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?

Figures & data

Table 1. Overview of thyroid-related AOPs including neurodevelopmental outcomes in mammals in the AOP Wiki (as per 16 October 2020).

Abbreviations: AO: adverse outcome; AOP: adverse outcome pathway; fT4: free thyroxine; GABA: gamma amino-butyric acid; KE: Key event; KER: key event relationship; MIE: molecular initiating event; NIS: sodium iodide symporter; PXR: pregnane X Receptor; T4: thyroxine; TPO: thyroid peroxidase; TTR: transthyretin; UGT: uridine diphosphate glucuronyltransferase.

Colour legend: Text colour = strength of evidence of KER: Green = strong; brown: moderate; blue: weak; black: MIE (since 2nd KE of respective KER is marked); OR: Information unavailable.

Shading of cell = quantitative evidence for KER (always marking 2nd KE of the respective KER): Green = strong; brown: moderate; blue: weak; white: MIE (since 2nd KE of respective KER is marked); OR: Information unavailable.

The bold boxes around “T4 in serum; decrease” and “T4 in neuronal tissue; decrease” serve to highlight that these key events are central to five of the six AOPs.

[a] Adverse outcome “cochlear function, decreased / loss” indicated in AOP Wiki as per 13 September 2019, and replaced by “cognitive function, decreased” as per 15 October 2019.

Table 2. Measurements of triiodothyronine, thyroxine or thyroid stimulating hormone in standard rodent toxicity studies.

Test guideline	Total T3	Total T4	TSH	Assessments of the thyroid gland [a]
OECD TG 408: Repeated dose 90-day oral toxicity study in rodents [b]	At study termination
OECD TG 414: Prenatal developmental toxicity study	GD 20/21: Adults
OECD TG 421: Reproduction/developmental toxicity screening	–	Adults; PND 4 pups optional; PND 13 pups; PND 13 dams optional		Adults; PND 13 pups optional
OECD TG 422: Combined repeated dose toxicity study & reproduction/developmental toxicity screening	–	Adults; PND 4 pups optional; PND 13 pups; PND 13 dams optional		Adults (n = 5); pups and remaining adults optional
OECD TG 443: Extended one-generation reproductive toxicity study [c]	–	P0/F1 adults at termination; PND 4 pups optional; PND 22 pups	P0/F1 adults at termination; PND 22 pups	P0/F1 adults at termination; PND 22 pups optional
OCSPP 890.1450: Pubertal development and thyroid function in intact juvenile/Peripubertal female rats	–	PND 42
OSCPP 890.1500: pubertal development and thyroid function in intact juvenile/Peripubertal male rats	–	PND 53
US EPA (2005) comparative thyroid assay	GD 20 dams/foetuses; PND 21 dams/pups

Table modified from a presentation by Sue Marty, Dow Chemical Company, at the HESI DART Committee Thyroid Hormone Assessment Workshop in Washington DC, USA; 9-10 May 2019; https://hesiglobal.org/wp-content/uploads/sites/11/2019/05/Marty-HESI-Regulatory-Requirements-for-Evaluation-9-May-2019-v-1A.pdf.

GD: gestation day; PND: postnatal day; -: not required or not mentioned.

Note: (Optional) can also be interpreted to mean ‘if relevant’, which is undefined.

[a] Assessments of the thyroid gland include necropsy (gross inspection), measurement of absolute and relative weight and histopathological investigations.

[b] Thyroid endpoints are optional as per OECD 407 Repeated Dose 28-day Oral Toxicity Study

[c] Relates to Cohort 1 A.

Table 3. Neurological and neurodevelopmental parameters included in standard rodent toxicity studies.

Test guideline	Pathological and histopathological evaluation of the brain	Further neuropathological evaluations	Neurological / neurobehavioural evaluations
OECD TG 408: 90-day oral repeated-dose toxicity study in rodents	Brain necropsy and HP (representative regions of cerebrum, cerebellum, medulla/pons)	Eyes (retina, optic nerve) peripheral nerve, muscle, spinal cord	Sensory reactivity to various stimuli (e.g. visual, auditory, proprioceptive); grip strengths, motor activity (at 11 weeks or later)
OECD TG 414: prenatal developmental toxicity study	Soft tissue alteration (foetuses)	None	None (study termination before delivery)
OECD TG 421: reproduction/developmental toxicity screening	None	None	None in the pups (apart from general clinical observations)
OECD TG 422: combined repeated dose toxicity study & reproduction/developmental toxicity screening	Brain weight and HP (representative regions of cerebrum, cerebellum und medulla/pons) adults	Eyes (retina, optic nerve) peripheral nerve, muscle, spinal cord	None in the pups (apart from general clinical observations)
OECD TG 426: developmental neurotoxicity study	Brain necropsy, weight, HP and morphometry at PND 22 and approx. PND 70 (termination); all major brain regions [a]	Neuropathology at PND 22 and approx. PND 70 (termination); representative sections of spinal cord and peripheral nerves [b]	Detailed clinical observations, behavioural ontogeny (2× before weaning), motor activity (1–3× at or before weaning; once in adults), motor and sensory function (1× post-weaning PND 25; 1× adults PND 60–70), learning and memory (1× post-weaning PND 25; 1× adults PND 60–70)
OECD TG 443: extended one-generation reproductive toxicity study	P0 and F1 (Cohort 1A) adults (PND 90): Brain necropsy, weight, HP F1 (Cohort 2A) adults (PND 90): Brain necropsy, weight, HP [c], morphometry (perfusion) F1 (Cohort 2B) weanlings (PND 21 or 22): Brain necropsy, weight, HP [c] (optional perfusion) Unselected F1 weanlings (PND 21 or 22): Brain necropsy, weight (preserved)	P0 and F1 (Cohort 1A) adults: HP: peripheral nerve, muscle, spinal cord, eye plus optic nerve F1 (Cohort 2A) adults: HP: eyes (retina, optic nerve) peripheral nerve, muscle, spinal cord	P0 and F1: detailed clinical observations F1 (Cohort 2A): Auditory startle (PND 24), functional observational battery (PND 63–75), motor activity (PND 63–75)
OCSPP 890.1450/1500: pubertal development and thyroid function in intact juvenile/peripubertal female/male rats	None (apart from pituitary weight)	None	None (apart from clinical observations)
US EPA comparative thyroid assay	None	None	None (apart from clinical observations)

GD: gestation day; HP: histopathology; PND: postnatal day.

Note: (Optional) can also be interpreted to mean ‘if relevant’, which is undefined.

[a] Olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, midbrain (tectum, tegmentum, and cerebral peduncles), pons, medulla oblongata, cerebellum.

[b] Including eye with optic nerve and retina, spinal cord at cervical and lumbar swellings, dorsal and ventral root fibres, proximal sciatic nerve, proximal tibial nerve (at knee), and tibial nerve calf muscle branches (transverse and longitudinal for spinal cord and peripheral nerves).

[c] For Cohorts 2A and 2B, brain HP includes olfactory bulbs, cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus, mid-brain (thecum, tegmentum, and cerebral peduncles), brain-stem and cerebellum.

Figure 4. Free T4 and tT4 reference ranges for humans (males, pregnant women, infants) as compared to tT4 normal distribution for Wistar rats (males, lactating dams, pups): Ranges from median to 2.5th percentile, relative to the median. fT4: free thyroxine; GD: gestational day; LD: lactational day; N: number of individuals; Trim: trimester; tT4: total thyroxine. Colour legend: grey columns: human fT4 data; striped columns: human tT4 data; black columns: rat tT4 data. Note that T4 variation only attains -50% as compared to TSH variation attaining up to 210% (Figure 3). Age/life stages (number of individuals): human males: 20–39 years (fT4/tT4: N = 286/130); Wistar rat males: 20 weeks (N = 486); pregnant women: 1st trimester (fT4/tT4: N = 418/417) and 3rd trimester (fT4/tT4: N = 169/169); Wistar rat dams: GD20 (N = 368) LD14 (N = 56); infants: 6 days to 3 months (fT4/tT4: N = 111/99); children: 1–6 years (fT4/tT4: N = 344/341); Wistar rat pups: postnatal day 13 (males/females: N = 398/399). The human fT4/tT4 reference ranges were taken from Roche (2009). The rat normal tT4 distribution ranges were measured in control rats at BASF SE, Ludwigshafen (Germany); see Supplementary Information SI-2 for methodological details and absolute data.

Figure 1. Reasonably detectable T4 and TSH level change [%] – dependent on group size. The percent T4 and TSH level change that is expected to be significant (and hence “reasonably detectable”) is shown for different group sizes. It is presumed that the concurrent control group and the treatment groups have the same maximal allowed coefficient of variation (CV) as per OECD TG 407 (25% for T3/T4 and 35% for TSH); statistical analysis: two-sided Wilcoxon test, power 75%; p < 0.05; software NQUERY.

Figure 2. Reasonably detectable T4 and TSH level change [%] – dependent on coefficient of variation (CV). The percent T4 and TSH level change that is expected to be significant (and hence “reasonably detectable”) assuming group sizes of N = 10 (for both the concurrent control group and the treatment group) was calculated using the formula p = effect size × CV, where p = percent decrease and effect size for N = 10 is 1.406. Statistical analysis: two-sided Wilcoxon test, power 75%; p < 0.05; software NQUERY); see Li et al. (2019) for further details.

Figure 3. TSH reference ranges for humans (males, pregnant women, infants) compared to TSH normal distributions for Wistar rats (males, lactating dams, pups): Ranges from median to 97.5th percentile, relative to the median. GD: gestational day; LD: lactational day; N: number of individuals; Trim: trimester; TSH: thyroid stimulating hormone. Colour legend: Striped columns: human data; black columns: rat data. Note that TSH variation attains up to 210% as compared to T4 variation attaining only -50% (Figure 4). Numbers of individuals; age/life stages: Human males: 20–39 years (N = 286); Wistar rat males: 20 weeks (N = 304); pregnant women: 1st trimester (N = 418) and 3rd trimester (N = 170); Wistar rat dams: GD20 (N = 364) and LD14 (N = 48); infants: 6 days to 3 months (N = 119); children: 1–6 years (N = 346); Wistar rat pups: postnatal day 13 (males/females: N = 222/222). The human TSH reference ranges were adapted from Roche (2009). The rat normal TSH distribution ranges were measured in control rats at BASF SE, Ludwigshafen (Germany); see Supplementary Information SI-2 for methodological details and absolute data.

US Environmental Protection Agency [US EPA]. 2005. Guidance for thyroid assays in pregnant animals, fetuses and postnatal animals, and adult animals. Washington (DC): US EPA, Office of Pesticide Programs, Health Effects Division.

 Google Scholar

Roche. 2009. Elecsys thyroid tests. Reference intervals for children and adults. D-Mannheim: Roche Diagnostics GmbH.

 Google Scholar

Li AA, Makris SL, Marty MS, Strauss V, Gilbert ME, Blacker A, Zorrilla LM, Coder PS, Hannas B, Lordi S, et al. 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.

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