Investigations on learning and memory function in extended one-generation reproductive toxicity studies – when considered needed and based on what?

Figures & data

Table 1. Summary of the three publications in which L&M was investigated.

	Axelstad et al. (2008)	van Wijk et al. (2008)	Amano et al. (2018)
Aim of the test	To link L&M findings to rat maternal or developmental T4 levels via linear regression analysis	To investigate effects of perinatal and chronic hypothyroidism during development on L&M in both male and female offspring of hypothyroid rats	To investigate L&M in adult male mice offspring exposed during the perinatal period
Test substance	Propylthiouracil (PTU)	To induce hypothyroidism, dams and offspring were fed an iodide-poor diet and drinking water with 0.75% sodium perchlorate	Propylthiouracil (PTU)
Animal species	Rat	Rat	Mouse
Test set-up	22 dams/group were dosed via gavage once a day with 0, 0.8, 1.6, or 2.4 mg PTU/kg bw. At PND 16, litter size was standardized to 3 males and 3 females, where possible. From the offspring, 1–3 males and 1–2 females from each litter were weaned on PND 27. After weaning each of the 4 dose groups consisted of 2 subgroups of animals: a group (1) consisting of 18–20 male and 18–20 female pups from 16 to 17 different litters per group; these animals were used for motor activity tests, Morris test and assessment of hearing. And a group (2) consisting of 18–20 male rats per dose group (from 17 litters per group); these animals were tested in the RAM test	Two weeks prior to mating, in one group of 6 females, hypothyroidism was induced; another group of 6 females served as control. Four days after birth, the litters were sexed and subsequently divided between 6 foster mothers (4 hypothyroid dams and 2 control dams). From PND 14 onward, 2 hypothyroid dams received the control diet (“hypo-normal” group). Thus, 3 treatment groups were formed for both sexes: a control group (“C” group), a hypothyroid group (“H” group) and a hypo-normal group (“H-N” group), each consisting of 2 dams with 8 pups each (4 males and 4 females). Offspring was dosed either until the day of euthanasia on PND 76 (“H” group) or only until weaning on PND 29 (“H-N” group) to test for reversibility of effects observed. Cognitive function was compared with the (“C” group)	Pregnant and lactating mice (number not indicated) received PTU via drinking water, 0 ppm for control, 5 ppm for the mild and 50 ppm for the moderate hypothyroid model
Dosing/treatment period	From GD 7 to PND 17	Dams: 2 weeks prior to mating until PND 14 or until PND 29 Pups: either until weaning on PND 29 or until PND 76	From GD 14 to PND 21
Type of L&M testing used	Morris water maze test and RAM test	Morris water maze test	ORT (Object Recognition Test) and OLT (Object in-Location Test)

Table 2. Table of deficiencies of the three publications in which L&M was investigated, including deviations from OECD TG 426.

	Axelstad et al. (2008)	van Wijk et al. (2008)	Amano et al. (2018)
OECD TG 426/443 recommend a total of 20 litters at each dose level; in OECD TG 426 it is also indicated that selection of pups should be performed so that, to the extent possible, both sexes from each litter in each dose group are equally represented in all tests	Although the study was started with 22 dams/group, tests were performed with pups from 16 to 17 litters per group. No information/explanation was provided on this discrepancy. Were the other dams not pregnant? It is therefore remarkable that in the abstract of the study it is simply, and in fact incorrectly, mentioned that “16–17 pregnant rats were dosed with PTU”	Three treatment groups were used: a control “C” group, a hypothyroid “H” group and a hypo-normal “H-N” group, each consisting of 2 dams with 8 pups each (4 males and 4 females)	The number of mice used is not stated explicitly. Only in the Results section (in Fig. 4 of the paper) it is mentioned that 6 control, 5 low dose and 8 high dose dams were used, and 20 control, 14 low dose and 17 high dose pups. It is not clear how many pups were used for ORT and OLT testing and n is not stated in any of the other figures and tables. The only indication is that 1–3 male pups per litter were used in each group and that pups from at least 3 dams were used in each experiment. In Table 2 of the paper, it is indicated that serum samples from 3 pups were collected in one tube, and 5 tubes were used for the measurement; this is in contradiction with the number of 20, 14, and 17 pups indicated in Fig. 4 of the paper
Information on test substance intake and/or deficiency status, and analytical verification of the concentrations in gavage solutions, diet or drinking water	No analytical verification of the gavage solutions (not reported)	No analytical verification and no measurement of dietary and drinking water intake (not reported); thus, except for information on thyroid hormone (TH) status, there is no information on possible variations in thyroid toxicity in dams and pups related to differences in intake	No analytical verification of concentrations in drinking water and no measurement of drinking water intake (not reported); in this way, there is no information on actual PTU intake
Required dosing period of dams (OECD TG 426): minimum from the time of implantation (GD 6) throughout lactation (PND 21)	Dosing from GD 7 to PND 17	Dams were fed an iodide-poor diet already starting 2 weeks prior to mating until PND 14 or PND 29	Dosing from GD 14 to PND 21
According to OECD TG 426: only dams should be dosed	No dosing of offspring	Offspring was fed an iodide-poor diet and drinking water with 0.75% sodium perchlorate until weaning (“H-N” group) on PND 29 or until the day of euthanasia (“H” group) on PND 76	No dosing of offspring
Randomization and blinding	Both the selection method and blind testing were not reported	Both the selection method and blind testing were not reported	Both the selection method and blind testing were not reported
Use of both sexes	Male rats were used in the RAM test without any further explanation why only one sex was used and not both sexes	Both male and female rats were used	Male mice were used without any further explanation
TH measurements in serum samples	T4	TH (T3, T4, TSH) measured in plasma; however, no information provided on validation or historical control data	TSH, free T3 (fT3), free T4 (fT4)
Time of blood sampling	Not reported	Not reported	Not reported
Storage of collected samples for TH analysis and TH assay validation	No stability testing of T4 under selected storage conditions reported	No stability testing of T3, T4, and TSH under selected storage conditions reported	No stability testing of T3, T4, and TSH under selected storage conditions reported
TH analysis	Only total serum T4 was measured. In addition, on PND 16 blood was pooled for all male and all female pups within each litter, thereby not taking individual variation into account. Also, pregnant F0 rats were set on a reverse light–dark cycle at their arrival on GD 4	Plasma TSH concentrations were significantly higher in the “H” pups and similar to the control values in the “H-N” pups; Plasma T4 and T3 levels were significantly lower in the “H” pups and comparable to control values in the “H-N” pups	Statistically significantly differences in TH but results were contradicting. In dams of the high dose group, TSH was higher and fT3 and fT4 lower than control; in dams of the low dose group only fT4 was lower than control. However, in pups of the high dose group, fT4 was lower than control, whereas in the low dose group fT4 was higher than control
Period of L&M investigations not in line with OECD TG 426: TG requires testing at two time points, viz. at PND 25 ± 2 for young pups and at PND 60–70 for testing young adults	Morris water maze test: performed at or around the 2nd time point (8–9 weeks of age) RAM test: performed at 5–6 months	Investigations only performed at or around the 2nd time point (PND 61–65)	Investigations only performed at or around the 2nd time point (PND 56 and PND 84)

Table 3. Summary of cognitive findings in the three publications.

	Axelstad et al. (2008)	van Wijk et al. (2008)	Amano et al. (2018)
Findings on cognitive performance are contradicting within and between the studies	The Morris water maze test (with males and females) did not show any changes in cognitive performance whereas in the RAM test (with males only) statistically significant changes were noted	All rats, from all experimental groups and of both sexes, learned to find the platform, but all “H” pups and the “H-N” female pups (but not the males of this group) showed a statistically significantly increased cumulative search error. The rate of learning was found to be different between the female experimental groups (not in males), with “H” female pups underperforming compared with their peers	On PND 56, OLT performance was statistically significantly lower in both PTU-treated groups compared to controls; however, a dose–response relationship was lacking regardless the 10 times difference in PTU concentration between the two dose groups; no differences in ORT performance were seen. On PND 84, there was only a statistically significant difference at 50 ppm PTU in the OLT, and again there was no statistically significant difference in the ORT

Table 4. Correlation between findings on thyroid histopathology and weight, and cognitive testing in the three publications.

	Axelstad et al. (2008)	van Wijk et al. (2008)	Amano et al. (2018)
Correlation between findings on thyroid histopathology and weight, and cognitive testing	Even in the presence of moderate to marked thyroid histopathological changes and strong increases in thyroid weight (up to more than 2× control) with the known thyroid toxicant PTU, L&M testing in the offspring showed inconsistent findings, viz. a negative Morris water maze test and a RAM test with only weak associations to T4 levels	No histopathological examination of the thyroid and no thyroid weight determined; thus, no correlation possible	No histopathological examination of the thyroid and no thyroid weight determined; thus, no correlation possible

Axelstad M, Hansen PR, Boberg J, Bonnichsen M, Nellemann C, Lund SP, Hougaard KS, Hass U. 2008. Developmental neurotoxicity of propylthiouracil (PTU) in rats: relationship between transient hypothyroxinemia during development and long-lasting behavioural and functional changes. Toxicol Appl Pharmacol. 232(1):1–13. doi: 10.1016/j.taap.2008.05.020.

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van Wijk N, Rijntjes E, van de Heijning BJ. 2008. Perinatal and chronic hypothyroidism impair behavioural development in male and female rats. Exp Physiol. 93(11):1199–1209. doi: 10.1113/expphysiol.2008.042416.

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Amano I, Takatsuru Y, Khairinisa MA, Kokubo M, Haijima A, Koibuchi N. 2018. Effects of mild perinatal hypothyroidism on cognitive function of adult male offspring. Endocrinology. 159(4):1910–1921. doi: 10.1210/en.2017-03125.

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