Fisher’s exact probability test or Wilcoxon-Mann-Whitney test (α=0.05 or 0.01) was also used for the incidences of pathological findings.
RESULTS
Group mean values or incidence of each examination in dams and offspring are summarized in Supplemental Tables 2-5, respectively.
Key Event 1: NaPB intake and plasma PB concentrations in dams
The group mean NaPB intake, estimated from food intake data (shown in Fig. 3A), is presented in Fig. 3B. Food consumption in the NaPB 1000 ppm group was slightly higher than that of the controls during the late gestation period, while it was generally lower during the lactation period. In the 1500 ppm group, food consumption was lower than that of the controls during the early treatment period (GD6-GD12) and throughout the lactation period. NaPB intake was generally stable during the gestation period, reflecting the stability in food consumption. The average NaPB intakes for the 1000 and 1500 ppm groups during GD6-GD20 were 69 and 83 mg/kg/day, respectively, which were consistent with the results from the previous prenatal test cohort study (60 and 84 mg/kg/day, respectively) (Minami et al., 2024).
The NaPB intake during the lactation period gradually increased from LD0 to LD10, as the dams consumed more food relative to their body weight, presumably to meet the increasing demands for milk production as the pups grew. The decrease in NaPB intake in the 1000 ppm group after PND13 was due to the reduction of the concentration in the test material to 500 ppm. The average NaPB intakes for the 1000 ppm and 1500 ppm groups during LD0-LD21 were 92 mg/kg/day and 101 mg/kg/day, respectively, which were slightly higher than those during the gestation period by 33% and 22%, respectively. The overall average NaPB intakes for the 1000 ppm and 1500 ppm groups were 81 mg/kg/day and 93 mg/kg/day, respectively. Compared with the 1000 ppm group, the NaPB intake during gestation and lactation in the 1500 ppm group increased by approximately 20% and 10%, respectively.
Plasma PB concentrations in LD21 dams from the 1000 ppm and 1500 ppm groups were 63.2 ± 6.5 mg/L and 124 ± 18.7 mg/L, respectively. Therefore, the plasma PB concentration in the 1500 ppm group was approximately twice that of the 1000 ppm group, which was a greater difference than the nominal dose difference (i.e., 1500 ppm/1000 ppm: a 1.5-fold increase). Moreover, considering that the PB concentrations in GD20 dams from the prenatal test cohort study were 86.4 ± 19.5 mg/L and 139 ± 29.9 mg/L, respectively (Minami et al., 2024), it can be inferred that the maternal NaPB exposure during the lactation period was generally comparable to that during the pregnancy period for both groups.
General condition and reproduction performance of dams
In the NaPB 1000 ppm group, one maternal animal died in the lactation period. Another maternal rat showed signs of poor health, including piloerection and decreased spontaneous motor activity during the prepartum period, and this animal was found dead on GD23, with some deceased fetuses remaining inside the vagina and uterus. No other maternal deaths were observed at any dose level during the study.
There were two animals with staggering gait recorded for the entire study at 1000 ppm: one case occurring transiently during GD7-GD9 and another case occurring in the deceased animal that had symptoms shortly before death (GD21). In the NaPB 1500 ppm group, 6 of 10 maternal rats exhibited staggering gait immediately after the start of dosing (i.e., during GD7-GD9), and by the end of the gestation period the incidence reached 10 of 10 animals. Staggering gait did not occur in any animals of this group after delivery (maybe due to reduction of dosing concentration of NaPB to 1000 ppm, as shown in Fig. 2), except one case noted during LD14-LD17.
During the gestation period, both NaPB groups had no significant alterations of maternal body weight, with the exception for the decrease of the NaPB 1500 ppm group on GD12 (Fig. 3C). However, maternal body weights at 1000 ppm were higher than control values, with statistical significance on LD0, LD4, and LD21. Maternal body weights at 1500 ppm were also higher than control values during the late lactation period, with statistical significance on LD21.
No treatment effect was detected for reproductive data (fertility index, gestation index, duration of gestation, number of implantation sites, number of pups delivered, and sex ratio) (Supplemental Table 2). However, significant mortality of offspring by PND4, which appeared to be an effect of acute toxicity caused by NaPB exposure, was observed in the NaPB-treated groups. The number of dead offspring in the control, 1000 ppm, and 1500 ppm groups was 9/193 (5%), 35/113 (31%), and 62/119 (52%), respectively. Moreover, mortality from PND5 to PND21 remained at 1/80 (1%), 4/56 (7%), and 4/49 (8%) for the control, 1000 ppm, and 1500 ppm groups, respectively.
Key Event 2: CAR activation as the Molecular Initiating Event (MIE), inferred from hepatic Cyp2b1/2 mRNA expression
NaPB treatment at both 1000 and 1500 ppm significantly induced hepatic Cyp2b1/2 mRNA levels in LD21 dams and PND21 pups, with both showing an increase of more than approximately 100-fold (Supplemental Tables 2 and 3, and Supplemental Fig. 2). These data suggest that CAR was significantly activated with equal potency by NaPB in both LD21 dams and PND21 pups. This was also supported by the increased incidence of centrilobular hepatocellular hypertrophy (a typical liver histopathology finding frequently observed with CAR activators) in LD21 dams and PND21 pups (Table 1). The increase in mRNA levels was substantially greater in PND21 pups than in GD20 fetuses (Supplemental Fig. 2, with data for GD20 fetuses adopted from the prenatal test cohort study; Minami et al., 2024), suggesting that the liver of PND21 pups exhibits expanded reactivity to exogenous chemicals. The CAR activation by NaPB was also observed 1 week after cessation of NaPB treatment, as evidenced by increased hepatic Cyp2b1/2 mRNA levels and increased incidence of centrilobular hepatocellular hypertrophy in PND28 pups of the NaPB group. (Table 1 and Supplemental Table 5).
Table 1. Summary of histopathology.
Key Event 3: Hepatic Ugt2b1 mRNA levels and UDPGT activity
NaPB treatment at both 1000 and 1500 ppm significantly induced hepatic Ugt2b1 mRNA levels in LD21 dams and PND21 and PND28 pups, with both showing an increase of more than 2-fold (Supplemental Tables 2, 3 and 5). Both doses of NaPB also significantly induced UDPGT activities with dose dependency in LD21 dams (Fig. 4A). Hepatic UDPGT activity in PND21 pups was increased by NaPB treatment with significant treatment effect [F(2,95)=138, p<0.001], sex [F(1,97)=42, p<0.001] and treatment x sex interactions [F(2,92)=4.8, p=0.01026], as determined by ANOVA (Supplemental Table 6). NaPB treatment at both 1000 and 1500 ppm significantly induced hepatic UDPGT activity in both male and female PND21 pups in a dose-dependent manner, which was higher than LD21 dam (Fig. 4B). Scatter plots of individual values of UDPGT activities revealed higher correlation between LD21 dams and PND21 male pups (R2=0.56) than PND21 female pups (R2=0.41) (Figs. 4C and 4D). However, scatter plots of individual values of UDPGT activities in PND21 pups revealed a much higher correlation between male and female PND21 pups (i.e., littermates) (R2=0.67; Fig. 4E), suggesting the sex difference in NaPB-induced hepatic UDPGT activity observed as significant interaction effect of treatment x sex in ANOVA in PND21 pups has little impact for evaluation of the effect of NaPB on TH status in PND21 pups. The increase in UDPGT activity was also observed even one week after the termination of the treatment on PND28 (Supplemental Table 5).
In LD21 dams, NaPB treatment significantly decreased both serum T3 and T4 concentrations with dose-dependency, but statistical significance was not observed in serum T3 of the 1000 ppm group (Figs. 5A and 5B). Serum TSH concentrations were not significantly changed (Fig. 5C).
Regarding the PND21 pup data, combined data from males and females were used to assess the effect of NaPB treatment on serum hormone concentrations. This was because ANOVA revealed no significant interaction between treatment and sex on serum T3, T4 or TSH concentrations (all p>0.05) (see Supplemental Table 6). Additionally, ANOVA showed no significant effects of sex, however, there were significant effects of treatment on serum T4 concentrations only [F(2,50) =9.72, p<0.001]. Unlike LD21 dams, NaPB treatment did not decrease serum T3 or T4 concentrations in PND21 pups (Figs. 5A and 5B). Instead, serum T4 concentrations in the NaPB groups were significantly higher than those of the controls (Fig. 5B). Scatter plots of individual serum T3 and T4 concentrations showed a high correlation (R2=0.50, Fig. 5D) in LD21 dams, suggesting that serum T3 and T4 concentrations increased concomitantly in response to maternal NaPB treatment. However, no such correlation was found in PND21 pups (R2=0.03, Fig. 5E), consistent with the observation that maternal NaPB exposure increased serum T4 but not T3 concentrations in their pups (Figs. 5A and 5B).
To understand the biological relevance of the statistically significant increases in serum T4 concentrations observed in PND21 pups from the NaPB group, we expanded the TH evaluation to include other testing time points such as PND4 and PND28, in addition to PND21. For serum T4 concentration in offspring, ANOVA revealed significant treatment effect [F(2,123) =8.83, p<0.001], but no significant sex effect [F(1,124) =1.93, p=0.17] or treatment by sex interactions [F(2,120)=1.46, p=0.24] (see Supplemental Table 6)). Therefore, a combined data set from male and female pups was acceptable. For serum T3 and TSH concentration in offspring, ANOVA revealed no significant treatment effect, sex effect and treatment by sex interactions.
Effects of NaPB on serum THs on different test points including GD20 (data from previous study; Minami et al., 2024) are plotted in Figs. 6A and 6B. Statistically significant reductions of serum T3 and T4 concentrations by NaPB were observed in GD 20 fetuses, while NaPB treatment no longer induced a significant decrease in serum TH concentrations in PND4 pups. The 1500 ppm group showed lower values than controls, but the difference was not statistically significant. This was contributed by the small number (N=3) of animals tested on PND4. In PND21 pups, as stated above, a statistically significant increase in serum T4 concentrations was observed in both NaPB groups. However, these increases in serum T4 were not observed one week after the termination of the treatment on PND28 (Figs. 6B). Effects of NaPB on serum TSH on different test points were also plotted in Fig. 6C. Statistically significant increases of serum TSH concentrations by NaPB were observed in GD 20 fetuses, while NaPB treatment no longer induced a significant alteration at postnatal phase.
The concentrations of brain T3 in PND4, PND21, and PND28 pups revealed a significant sex effect [F(1,124) =6.24, p=0.014], with no significant treatment effects [F(2,123)=0.81, p=0.45] or treatment by sex interactions [F(2,120)=0.41, p=0.67], as determined by ANOVA (Supplemental Table 6). Conversely, brain T4 concentrations in the same pups showed a significant treatment effect [F(2,123)=11.5, p<0.001], but no significant sex effects [F(1,124)=0.51, p=0.48] or treatment x sex interactions [F(2,120)=2.05, p=0.13]. Consequently, for the analysis of brain TH concentrations in these pups, the data from both sexes were pooled to evaluate the effect of NaPB treatment.
As previously reported (Minami et al., 2024), statistically significant reductions in brain T3 and T4 concentrations due to NaPB were observed in GD 20 fetuses (Figs. 6D and 6E). However, no significant decreases in brain T3 and T4 concentrations were noted in PND4 and PND21 pups. In PND21 pups, as shown in Fig. 6E, both NaPB-treated groups exhibited statistically significant increases in brain T4, but not in T3 concentrations (Fig. 6D), consistent with the serum TH status for PND21 pups (Fig. 6A and 6B). In PND28 pups, T4 concentrations in both NaPB-treated groups returned to control levels (Fig. 6B and 6E). Although the brain T3 concentration in PND28 pups of the 1500 ppm group was statistically decreased, no such significant decrease was observed in PND21 pups (Fig. 6D). The biological relevance of the observed reduction in brain T3 at PND28 remains unclear, as individual brain T3 concentration values showed no correlation with either serum T3 (R2=0.17) or brain T4 (R2=0.002) in PND28 pups.
Scatter plots of individual serum and brain T4 concentrations revealed a higher correlation in PND21 pups (R2=0.65) (Fig. 7B), indicating that the increase in T4 concentrations in the serum and brain of PND21 pups is biologically significant, but no correlations were observed in PND4 (R2=0.36) (Fig. 7A) or PND28 pups (R2=0.10) (Fig. 7C).
In the qualitative histopathological analysis, no treatment-related abnormalities were detected in the cerebrum and cerebellum of either sex in PND21 and PND28 pups treated with NaPB (Table 1).
DISCUSSION
In the current study, we evaluated the impact of maternal dietary exposure to NaPB, a well-known prototypical mild to moderate TH disruptor in adult rats (Barter and Klaassen, 1994; Capen, 1997; Finch et al., 2006; Haines et al., 2019; Hood et al., 1999b; Liu et al., 1995; McClain et al., 1989; O’Connor et al., 1999), on the postnatal test cohort of the modified CTA we proposed. The study incorporated the following modifications: 1) a 50% reduction in group size (i.e., from 20 rats per group in the standard CTA to 10 rats); and 2) additional endpoints, including brain TH concentrations and a qualitative assessment of brain histopathology in the offspring. Previously, we observed that a prenatal test cohort of the modified CTA could detect NaPB-induced dose-dependent reductions in serum T3 (up to -26%) and T4 (up to -44%) in GD20 dams, with corresponding decreases in serum (up to -26%) and brain (up to -18%) T3, and in serum (up to -26%) and brain (up to -29%) T4 in GD20 fetuses, although without a clear dose-response relationship (Minami et al., 2024). However, the current study revealed that NaPB treatment throughout pre- and post-natal periods (GD6-PND21) did not result in a significant reduction of TH in the serum and brain of the pups, despite a marked reduction in maternal serum TH concentrations in the postnatal test cohort as well as in the prenatal test cohort. The biological relevance of the negative findings in pups is discussed below, considering the validity of the NaPB dose and endpoints employed in this study, in light of information from published literature. The acceptability of the modifications of CTA protocol is then addressed.
Effects of NaPB
Suitability of exposure levels of NaPB
Food consumption in maternal rats of the NaPB group exhibited minor variations from the control at only a few points during the gestation period, while it generally decreased throughout the lactation period. Plasma NaPB concentration was observed to increase dose-dependently in LD21 dams. The difference in the plasma NaPB concentration between the 1000 and 1500 ppm groups was 2-fold, which was greater than the nominal target dose difference (i.e., 1.5-fold). This discrepancy may be attributed to a decrease in dietary concentration from 1000 ppm to 500 ppm during LD13 to LD21 in the 1000 ppm group.
During the gestation period, there were no significant changes in maternal body weight in both NaPB groups. However, maternal body weights during the lactation period were higher than control values at several points, which was consistent with a previous study (Minami et al., 2023). Regarding the general condition of the maternal rats, a staggering gait (a typical sign of neurotoxicity from NaPB) was observed during the gestation period but not during the lactation period (except one case in the 1500 ppm group). Overall, the NaPB dose levels tested (i.e., 1000 and 1500 ppm) did not cause excessive toxicities (e.g., maternal death, severely reduced food consumption due to drowsiness), and thus, they did not confound the findings concerning potential TH disruption in dams.
Findings from each key event in the proposed adverse outcome pathway (AOP) associated with TH disruption induced by NaPB
In this study, exposure to NaPB (Key Event 1) significantly affected the maternal liver, consistent with the findings documented in previous studies on adult rats (Finch et al., 2006; Haines et al., 2019; Hood et al., 1999a, 1999b; Liu et al., 1995; Yamada et al., 2021). As a molecular initiating event (MIE, Key Event 2), NaPB exposure led to the activation of the CAR in the liver of LD21 maternal rats. This was evident from the upregulation of hepatic Cyp2b1/2 mRNA, a recognized biomarker for CAR activation (Okuda et al., 2017; Yamada et al., 2021). A similar upregulation was noted in PND21 pups, suggesting that CAR activation in offspring may result from both the transferred NaPB through breast milk (Moriyama et al., 1999) and ingestion by the pups themselves during the later stages of lactation. Although hepatic Cyp2b1/2 mRNA expression was not assessed in PND4 pups within this study, previous study has indicated significant induction at 1000 ppm NaPB (Minami et al., 2023). Consequently, it appears that CAR activation by NaPB persists in both maternal rats and their offspring throughout the conditions of this experiment.
The activation of CAR leads to the induction of UDPGT activity (Key Event 3), thereby enhancing the catabolism of TH (Crofton, 2008; Noyes et al., 2019). Since RNAs for UDPGTr2 remained relatively stable from late gestation through to adulthood (Marie and Cresteil, 1989), it is plausible that NaPB could stimulate fetal hepatic UDPGT activity starting from late gestation. The previous study confirmed NaPB-induced hepatic UDPGT activity and/or Ugt2b1 mRNA in GD20 dams and GD20 fetuses (Minami et al., 2023, 2024), a finding that is echoed in the current study with LD21 dams and PND21 pups. These results suggest that NaPB continuously induces hepatic UDPGT activity in both maternal rats and their offspring under the conditions of this experiment. This consideration is further supported by the previous observation that hepatic mRNA expression of Ugt1a1, Ugt1a6, and Ugt2b1 in PND4 pups was significantly increased following exposure to 1000 ppm NaPB (Minami et al., 2023).
Due to the low concentrations of TH in maternal milk (Mizuta et al., 1983), early disruption of TH in newborn rats (if it exists) may primarily result from increased elimination of TH due to exposure to NaPB in the milk, rather than from a reduced transfer of TH via the milk. Pups during the later stages of lactation could be exposed to an increased amount of chemical through their own feeding habits. This additionally increased exposure of NaPB might explain the observed higher induction of hepatic UDPGT activity in pups compared to their mothers. Therefore, the intake of NaPB by the pups through their own feeding does have some effect, while it does not appear to cause a large enough variation to mask the effects passed through the mother rats, including dose-response relationships. This was supported by a correlation between the individual hepatic UDPGT activities of the mother rats and their offspring. Thus, these findings suggest that the degree of NaPB exposure in mother rats may be a significant factor influencing the activation of hepatic enzymes in their offspring during lactation. Since this study design does not involve direct administration to the young animals, but rather investigates the effects of substances through exposure to the dams, it allows for the observation of a chemical’s impact in a situation that more closely mimics human real-life exposure scenarios with pesticides. Although this modified CTA has a strong emphasis on utilization as a DNT screening test, it is also expected to be useful as a mechanistic test or risk characterization test that takes pharmacokinetics into account. In that sense, it has the advantage of being closer to human exposure conditions.
In LD21 dams, a significant decrease in serum concentrations of THs (Key Event 4) was observed, which was accompanied by a notable increase in hepatic UDPGT activity (Key Event 3). Conversely, PND4 and PND21 pups showed no significant decrease in serum TH concentrations (Key Event 4) despite the significant induction of hepatic UDPGT activity (Key Event 3). Instead, PND21 pups exhibited elevated TH concentrations in both serum and brain (Key Events 4 and 5). This pattern of increased T4 was also identified in a previous study involving 1000 ppm NaPB (Minami et al., 2023), indicating that the results are consistent with known effects rather than being due to experimental error. In other research, exposure to perchlorate led to significant reductions in maternal and fetal THs (Gilbert et al., 2022), yet postnatal pups did not show substantial alterations in serum hormone concentrations (Gilbert and Sui, 2008; Gilbert et al., 2023, 2024). Exposure to perfluorohexane sulfonate and triclosan resulted in decreased brain T4 concentrations on PND0 and PND2, and brain T3 concentrations were significantly lower in triclosan-exposed pups on PND2. However, by PND6, no differences from control values were observed in either hormone (Gilbert et al., 2021). These findings, including those from our study, suggest that while significant reductions in serum and/or brain TH concentrations are evident in fetuses, such reductions appear to be less pronounced or absent in pups (beyond PND4), even when maternal TH concentrations are significantly and persistently reduced. This contrasted with the effects observed with 6-PTU (Minami et al., 2023; O’Shaughnessy et al., 2018; Ogata et al., 2024).
In the current study, a strong positive correlation was observed between serum and brain T4 concentrations in PND21 rat pups, indicating that an increase in serum T4 is reflected in an increase in brain T4 levels. Notably, the elevation in T4 concentrations subsided by PND28 (i.e., one week after the cessation of dosing). These observations suggest that the rise in T4 concentrations in serum and brain observed in the NaPB group of PND21 pups is a biological response to the NaPB exposure. This compensatory increase (following decreases in serum and brain T4 concentrations on GD20) may represent a homeostatic attempt by the neonatal brain to maintain TH supply, in the context of the actively developing hypothalamic-pituitary-thyroid axis postnatally (Howdeshell, 2002). However, since serum TSH concentrations were not increased in the NaPB groups, the exact mechanism of the increased T4 concentration remains unknown. In rats, it is possible that the transient abundance of Thyroxine Binding Globulin (TBG) (Savu et al., 1987) from birth to the weaning period may also contribute to the reduced metabolism of blood TH.
Finally, in the current experiment, heterotopia formation (Key Event 5; downstream indicator for TH insufficiency) did not significantly increase in PND21 and PND28 pups of the 1000 ppm NaPB group (as explained in the Materials and Methods section, only the 1000 ppm group was tested considering that the decrease in brain TH concentration was equivalent in both the 1000 and 1500 ppm groups). The inability of exposure of NaPB during the perinatal period (i.e., GD20 to LD0) to avoid neonatal mortality, due to its acute toxicity, cannot be completely ruled out as a factor contributing to this negative result. However, Hassan et al. reported that the previous quantitative morphometric analyses revealed that the presence of small heterotopias (approximately 0.005 mm3) just above background levels was associated with fetal serum T4 decrements of approximately -35% in GD20 fetus (Hassan et al., 2017). Thus, it was not surprising that the decreases in brain T3 (up to -18%) and T4 (up to -29%) in GD20 fetuses induced by 1000 and 1500 ppm NaPB did not induce periventricular heterotopia formation. Furthermore, periventricular heterotopia became larger as a result of further enhancing the postnatal TH reduction at least until PND6 (Gilbert et al., 2023). However, in our current experiment, we did not observe a significant decrease in serum and brain TH concentrations in the NaPB group at PND4 or PND21, which is consistent with the absence of significant periventricular heterotopia increase even under continuous NaPB exposure and maternal TH reductions until PND21. Overall, although further studies will be required, approximately 30% reduction in fetal brain TH concentrations in NaPB-exposed rats may have little impact on postnatal brain heterotopia formation.
Evaluation of the modified CTA protocol
Number of test animals
The prenatal test cohort of the CTA suggests that reducing the group size from 20 to 10 animals per group per cohort appears capable of detecting even mild TH disruption (approximately 20-30% TH reduction) when assessing fetal serum and brain T4 concentrations (Minami et al., 2023, 2024). This level of statistical power should be acceptable based on a previous report by Crofton, which demonstrated that a 50-60% decrease in circulating T4 was necessary to significantly impact hearing function in rats (Crofton, 2004). Additionally, a recent review indicated that offspring serum T4 reductions of >60%/>50% (at top-/lower-dose groups) were associated with an increased likelihood of statistically significant neurodevelopmental effects (Marty et al., 2022).
The absence of TH reduction in the pups of the NaPB groups in the present study is unlikely to be due to the small group size (N=10 per group), as the coefficient of variance of control TH concentrations in the PND21 animals was similar to or smaller than those in the control GD20 fetuses, in which a mild but significant TH reduction by NaPB could be detected with a small number (N=10 per group) of GD20 fetuses (Minami et al., 2023, 2024). Moreover, the control values of serum and brain TH concentrations in PND21 pups are much higher than those of GD20 fetuses (Minami et al., 2023, 2024), which should make it easier to detect any reduction in TH. Therefore, the lack of TH reduction in the pups from NaPB-treated dams cannot be attributed to a poor detection sensitivity issue, but rather suggests a biological response to a moderate TH disruptor in maternal rats. Overall, this indicates that the absence of TH reduction in the pups from NaPB-treated dams is not a valid reason to dismiss the reduction of animal numbers in the modified CTA.
Lack of sex-effects on fetal TH disruption
The USEPA CTA guidance (USEPA, 2005) apparently recommends separate blood collection from male and female offspring. However, our findings showed that male and female littermates in the NaPB group exhibited similar induction of hepatic UDPGT activity. This suggests that evaluating one pup per litter, regardless of sex, is sufficient to assess downstream events of hepatic UDPGT activity induction in the pups. This conclusion is supported by both the prenatal test cohort (Minami et al., 2024) and the postnatal test cohort (present study), which found no interaction effects between NaPB treatment and sex on serum and brain TH concentrations. Additionally, our previous studies on periventricular heterotopia formation using 6-PTU also showed no sex differences (Minami et al., 2023; Ogata et al., 2024). Other publications have also reported no evidence of a differential effect of sex on TH alterations and heterotopia formation (Gilbert and Sui, 2008; Gilbert et al., 2023; Marty et al., 2022; O’Shaughnessy et al., 2018). Therefore, although further research using other chemicals with different AOP is necessary, we believe that separate assessment of TH concentration and heterotopia formation based on sex is unnecessary. Instead, pooling fetal or PND4 pup samples by litter, regardless of sex, would be acceptable. This supports the idea that obtaining additional applications, such as evaluating the toxicokinetics of test substances, especially in fetuses and PND4 pups, would be beneficial.
Qualitative heterotopia assessment in postnatal test cohort of the modified CTA
Because of potential compensatory mechanisms and the sensitivity of TH and TSH concentrations to possible factors (stress, circadian rhythm, etc.), determination of serum TH alone may have limitations for assessment of thyroid function and TH action (DeVito et al., 1999). This is true for the CTA even including measurement of brain TH concentrations because of technical difficulty. Since clear morphological changes occur during the first two postnatal weeks and by PND14 (O’Shaughnessy et al., 2019), brain histology in the postnatal cohort of the CTA would be valuable. As stated in the Introduction section, assessment of periventricular heterotopia in postnatal rat brain from PND14 onwards is informative for assessing TH insufficiency, as well reported by the group of Gilbert and O’Shaughnessy et al. (Gilbert et al., 2014, 2023; Goodman and Gilbert, 2007; O’Shaughnessy et al., 2018, 2019). Considering the feasibility of implementation at CROs, we are exploring whether it is possible to determine results without significant error using the qualitative pathological examinations on slides with hematoxylin and eosin staining that are commonly conducted at many CROs, rather than performing morphometry on slides with immunostaining of a neuronal cell specific protein such as NeuN. In this case, step section with grading is necessary (Minami et al., 2023; Ogata et al., 2024). We have determined that PND21, rather than PND4, is the optimal time to assess periventricular heterotopia in the rat CTA (Ogata et al., 2024).
The absence of heterotopia formation was observed not only in our experiments using NaPB but also in other cases via “extrathyroidal” action. Perfluorohexane sulfonate (50 mg/kg/day) and triclosan (300 mg/kg/day) also induced mild TH alterations (<40%) in the brain of the PND0 and PND2 but did not produce any changes in qualitative brain morphology (including heterotopia) in PND14 pups or behavioral changes examined during PND60 to PND80 (Gilbert et al., 2021). Based on these findings, including our study, alongside brain TH concentration assessments, incorporating the qualitative investigation of periventricular heterotopia appears to enhance the CTA (Minami et al., 2023; Ogata et al., 2024). The USEPA’s CTA guidance proposes that dosing should be avoided, when possible, on the day of parturition (USEPA, 2005). However, since the day of birth falls within the critical time window for heterotopia formation (GD19-LD2) (O’Shaughnessy et al., 2019), no treatment at parturition can complicate the interpretation of heterotopia formation results. Therefore, when evaluating heterotopia formation as an endpoint, it is suggested to avoid no-dosing on the day of parturition as much as possible.
Conclusions
We are currently evaluating the feasibility, sensitivity, and reliability of the modified CTA, which includes the examination of brain THs and periventricular heterotopia formation, while reducing the number of test animals, even for chemicals with mild TH disruption. The previous study confirmed that the prenatal test cohort of the modified CTA protocol successfully detected mild but statistically significant TH disruption induced by NaPB in pregnant dams and their GD20 fetuses (Minami et al., 2024). However, we did not observe this TH disruption in the pups, despite continuous TH reduction in maternal rats, in the present study. Additionally, no heterotopia formation was observed on PND21 (and PND28), indicating that an approximately 30% reduction in fetal brain TH reduction was insufficient for heterotopia formation, consistent with previous studies (Gilbert et al., 2023, 2024; Hassan et al., 2017). Crofton reported that a 50–60% decrease in circulating T4 was needed to significantly impact hearing function in rats (Crofton, 2004). Additionally, an analysis of multiple studies concluded that for a statistically significant impact on neurodevelopment, a reduction of >60%/>50% (high-dose group/low-dose group) in serum T4 levels and a statistically significant reduction of >20% in serum T3 levels are required (Marty et al., 2022). Therefore, considering these findings (Crofton, 2004; Gilbert et al., 2023, 2024; Marty et al., 2022), an approximately 30% reduction in brain TH concentrations in NaPB-exposed fetuses may have little impact on postnatal brain development.
We have suggested several modifications to the CTA design: 1) reducing the group size by 50% (N=10 rats per group); 2) including additional endpoints such as measuring brain TH concentrations and qualitatively assessing periventricular heterotopia in offspring; 3) pooling fetal or PND4 pup blood samples; 4) assessing combined data from both male and female animals for heterotopia formation; and 5) avoiding “no-dosing” on the day of parturition as much as possible. The present study suggests that these modifications may offer a useful alternative to the original CTA protocol. However, further studies using other compounds or conducted in different laboratories will be necessary to validate these findings.
ACKNOWLEDGMENTS
The study was supported by Sumitomo Chemical Company, Ltd. and the Institute of Environmental Toxicology and partly through a grant of LRI (The Long-range Research Initiative, #20-3-02) by Japan Chemical Industry Association. The authors would like to express deep gratitude to Dr. Kevin M. Crofton (R3Fellows LLC, Durham, North Carolina, 27705, United States), Dr. Mary E. Gilbert, Dr. Katherine L. O’Shaughnessy and Dr. Tammy E. Stoker (United States Environmental Protection Agency, Research Triangle Park, NC, United States) for useful comments and advice in scientific discussions. The authors thank Prof. Samuel M. Cohen (University of Nebraska Medical Center, Omaha, Nebraska, USA) for review of the manuscript and the reviewers selected by the Editor who provided valuable comments anonymously to the authors, which were instrumental in revising and refining the manuscript. The authors also thank the other contributors to this research project from Sumitomo Chemical Co., Ltd., the Institute of Environmental Toxicology, and Sumika Technoservice Corp.
Supplemental data
Supplemental information (including 6 Tables and 2 Figures) to this article can be found online.
Conflict of interest
The authors declare that there is no conflict of interest.
REFERENCES
- Barter, R.A. and Klaassen, C.D. (1994): Reduction of thyroid hormone levels and alteration of thyroid function by four representative UDP-glucuronosyltransferase inducers in rats. Toxicol. Appl. Pharmacol., 128, 9-17.
- Bartsch, R., Brinkmann, B., Jahnke, G., Laube, B., Lohmann, R., Michaelsen, S., Neumann, I. and Greim, H. (2018): Human relevance of follicular thyroid tumors in rodents caused by non-genotoxic substances. Regul. Toxicol. Pharmacol., 98, 199-208.
- Bernal, J. (2017): Thyroid hormone regulated genes in cerebral cortex development. J. Endocrinol., 232, R83-R97.
- Capen, C.C. (1997): Mechanistic data and risk assessment of selected toxic end points of the thyroid gland. Toxicol. Pathol., 25, 39-48.
- Coecke, S., Goldberg, A.M., Allen, S., Buzanska, L., Calamandrei, G., Crofton, K., Hareng, L., Hartung, T., Knaut, H., Honegger, P., Jacobs, M., Lein, P., Li, A., Mundy, W., Owen, D., Schneider, S., Silbergeld, E., Reum, T., Trnovec, T., Monnet-Tschudi, F. and Bal-Price, A. (2007): Workgroup report: incorporating in vitro alternative methods for developmental neurotoxicity into international hazard and risk assessment strategies. Environ. Health Perspect., 115, 924-931.
- Crofton, K.M. (2004): Developmental disruption of thyroid hormone: correlations with hearing dysfunction in rats. Risk Anal., 24, 1665-1671.
- Crofton, K.M. (2008): Thyroid disrupting chemicals: mechanisms and mixtures. Int. J. Androl., 31, 209-223.
- Crofton, K.M., Mundy, W.R. and Shafer, T.J. (2012): Developmental neurotoxicity testing: a path forward. Congenit. Anom. (Kyoto), 52, 140-146.
- Crofton, K.M., Bassan, A., Behl, M., Chushak, Y.G., Fritsche, E., Gearhart, J.M., Marty, M.S., Mumtaz, M., Pavan, M., Ruiz, P., Sachana, M., Selvam, R., Shafer, T.J., Stavitskaya, L., Szabo, D.T., Szabo, S.T., Tice, R.R., Wilson, D., Woolley, D. and Myatt, G.J. (2022): Current status and future directions for a neurotoxicity hazard assessment framework that integrates in silico approaches. Comput. Toxicol., 22, 100223.
- de Escobar, G.M., Obregón, M.J. and del Rey, F.E. (1989): Transfer of thyroid hormones from the mother to the fetus. In: Research in congenital hypothyroidism (Delange, F., Fisher, D.A. and Glinoer, D., eds.), pp. 15-29, Springer US, Boston, MA.
- Dellarco, V.L., McGregor, D., Berry, S.C., Cohen, S.M. and Boobis, A.R. (2006): Thiazopyr and thyroid disruption: case study within the context of the 2006 IPCS Human Relevance Framework for analysis of a cancer mode of action. Crit. Rev. Toxicol., 36, 793-801.
- DeVito, M., Biegel, L., Brouwer, A., Brown, S., Brucker-Davis, F., Cheek, A.O., Christensen, R., Colborn, T., Cooke, P., Crissman, J., Crofton, K., Doerge, D., Gray, E., Hauser, P., Hurley, P., Kohn, M., Lazar, J., McMaster, S., McClain, M., McConnell, E., Meier, C., Miller, R., Tietge, J. and Tyl, R. (1999): Screening methods for thyroid hormone disruptors. Environ. Health Perspect., 107, 407-415.
- Dingemanse, J., van Bree, J.B. and Danhof, M. (1989): Pharmacokinetic modeling of the anticonvulsant action of phenobarbital in rats. J. Pharmacol. Exp. Ther., 249, 601-608.
- European Commission. Directorate-General for Environment, Axelstand, M., Ramhøj, L., Scholze, M., Martin, O., Köhrle, J., and Kortenkamp, A. (2019): Development of a study protocol for thyroid disruptor testing in the mammalian system - Deliverable 16 - Final feasibility study report, Publications Office. https://data.europa.eu/doi/10.2779/194307. Accessed Aug 7, 2024.
- Finch, J.M., Osimitz, T.G., Gabriel, K.L., Martin, T., Henderson, W.J., Capen, C.C., Butler, W.H. and Lake, B.G. (2006): A mode of action for induction of thyroid gland tumors by Pyrethrins in the rat. Toxicol. Appl. Pharmacol., 214, 253-262.
- Foster, J.R., Tinwell, H. and Melching-Kollmuss, S. (2021): A review of species differences in the control of, and response to, chemical-induced thyroid hormone perturbations leading to thyroid cancer. Arch. Toxicol., 95, 807-836.
- Garman, R.H., Li, A.A., Kaufmann, W., Auer, R.N. and Bolon, B. (2016): Recommended methods for brain processing and quantitative analysis in rodent developmental neurotoxicity studies. Toxicol. Pathol., 44, 14-42.
- Gilbert, M.E. and Sui, L. (2008): Developmental exposure to perchlorate alters synaptic transmission in hippocampus of the adult rat. Environ. Health Perspect., 116, 752-760.
- Gilbert, M.E., O’Shaughnessy, K.L. and Axelstad, M. (2020): Regulation of thyroid disrupting chemicals to protect the developing brain. Endocrinology, 161, 1-17.
- Gilbert, M.E., Rovet, J., Chen, Z. and Koibuchi, N. (2012): Developmental thyroid hormone disruption: prevalence, environmental contaminants and neurodevelopmental consequences. Neurotoxicology, 33, 842-852.
- Gilbert, M.E., Ramos, R.L., McCloskey, D.P. and Goodman, J.H. (2014): Subcortical band heterotopia in rat offspring following maternal hypothyroxinaemia: structural and functional characteristics. J. Neuroendocrinol., 26, 528-541.
- Gilbert, M.E., O’Shaughnessy, K.L., Bell, K.S. and Ford, J.L. (2023): Structural malformations in the neonatal rat brain accompany developmental exposure to ammonium perchlorate. Toxics, 11, 1027.
- Gilbert, M.E., Hassan, I., Wood, C., O’Shaughnessy, K.L., Spring, S., Thomas, S. and Ford, J. (2022): Gestational exposure to perchlorate in the rat: thyroid hormones in fetal thyroid gland, serum, and brain. Toxicol. Sci., 188, 117-130.
- Gilbert, M.E., Hassan, I., O’Shaughnessy, K.L., Wood, C., Stoker, T.E., Riutta, C. and Ford, J.L. (2024): Ammonium perchlorate: serum dosimetry, neurotoxicity, and resilience of the neonatal rat thyroid system. Toxicol. Sci., 198, 113-127.
- Gilbert, M.E., O’Shaughnessy, K.L., Thomas, S.E., Riutta, C., Wood, C.R., Smith, A., Oshiro, W.O., Ford, R.L., Hotchkiss, M.G., Hassan, I. and Ford, J.L. (2021): Thyroid disruptors: extrathyroidal sites of chemical action and neurodevelopmental outcome - an examination using triclosan and perfluorohexane sulfonate. Toxicol. Sci., 183, 195-213.
- Goodman, J.H. and Gilbert, M.E. (2007): Modest thyroid hormone insufficiency during development induces a cellular malformation in the corpus callosum: a model of cortical dysplasia. Endocrinology, 148, 2593-2597.
- Haines, C., Chatham, L.R., Vardy, A., Elcombe, C.R., Foster, J.R. and Lake, B.G. (2019): Comparison of the hepatic and thyroid gland effects of sodium phenobarbital and pregnenolone-16α-carbonitrile in wild-type and constitutive androstane receptor (CAR)/pregnane X receptor (PXR) knockout rats. Xenobiotica, 49, 227-238.
- Hassan, I., El-Masri, H., Kosian, P.A., Ford, J., Degitz, S.J. and Gilbert, M.E. (2017): Neurodevelopment and thyroid hormone synthesis inhibition in the rat: quantitative understanding within the adverse outcome pathway framework. Toxicol. Sci., 160, 57-73.
- Hood, A., Liu, J. and Klaassen, C.D. (1999a): Effects of phenobarbital, pregnenolone-16alpha-carbonitrile, and propylthiouracil on thyroid follicular cell proliferation. Toxicol. Sci., 50, 45-53.
- Hood, A., Hashmi, R. and Klaassen, C.D. (1999b): Effects of microsomal enzyme inducers on thyroid-follicular cell proliferation, hyperplasia, and hypertrophy. Toxicol. Appl. Pharmacol., 160, 163-170.
- Howdeshell, K.L. (2002): A model of the development of the brain as a construct of the thyroid system. Environ. Health Perspect., 110 (Suppl 3), 337-348.
- Kortenkamp, A., Axelstad, M., Baig, A.H., Bergman, Å., Bornehag, C.G., Cenijn, P., Christiansen, S., Demeneix, B., Derakhshan, A., Fini, J.B., Frädrich, C., Hamers, T., Hellwig, L., Köhrle, J., Korevaar, T.I., Lindberg, J., Martin, O., Meima, M.E., Mergenthaler, P., Nikolov, N., Du Pasquier, D., Peeters, R.P., Platzack, B., Ramhøj, L., Remaud, S., Renko, K., Scholze, M., Stachelscheid, H., Svingen, T., Wagenaars, F., Wedebye, E.B. and Zoeller, R.T. (2020): Removing critical gaps in chemical test methods by developing new assays for the identification of thyroid hormone system-disrupting chemicals-The ATHENA Project. Int. J. Mol. Sci., 21, 3123.
- Landers, K. and Richard, K. (2017): Traversing barriers - How thyroid hormones pass placental, blood-brain and blood-cerebrospinal fluid barriers. Mol. Cell. Endocrinol., 458, 22-28.
- Lein, P., Locke, P. and Goldberg, A. (2007): Meeting report: alternatives for developmental neurotoxicity testing. Environ. Health Perspect., 115, 764-768.
- Li, A.A., Makris, S.L., Marty, M.S., Strauss, V., Gilbert, M.E., Blacker, A., Zorrilla, L.M., Coder, P.S., Hannas, B., Lordi, S. and Schneider, S. (2019): Practical considerations for developmental thyroid toxicity assessments: what’s working, what’s not, and how can we do better? Regul. Toxicol. Pharmacol., 106, 111-136.
- Liu, J., Liu, Y., Barter, R.A. and Klaassen, C.D. (1995): Alteration of thyroid homeostasis by UDP-glucuronosyltransferase inducers in rats: a dose-response study. J. Pharmacol. Exp. Ther., 273, 977-985.
- Lumley, L.A., Marrero-Rosado, B., Rossetti, F., Schultz, C.R., Stone, M.F., Niquet, J. and Wasterlain, C.G. (2021): Combination of antiseizure medications phenobarbital, ketamine, and midazolam reduces soman-induced epileptogenesis and brain pathology in rats. Epilepsia Open, 6, 757-769.
- Marie, S. and Cresteil, T. (1989): Phenobarbital-inducible gene expression in developing rat liver: relationship to hepatocyte function. Biochim. Biophys. Acta, 1009, 221-228.
- Marty, M.S., Sauer, U.G., Charlton, A., Ghaffari, R., Guignard, D., Hallmark, N., Hannas, B.R., Jacobi, S., Marxfeld, H.A., Melching-Kollmuss, S., Sheets, L.P., Urbisch, D., Botham, P.A. and van Ravenzwaay, B. (2022): Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny-part III: how is substance-mediated thyroid hormone imbalance in pregnant/lactating rats or their progeny related to neurodevelopmental effects? Crit. Rev. Toxicol., 52, 546-617.
- Marty, S., Beekhuijzen, M., Charlton, A., Hallmark, N., Hannas, B.R., Jacobi, S., Melching-Kollmuss, S., Sauer, U.G., Sheets, L.P., Strauss, V., Urbisch, D., Botham, P.A. and van Ravenzwaay, B. (2021): Towards a science-based testing strategy to identify maternal thyroid hormone imbalance and neurodevelopmental effects in the progeny - part II: how can key events of relevant adverse outcome pathways be addressed in toxicological assessments? Crit. Rev. Toxicol., 51, 328-358.
- McClain, R.M., Levin, A.A., Posch, R. and Downing, J.C. (1989): The effect of phenobarbital on the metabolism and excretion of thyroxine in rats. Toxicol. Appl. Pharmacol., 99, 216-228.
- Miller, M.D., Crofton, K.M., Rice, D.C. and Zoeller, R.T. (2009): Thyroid-disrupting chemicals: interpreting upstream biomarkers of adverse outcomes. Environ. Health Perspect., 117, 1033-1041.
- Minami, K., Sato, A., Tomiyama, N., Ogata, K., Kosaka, T., Hojo, H., Takahashi, N., Suto, H., Aoyama, H. and Yamada, T. (2024): Prenatal test cohort of a modified rat comparative thyroid assay adding brain thyroid hormone measurements and histology but lowering group size appears able to detect disruption by sodium phenobarbital. Curr. Res. Toxicol., 6, 100168.
- Minami, K., Suto, H., Sato, A., Ogata, K., Kosaka, T., Hojo, H., Takahashi, N., Tomiyama, N., Fukuda, T., Iwashita, K., Aoyama, H. and Yamada, T. (2023): Feasibility study for a downsized comparative thyroid assay with measurement of brain thyroid hormones and histopathology in rats: case study with 6-propylthiouracil and sodium phenobarbital at high dose. Regul. Toxicol. Pharmacol., 137, 105283.
- Mizuta, H., Amino, N., Ichihara, K., Harada, T., Nose, O., Tanizawa, O. and Miyai, K. (1983): Thyroid hormones in human milk and their influence on thyroid function of breast-fed babies. Pediatr. Res., 17, 468-471.
- Moriyama, M., Yamashita, S., Domoto, H., Furuno, K., Araki, H. and Gomita, Y. (1999): Determination of plasma phenobarbital concentration by high-performance liquid chromatography in rat offspring. J. Chromatogr. B Biomed. Sci. Appl., 723, 301-305.
- Murk, A.J., Rijntjes, E., Blaauboer, B.J., Clewell, R., Crofton, K.M., Dingemans, M.M., Furlow, J.D., Kavlock, R., Köhrle, J., Opitz, R., Traas, T., Visser, T.J., Xia, M. and Gutleb, A.C. (2013): Mechanism-based testing strategy using in vitro approaches for identification of thyroid hormone disrupting chemicals. Toxicol. In Vitro, 27, 1320-1346.
- Noyes, P.D., Friedman, K.P., Browne, P., Haselman, J.T., Gilbert, M.E., Hornung, M.W., Barone, S. Jr., Crofton, K.M., Laws, S.C., Stoker, T.E., Simmons, S.O., Tietge, J.E. and Degitz, S.J. (2019): Evaluating chemicals for thyroid disruption: opportunities and challenges with in vitro testing and adverse outcome pathway approaches. Environ. Health Perspect., 127, 95001.
- O’Connor, J.C., Frame, S.R., Davis, L.G. and Cook, J.C. (1999): Detection of thyroid toxicants in a tier I screening battery and alterations in thyroid endpoints over 28 days of exposure. Toxicol. Sci., 51, 54-70.
- O’Shaughnessy, K.L. and Gilbert, M.E. (2020): Thyroid disrupting chemicals and developmental neurotoxicity - New tools and approaches to evaluate hormone action. Mol. Cell. Endocrinol., 518, 110663.
- O’Shaughnessy, K.L., Thomas, S.E., Spring, S.R., Ford, J.L., Ford, R.L. and Gilbert, M.E. (2019): A transient window of hypothyroidism alters neural progenitor cells and results in abnormal brain development. Sci. Rep., 9, 4662.
- O’Shaughnessy, K.L., Kosian, P.A., Ford, J.L., Oshiro, W.M., Degitz, S.J. and Gilbert, M.E. (2018): Developmental thyroid hormone insufficiency induces a cortical brain malformation and learning impairments: A cross-fostering study. Toxicol. Sci., 163, 101-115.
- OECD (2018): Revised Guidance Document 150 on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption. OECD Series on Testing and Assessment, No. 150, OECD Publishing, Paris. https://doi.org/10.1787/9789264304741-en. Accessed Aug 7, 2024.
- OECD. (2020): Overview of Concepts and Available Guidance Related to Integrated Approaches to Testing and Assessment (IATA); OECD Series on Testing and Assessment, No 329; OECD, Paris, France. https://doi.org/10.1787/cd920ca4-en. Accessed Aug 7, 2024.
- Ogata, K., Suto, H., Sato, A., Maeda, K., Minami, K., Tomiyama, N., Kosaka, T., Hojo, H., Takahashi, N., Aoyama, H. and Yamada, T. (2024): Optimal testing time for cerebral heterotopia formation in the rat comparative thyroid assay, a downstream indicator for perinatal thyroid hormone insufficiency. J. Toxicol. Pathol., 37, 173-187.
- Okuda, Y., Kushida, M., Sumida, K., Nagahori, H., Nakamura, Y., Higuchi, H., Kawamura, S., Lake, B.G., Cohen, S.M. and Yamada, T. (2017): Mode of action analysis for rat hepatocellular tumors produced by the synthetic pyrethroid momfluorothrin: evidence for activation of the constitutive androstane receptor (CAR) and mitogenicity in rat hepatocytes. Toxicol. Sci., 158, 412-430.
- Reyns, G.E., Venken, K., Morreale de Escobar, G., Kühn, E.R. and Darras, V.M. (2003): Dynamics and regulation of intracellular thyroid hormone concentrations in embryonic chicken liver, kidney, brain, and blood. Gen. Comp. Endocrinol., 134, 80-87.
- Rice, D. and Barone, S. Jr. (2000): Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environ. Health Perspect., 108 (Suppl 3), 511-533.
- Savu, L., Vranckx, R., Maya, M. and Nunez, E.A. (1987): A thyroxine binding globulin (TBG)-like protein in the sera of developing and adult rats. Biochem. Biophys. Res. Commun., 148, 1165-1173.
- USEPA. (2005): Guidance for thyroid assays in pregnant animals, fetuses and postnatal animals, and adult animals, Office of Pesticide Programs Health Effects Division, Washington DC. https://www.epa.gov/pesticide-registration/guidance-thyroid-assays-pregnant-animals-fetuses-and-postnatal-animals-and. Accessed Aug 7, 2024.
- Yamada, T., Cohen, S.M. and Lake, B.G. (2021): Critical evaluation of the human relevance of the mode of action for rodent liver tumor formation by activators of the constitutive androstane receptor (CAR). Crit. Rev. Toxicol., 51, 373-394.
- Zoeller, R.T. and Rovet, J. (2004): Timing of thyroid hormone action in the developing brain: clinical observations and experimental findings. J. Neuroendocrinol., 16, 809-818.
- Zoeller, R.T. and Crofton, K.M. (2005): Mode of action: developmental thyroid hormone insufficiency--neurological abnormalities resulting from exposure to propylthiouracil. Crit. Rev. Toxicol., 35, 771-781.
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