This is a paper for discussion.

This does not represent the views of the committee and should not be cited.

Introduction

1.             This paper is part of a series of papers supporting the COT assessment of the toxicological assessment of PFAS. This paper provides the evidence on in vivo thyroid toxicity. Later papers will include new approach methodologies (NAMS) and human evidence, and other endpoints including developmental, liver toxicity and immunotoxicity.

Background

2.             In 2020 the European Food Safety Agency (EFSA) published an opinion “Risk to human health related to the presence of perfluoroalkyl substances in food” in which tolerable weekly intake (TWI) for perfluoroalkyl substances (PFASs) of 4.4 ng/kg bw/week was determined (EFSA., 2020). This was based on a human study from which the lowest benchmark dose of 17.5 ng/mL serum was calculated for 1-year old children, based on the sum of four PFAS. This serum value was extrapolated to long-term maternal exposure of 0.63 ng/kg bw/day using PBPK modelling, which was converted to the TWI due to the accumulation of PFAS over time.

3.             This TWI is lower than EFSA’s previous tolerable intake of 13 ng/kg bw/week for PFOS and 6 ng/kg bw/week for perfluorooctanoic acid (PFOA) (EFSA., 2018) based on increased serum cholesterol in adults and a decrease in antibody response at vaccination in children, and increased serum cholesterol, respectively. Liver toxicity and a reduction in birth weight were also considered for PFOA.

4.             The COT has previously considered PFAS on a number of occasions (see summary in TOX/2022/53), and has recently published a statement on the EFSA opinion. A paper summarising health-based guidance values (HBGV) was presented in December 2022 (TOX/2022/67) and following agreement in March 2023 the PFAS subgroup was established and an interim position published outlining future work.

5.             This paper is the first in a series of papers supporting the COT subgroup on the risk assessment of PFAS.

Literature search

6.             Search terms used previously by (EFSA, 2018) and EFSA (2020) were replicated. Such search terms, inclusion and exclusion criteria and the search results are presented in Annex A to this paper.

Toxicity of PFAS

7.             In vivo thyroid toxicity data following acute exposure to perfluoroalkyl carboxylic acids (PFCAs) and perfluorosulfonic acids (PFSAs) are presented in Table 1 and Table 2, respectively, and thyroid toxicity effects following repeated exposure are presented in Table 3 and Table 4, respectively. Narrative summaries of the studies are provided below.

 

PFBS

In vivo toxicity data

Feng et al. 2017

8.             Feng et al. (2017) investigated the effects of perfluorobutanesulfonate (PFBS) on levels of thyroid hormones (TH) in pregnant mice and their female offspring. As part of a developmental study, pregnant ICR mice were administered daily doses of PFBS potassium salt (K⁺PFBS) at doses 0, 50, 200, and 500 mg/kg bw/day by gavage from gestation day 1 (GD1) to GD20. Upon delivery offspring were housed with dams until weaning on postnatal day 21 (PND21).

9.             Dams were split into two experimental groups examining thyroid effects. In the first experiment, TH levels and relative mRNA expression were analysed using orbital blood collected from dams on GD20 and female offspring on either PND1, PND30 or PND60. Hypothalami were removed from female offspring on PND1, PND30 or PND60.

10.             Total thyroxine (TT4), total triiodothyronine (TT3) and thyroid stimulating hormone (TSH) in serum were measured in dams (10/group) and 50 offspring: PND1 (30/group), PND30 (10/group) and PND60 (10/group). Free thyroxine (FT4) was also measured in dams (10/group). Gene expression for Trh (thyrotropin-releasing hormone) mRNA was determined from hypothalamus samples.

11.             In the second experiment, serum PFBS levels were measured in orbital blood collected from dams (10/group) on GD20.

12.             Mortality: No deaths were observed in offspring. No data were provided on maternal survival.

13.             General toxicity and body weight: No clinical signs of general toxicity in offspring were observed. No data regarding dams were reported. Female offspring body weight was significantly decreased at 200 and 500 mg/kg bw/day from PND1 to PND60, compared with controls. No data regarding male offspring were provided. Terminal body weight and body weight gain in dams was unaffected by treatment.

14.             Thyroid hormone levels: In offspring, TT3 and TT4 levels were significantly decreased at 200 and 500 mg/kg bw/day on PND1, PND30 and PND60. TSH levels were significantly elevated at 200 and 500 mg/kg bw/day on PND30. In dams, TT4, TT3 and FT4 levels were significantly reduced, and TSH levels were significantly increased at 200 and 500 mg/kg bw/day groups. Hypothalamic gene expression: In offspring, Trh mRNA levels in were significantly elevated at 200 and 500 mg/kg bw/day on PND30.

15.             Serum PFBS concentrations: PFBS treatment on GD1 – 20 led to a dose-dependent increase in serum PFBS concentration in dams. PFBS concentrations measured on GD20 were 1.73 ng/mL (control), 74.0 ng/mL (50 mg/kg bw/day), 332 ng/mL (200 mg/kg bw/day) and 721 ng/mL (500 mg/kg bw/day).

16.          The authors concluded that prenatal exposure to PFBS ≥ 200 mg/kg bw/day causes permanent hypothyroxinemia in female mice.

NTP, 2022b

17.          NTP (2022b) investigated the effects of PFBS on thyroid weight, histopathology and TH levels in rats. In a repeated dose study, SD rats (10/sex/group) were administered PFBS at doses 0, 62.6, 125, 250, 500 or 1000 mg/kg bw/day with half the dose being administered twice daily by gavage for 28 days. At necropsy on day 29, blood samples were collected for TT4, TT3, FT4, TSH and PFBS analysis, and thyroids were removed for histopathological evaluation.

18.          Mortality: In males, all ten rats died at 1000 mg/kg bw/day before scheduled necropsy (one from a dosing accident). In females, eight rats died at 1000 mg/kg bw/day, one rat at 500 mg/kg bw/day and one rat at 250 mg/kg bw/day. With the exception of the dosing accident, the observed mortality was attributed to treatment.

19.          General toxicity and body weight: Seizures were noted in one male rat at 1000 mg/kg bw/day. In females, seizures were noted in one rat at 1000 mg/kg bw/day, two rats at 500 mg/kg bw/day and one rat at 250 mg/kg bw/day. In females at 1000 mg/kg bw/day, lethargy was reported in one rat, ruffled hair was reported in two rats, and two were reported to be thin. In males, terminal body weights were unaffected by treatment, compared with controls. In females, terminal body weights were significantly reduced at 500 mg/kg bw/day.

20.          Gross pathology: Thyroid weights in males and females were unaffected by treatment.

21.          Histopathology: Histopathology in males and females was unaffected by treatment.

22.          Thyroid hormone levels: In males and females, TT4, FT4 and TT3 levels were significantly decreased at ≥ 62.6 mg/kg bw/day in a dose-response manner, compared with controls. TSH levels were unaffected by treatment.

23.          Plasma PFBS concentrations: In males, mean plasma PFBS concentrations on day 29 were 0.090 µg/mL (control), 2.222 µg/mL (62.6 mg/kg bw/day), 5.366 µg/mL (125 mg/kg bw/day), 12.430 µg/mL (250 mg/kg bw/day) and 43.160 µg/mL (500 mg/kg bw/day). There were no data for 1000 mg/kg bw/day due to mortality. In females, mean plasma PFBS concentrations on day 29 were ND (control), 0.154 µg/mL (62.6 mg/kg bw/day), 0.309 µg/mL (125 mg/kg bw/day), 0.931 µg/mL (250 mg/kg bw/day), 8.171 µg/mL (500 mg/kg bw/day) and 25.455 µg/mL (1000 mg/kg bw/day).

24.          The authors concluded that TT4, FT4 and TT3 decreased in a dose- response manner. TSH was unaffected, nor were there any histopathologic changes in the thyroid gland (hyperplasia/hypertrophy).

PFHxS

In vivo toxicity data

Butenhoff et al. (2009)

25.          Butenhoff et al. (2009) investigated the effect of potassium perfluorohexanesulfonate (K⁺PFHxS) on thyroid histopathology. In a modified OECD Test Guideline 422 study (Combined Repeated Dose Toxicity Study with Reproduction/Developmental Toxicity Screening), adult SD rats (18/sex/group) were administered K⁺PFHxS daily by gavage at doses 0, 0.3, 1, 3 or 10 mg/kg bw/day. Adult males were dosed for 14 days prior to cohabitation until day 43. Adult females were dosed for 14 days prior to cohabitation until PND21, or presumed GD25 for rats that did not deliver a litter. Offspring were not dosed directly but were exposed in utero or potentially via milk during the lactation period. In each treatment group, adults were assigned to the main study or for collection of serum samples for PFHxS concentration determination.

26.          Adults from the main study selected for histopathological evaluation were sacrificed on day 44 (males), or PND22 or GD25 (females). Thyroids were removed and submitted for histopathological analysis. Blood samples for serum PFHxS concentrations were collected from adults on day 14 (males and females). Adults selected for serum PFHxS concentrations were sacrificed on day 42 (males) and GD21 (females). Offspring selected for serum PFHxS concentrations were sacrificed on GD21 and PND22.

27.          Mortality: All adults survived to scheduled necropsy (no data on offspring reported).

28.          General toxicity and body weight: No clinical signs of general toxicity were observed in adults (no data on offspring reported). In males, significantly reduced body weight gain was observed at 10 mg/kg bw/day from day 1 to day 44, and at 0.3 and 3 mg/kg bw/day between day 29 to day 43, compared with controls. Regardless of these variations, body weights were unaffected by treatment. In females, significantly reduced body weights were observed at 10 mg/kg bw/day on PND8, compared with controls. Occasional significant reductions in body weight also occurred at 0.3, 3 and 10 mg/kg bw/day from PND4 to 14, compared with controls. However, body weight gain was unaffected by treatment. Food consumption was unaffected by treatment in adults.

29.          Histopathology: An increase in the incidence of mild to moderate hypertrophy was observed in the thyroid glands of males at 3 mg/kg bw/day and 10 mg/kg bw/day on day 44. The observed changes included hypertrophy and hyperplasia of the follicular epithelium cells. No treatment-related changes were observed in females at any dose.

30.          Serum PFHxS concentrations: Mean serum PFHxS concentrations in parental males on day 42 were 0.32 µg/mL (control), 44.2 µg/mL (0.3 mg/kg bw/day), 89.1 µg/mL (1 mg/kg bw/day), 129 µg/mL (3 mg/kg bw/day), and 202 µg/mL (10 mg/kg bw/day).

31.          The authors concluded that K⁺PFHxS-induced effects noted in parental males included increased hyperplasia of thyroid follicular cells at 3 mg/kg bw/day and 10 mg/kg bw/day.

Gilbert et al. 2021

32.          Gilbert et al. (2021) studied the effect of PFHxS on TH levels and thyroid-responsive genes. As part of a neurological developmental study in rats, pregnant Long-Evans rats (9/group and 6/control) were administered PFHxS at doses of 0 and 50 mg/kg bw/day daily by gavage from GD6 to PND21. Offspring were exposed in utero and during lactation only. Offspring were sacrificed on days PND0, PND2, PND6 and PND14 and tissues collected, dams were sacrificed on PND22 when pups were weaned.

33.          Blood was collected from dams on GD20 and PND22, and offspring on PND0, PND2, PND6 and PND14, and serum TT4, FT4, TT3 and TSH levels measured. Brain tissue T4 and T3 levels (assumed to be TT4 and TT3, respectively) were measured in offspring on PND0, PND2, PND6 and PND14. Samples collected for gene expression included thyroid glands from dams on PND22 and offspring on PND0, PND2 and PND14; liver from dams on PND22 and offspring on PND2, PND6 and PND14; and the anterior neocortex from offspring on PND14.

34.          Body weight: Body weights in dams were slightly but not significantly reduced by treatment. Body weights in offspring were unaffected.

35.          Gross pathology: Thyroid weight was not significantly different in offspring on PND14 compared with controls (no other data given). Thyroid hormone levels: In dams, TT4 and TT3 levels were significantly decreased on GD20 and PND22, and FT4 levels were significantly decreased on PND22, compared with controls (no data provided for GD20). No change was detected in TSH levels in dams on PND22 (no data provided for GD20). In offspring, TT4 levels were significantly decreased on PND0, PND2, PND6 and PND14; TT3 levels were significantly decreased on PND6 and PND14 (no data provided for PND0 and PND6); FT4 levels were significantly decreased on PND14 (no data provided for PND0, PND2, PND6), all compared with controls. No change was detected in TSH levels on PND14 (no data provided for PND0, PND2, PND6). A significant reduction in brain TT4 levels occurred in offspring on PND0, compared with controls. No changes were observed at subsequent ages. There were no treatment related effects in offspring brain TT3 levels at any age.

36.          Gene expression: The expression of thyroid gland genes involved in thyroid function (thyroid peroxidase, Tpo) or sodium-iodide symporter (Nis) was not significantly altered in samples collected from dams on PND22 or offspring on PND0, PND2 and PND14. No changes were seen in the relative expression of thyroid hormone responsive genes (Dio1, multidrug resistance 1(Mdra1), malic enzyme (ME), thyroid hormone-inducible hepatic protein (THRSP, or Spot14)) in livers taken from dams on PND22 and offspring on PND2, PND6 and PND14. Gene expression of eleven thyroid hormone- responsive genes remained unchanged in the neocortex of offspring on PND14.

37.          The authors concluded that PFHxS reduced serum TT4 but did not increase TSH, whilst thyroid-responsive genes in the liver, thyroid gland and brain were largely unchanged. Brain tissue TT4 was reduced in offspring on PND0, but despite persistent TT4 reductions in serum, had recovered in the PND2 offspring brain.

NTP, 2022b

38.          NTP (2022b) investigated the effects of PFHxS on thyroid weight, histopathology and TH levels in rats. In a repeated dose study, SD rats (10/sex/group) were administered K⁺PFHxS at doses 0, 0.625, 1.25, 2.5, 5 or 10 mg/kg bw/day) for males, or 0, 3.12, 6.25, 12.5, 25 or 50 mg/kg bw/day for females by gavage for 28 days. At necropsy on day 29, blood samples were collected for TT4, TT3, FT4, TSH and PFHxS analysis, and thyroids were removed for histopathological evaluation.

39.          Mortality: All rats survived to scheduled necropsy.

40.          General toxicity and body weight: No clinical signs of general toxicity were observed. Terminal body weights were unaffected by treatment, compared with controls.

41.          Gross pathology: Thyroid weights in males and females were unaffected by treatment.

42.          Histopathology: Histopathology in males and females was unaffected by treatment.

43.          Thyroid hormone levels: In males, TT4, FT4 and TT3 levels were significantly decreased at ≥ 0.625 mg/kg bw/day, compared with controls. TSH levels were unaffected by treatment. In females, TT4 levels were significantly decreased at ≥ 6.25 mg/kg bw/day, and FT4 levels were significantly decreased at ≥ 12.5 mg/kg bw/day. TT3 and TSH levels were unaffected by treatment.

44.          Plasma PFHxS concentrations: In males, mean plasma PFHxS concentrations on day 29 were 0.102 µg/mL (control), 66.760 µg/mL (0.625 mg/kg bw/day), 92.080 µg/mL (1.25 mg/kg bw/day), 129.000 µg/mL (2.5 mg/kg bw/day), 161.700 µg/mL (5 mg/kg bw/day) and 198.300 µg/mL (10 mg/kg bw/day). In females, concentrations on day 29 were 1.750 µg/mL (control), 37.030 µg/mL (3.12 mg/kg bw/day), 50.410 µg/mL (6.25 mg/kg bw/day), 63.820 µg/mL (12.5 mg/kg bw/day), 83.820 µg/mL (25 mg/kg bw/day) and 95.510 µg/mL (50 mg/kg bw/day). 

45.         The authors concluded that TT4 and FT4 decreased in a dose- response manner. TSH was unaffected, nor were there any histopathologic changes in the thyroid gland (hyperplasia/hypertrophy).

Ramhøj et al. 2018

46.          Ramhøj et al. (2018) investigated the effect of low doses of PFHxS on thyroid hormone levels in rats as part of two developmental studies evaluating PFHxS alone or in combination with an endocrine disruptor mix. In Study 1, pregnant Wistar rats (8/group) were administered PFHxS at doses 0, 25 or 45 mg/kg bw/day by gavage on GD7 to PND22. In Study 2, pregnant Wistar rats (16 – 20/group) were administered lower doses of PFHxS at doses 0, 0.05, 5 and 25 mg/kg bw/ day by gavage from GD7 to PND22.

47.          In Study 1, trunk blood was collected at sacrifice for TT4 measurement from offspring on PND16 and dams on PND22. Serum PFHxS concentrations were measured in dams (5 – 7/group) on PND22. In Study 2, tongue blood was collected from dams on GD15, and trunk blood was collected at sacrifice from male offspring on PND16, female offspring on PND17 and dams on PND22. TT4 levels were measured in these samples.

48.          General toxicity and body weight: No clinical signs of general toxicity were observed in dams or offspring in either study. Maternal weight and weight gain was also unaffected by treatment. In Study 2, male offspring body weight was slightly decreased at 25 mg/kg bw/day on PND0. There was no significant effect on female offspring birth weights in either study, or in males in Study 1. No significant effect was observed on offspring body weight or weight gain on PND6, PND14 or PND22 in either study.

49.          Thyroid hormone levels: Treatment with PFHxS reduced TT4 levels in both dams and offspring. In Study 1, TT4 levels in dams on PND22 were significantly reduced at both 25 and 45 mg/kg bw/day, compared with controls. TT4 levels in offspring on PND16 were significantly reduced at both 25 mg/kg bw/day and 45 mg/kg bw/day. In Study 2, TT4 levels in dams on GD15 and PND22 were significantly reduced at both 5 mg/kg bw/day and 25 mg/kg bw/day. TT4 levels in offspring on PND16/17 were significantly reduced at 5 and 25 mg/kg bw/day.

50.          Serum PFHxS concentrations: PFHxS concentrations in dams at PND22 were ND (controls), 139 µg/mL (25 mg/kg bw/day) and 174 µg/mL (45 mg/kg bw/day).

51.          Based on these results, the authors proposed that PFHxS is an effective thyroid hormone disruptor in rats as PFHxS administration significantly decreased serum TT4 levels in rat dams and their offspring. Significantly lower TT4 levels were seen at 5 mg/kg bw/day, after only 7 days of exposure.

52.          The authors concluded that PFHxS can induce marked reductions in circulating serum TT4 in rats, which at critical developmental stages can lead to altered brain morphology and adverse behaviour.

Ramhøj et al. 2020

53.          Ramhøj et al. (2020) investigated the effect of PFHxS on the thyroid system as part of a developmental study evaluating PFHxS alone or in combination with an endocrine disruptor mix. Pregnant Wistar rats (16 – 20/group) were administered K⁺PFHxS at doses 0, 0.05, 5 and 25 mg/kg bw/day by gavage on GD7 to PND22.

54.          Tongue blood was drawn from dams on PND15. Necropsy of one male and one female per litter took place on PND16 and PND17, respectively, and dams on PND22. Trunk blood was collected for measurement of TT3 and TSH. Thyroid glands were excised, weighed and saved for histopathology. Sections of thyroid glands from dams on PND22 and male offspring on PND16 were extracted for histopathological examination. A sub-set of female offspring was weaned and sacrificed at PND22 for collection of thyroid glands. Both lobes of the thyroid gland from female offspring on PND17 were extracted for RNA analysis. Expression levels of six gene transcripts involved in thyroid hormone synthesis and regulation were analysed, namely solute carrier family 5 (Slc5a5(NIS)), NK2 homeobox 1 (thyroid transcription factor 1, TTF-1) (Nkx2.1), Tpo, Thyroid stimulating hormone receptor (Tshr), Paired box 8 (Pax8) and Dio1. Serum PFHxS concentrations were not determined in this study.

55.          Note: TT4 levels reported in this study have been previously provided in the summary of Ramhøj et al. (2018) above, refer Paragraph 48. Ramhøj et al. (2018) reported exposure to PFHxS during gestation and lactation and showed dose-dependent reductions in serum TT4 levels in both offspring and dams.

56.          Gross pathology: Dose-dependent decreases in thyroid gland weight were observed in female offspring on PND22. Reductions were significant at 5 and 25 mg/kg bw/day compared with controls. There were no treatment- related effects on thyroid weights in dams on PND22 or female offspring on PND17. No data regarding males were presented.

57.          Histopathology: There were no treatment-related effects on maternal histopathology. Statistically significant mild histological changes were seen in male offspring on PND16 at 25 mg/kg bw/day compared with controls; however, these changes were no longer seen on PND22. No data regarding female offspring were presented.

58.          Thyroid hormone levels: In dams, a significant decrease in TT3 levels was observed at 25 mg/kg bw/day on PND22, compared with controls. TSH levels in dams were not altered by exposure to PFHxS. In offspring, a significant decrease in TT3 levels was observed at 25 mg/kg bw/day on PND16/17. TSH levels in offspring were not altered by exposure to PFHxS.

59.          Gene expression: There were no treatment related effects on expression levels of genes associated with thyroid hormone synthesis and regulation (Slc5a5(NIS), Nkx2.1, Tpo, Tshr, Pax8 and Dio1) in thyroid glands from dams on PND22. Similarly in female offspring on PND17, no treatment relate effects were detected in expression levels of four gene transcripts involved in thyroid hormone synthesis and regulation (Slc5a5 (NIS), Nkx2.1, Tpo, and Tshr).

60.          The study revealed that exposure to PFHxS resulted in marked reductions in TT4 levels and moderate reductions in TT3 in both dams and offspring, but had no significant effects on TSH levels in dams (PND22) or in offspring (PND16 and PND17). The authors propose that the results do not correspond to the classic view of the hypothalamic–pituitary–thyroid (HPT) axis, whereby TRH activates the pituitary to release TSH, which binds to TSH receptors on the thyroid gland, upregulating synthesis and release of thyroid hormone into the blood. The absence of effects on maternal TSH levels, thyroid gland weight, histopathology or gene expression indicate a lack of thyroid gland perturbation in response to exposure or as a compensatory reaction to decreases in serum T4.

61.          The authors concluded that PFHxS at doses up to 25 mg/kg bw/day lowered TH levels in both dams and offspring in a dose-dependent manner, but did not change TSH levels, thyroid weight, thyroid histology, or expression of marker genes of the thyroid gland. PFHxS reduced TT3 and TT4 in pregnant dams and their progeny, but did not appear to activate the HPT axis at doses up to 25 mg/kg bw/day.

In vivo toxicity data

Butenhoff et al. 2012

62.                Butenhoff et al. (2012) investigated the effects of perfluorooctane sulfonate (PFOS) exposure on thyroid toxicity and neoplastic potential in rats. In a combined toxicity and carcinogenicity study, SD rats (60-70/sex/group) were administered K⁺PFOS at doses 0, 0.5, 2, 5 or 20 mg/kg diet (equivalent to 0, 0.024, 0.098, 0.242 or 0.984 mg/kg bw/day for males, and 0, 0.029, 0.120, 0.299 or 1.251 mg/kg bw/day for females) in the diet for 104 weeks (103 weeks for females in the 0.120 mg/kg bw/day group); a subset of 10/sex/group of the high dose and controls were terminated at 52 weeks. A recovery group (40/sex) was administered 20 mg/kg diet (equivalent to 1.144 mg/kg bw/day for males, and 1.385 mg/kg bw/day for females) for the first 52 weeks of the study, after which they were administered a control diet until study termination. Necropsies were scheduled on week 53 after 52 weeks of treatment (10/sex/group) for controls, for males treated with 0.984 mg/kg bw/day group and females treated with 1.251 mg/kg bw/day and at study termination after 103 or 104 weeks of treatment for remaining rats for all groups. Thyroids were removed from males administered 0.984 mg/kg bw/day and from females treated with 1.251 mg/kg bw/day at scheduled necropsy during week 53. Thyroid tissue samples were collected at terminal necropsy for histopathological evaluation.

63.             Mortality: In males, mortality was significantly decreased at 0.299 and 1.251 mg/kg bw/day compared with controls. In females, mortality was significantly increased at 0.120 mg/kg bw/day. Mortality was unaffected by treatment in all other groups.

64.                General toxicity and body weight: No clinical signs of general toxicity were observed. In males, body weight was significantly decreased at 0.984 mg/kg bw/day (weeks 9 – 37), and at 1.144 mg/kg bw/day in the recovery group (weeks 9 – 37), compared with controls. In females, body weight was significantly decreased at 1.251 mg/kg bw/day (weeks 3 – 101), and at 1.385 mg/kg bw/day in the recovery group (weeks 3 – 61). In female rats sacrificed at 53 weeks, body weight was significantly decreased at 1.251 mg/kg bw/day. Body weights in males sacrificed at 53 weeks were unaffected by treatment. At terminal sacrifice, body weights in both males and females were unaffected by treatment.

65.                Gross pathology: In males, significantly decreased left thyroid/parathyroid weights were recorded at 0.984 mg/kg bw/day but were considered spurious by the authors due to there being no contralateral effect in the right thyroid/parathyroid and a lack of difference in organ-to-weight ratios between treated and control rats. In females, thyroid weights at 1.251 mg/kg bw/day were unaffected by treatment.

66.                Non-neoplastic lesions: There were no non-neoplastic microscopic observations attributed to treatment in thyroid tissues.

67.                Neoplastic lesions: In males, there was a significant increase in thyroid follicular cell adenoma and combined thyroid follicular cell tumours (adenoma and carcinoma) at 1.144 mg/kg bw/day in the recovery group, compared with controls. The authors noted that although the increased incidence of thyroid follicular cell tumours was outside the range of historical control values, there was no other microscopic evidence of thyroid abnormality. Further, the significantly increased incidence of thyroid follicular cell adenoma in the 1.144 mg/kg bw/day recovery group, without observation of similar increases at 0.984 mg/kg bw/day in males and/or at 1.251 mg/kg bw/day in females is contradictory and may represent a chance occurrence. In females, there was a significant increase in combined thyroid follicular cell adenoma and carcinoma at 0.299 mg/kg bw/day, compared with controls. The authors noted these tumours are known to occur in historical controls. In addition, the absence of either a dose–response or non-neoplastic thyroid findings suggests that the thyroid follicular cell tumours at 0.299 mg/kg bw/day in females were a spurious finding.

68.                Serum PFOS concentrations: In males, mean PFOS concentrations on week 105 were 0.012 µg/mL (control), 1.31 µg/mL (0.024 mg/kg bw/day), 7.60 µg/mL (0.098 mg/kg bw/day), 22.50 µg/mL (0.242 mg/kg bw/day) and 69.3 µg/mL (0.984 mg/kg bw/day). In females, mean PFOS concentrations on week 105 were 0.084 µg/mL (control), 4.35 µg/mL (0.029 mg/kg bw/day), 20.20 µg/mL on week 102 (0.120 mg/kg bw/day), 75.00 µg/mL (0.299 mg/kg bw/day) and 233.0 µg/mL (1.251 mg/kg bw/day).

69.                The authors concluded there were no anatomical indications of a response of the thyroid to dietary treatment with K⁺PFOS, including thyroid weight and microscopic histological changes. There were no treatment-related findings in thyroid tissue in rats fed K⁺PFOS through study termination.

Chang et al. 2008

70.                Chang et al. (2008) investigated the effect of PFOS on thyroid hormones and regulatory functions of the HPT axis in rats in a series of three experiments.

71.             In experiment 1, serum thyroid levels were collected to assess whether PFOS competes for thyroxine (T4) protein binding sites, and so resulting in transiently elevated FT4. Female Sprague-Dawley (SD) rats (5 – 15/group) were given either a single oral dose (assumed by gavage) of potassium-PFOS (K⁺PFOS) (15 mg/kg bw) or negative control. Rats were divided into three groups and sacrificed at either 2, 6 or 24 hours post-dosing and blood samples collected. Serum TSH, FT4, TT4, TT3, reverse triiodothyronine (rT3) levels and PFOS concentrations were measured. Liver tissues were collected, and hepatic biochemical markers, ME and UDP‑glucuronosyltransferase 1A (UGT1A) mRNA transcript levels as well as ME activity were measured as these can also reflect impacts on activity in the thyroid.

72.             Thyroid hormone levels: A transient significant increase of FT4 and decrease of TSH levels were seen after 6 hours. In contrast, these levels remained stable after 2 and 24 hours. A significant decrease in TT4 levels were seen after 2, 6 and 24 hours. TT3 and rT3 levels were relatively stable after 2 and 6 hours but then significantly decreased after 24 hours.

73.             Gene expression: ME mRNA transcripts were significantly increased after 2 hours returning to within control levels after 6 and 24 hours. ME activity was stable after 2 and 6 hours but significantly increased after 24 hours. Liver UGT1A mRNA transcripts were significantly elevated after 2 and 6 hours returning to control levels after 24 hours. The authors indicate that this may be representative of induction of increased glucuronidation and turnover of T4. Serum PFOS concentrations: Mean serum PFOS concentrations were significantly elevated at all time points compared with controls, peaking at 6 hours. Measured mean concentrations were <10 µg/mL (control), 37.28 µg/mL (2 hours), 66.90 µg/mL (6 hours) and 61.58 µg/mL (24 hours).

74.             The second experiment investigated whether transiently elevated levels of FT4 in response to PFOS exposure leads to increased turnover and elimination of T4. Male and female SD rats were injected with either 11 µCi (4/group, males) or 9.3 µCi (5/group, females) ¹²⁵I-labelled T4 (specific activity 1250 µCi/µg). This was followed by a single oral dose (assumed by gavage) of either 15 mg/kg bw K⁺PFOS or vehicle. Urine and faeces were collected over the 24-hour period following PFOS administration, whilst serum and liver were harvested after 24 hours. Serum TT4 levels were measured. Serum, liver, urine and faeces were all measured for ¹²⁵I radioactivity to determine elimination of T4.

75.             Thyroid hormone levels and turnover: TT4 levels were significantly reduced in males and females compared with controls after 24 hours and this correlated with a decrease in ¹²⁵I radioactivity in both sexes. Liver ¹²⁵I radioactivity was reduced in both males and females, urine and faecal ¹²⁵I radioactivity was significantly increased in males, and in faeces in females, indicating increased turnover and loss of thyroid hormones.

76.             In the third experiment, pituitary function was investigated by measuring release of TSH following inhibition of thyroid hormone synthesis in the thyroid. Propyl thiouracil (PTU) was used as a thyroid hormone synthesis inhibitor which leads to increased pituitary secretion of TSH. Adult male SD rats (6/group) were administered either negative control (gavage); K⁺PFOS only (3 mg /kg bw/day, gavage); PTU only (10 µg/mL, drinking water); or combined PTU (10 µg/mL, drinking water) and K⁺PFOS (3 mg /kg bw/day, gavage), all for 7 consecutive days. Interim serum samples were collected on days 1, 3 and 7, whilst terminal serum samples were collected post sacrifice on day 8, 24 hours after the last treatment. Samples were analysed for TT4, TT3 and TSH. Anterior pituitary samples were removed on day 8 and placed in static culture for assessment of TRH-mediated release of TSH.

77.             Thyroid hormone levels: In the combined PTU and PFOS treatment group, TT4, TT3 and TSH levels did not differ significantly from levels observed in the PTU-only treatment group. In the PFOS only treatment group, TSH levels were unchanged, whilst TT4 and TT3 levels were significantly decreased compared with controls. PFOS treatment had no effect on TRH- mediated release of TSH from the pituitary gland.

78.             Overall, the authors concluded that a single oral dose of PFOS in rats results in a transient increase in tissue availability of thyroid hormones and turnover of T4, with a resulting reduction in serum TT4. Under the dosing conditions of the study, PFOS does not induce a classical hypothyroid state nor does it alter hypothalamic-pituitary-thyroid activities.

Chang et al. 2009

79.             Chang et al. (2009) evaluated thyroid status and histomorphological factors associated with thyroid follicles following exposure to PFOS. As part of a developmental study in rats, groups of pregnant SD rats (25/group) were administered daily oral doses (assumed by gavage) of K⁺PFOS at 0, 0.1, 0.3 and 1.0 mg/kg bw/day from GD0 through to PND20. Offspring were nursed until PND21 and then observed until PND72. Maternal and foetal serum and tissue samples were obtained from a further group (10/group) administered daily oral doses (assumed by gavage) of K⁺PFOS at 0, 0.1, 0.3 and 1.0 mg/kg bw/day until GD19 and sacrificed on GD20.

80.             Serum was collected on GD20 (dams and foetuses), PND4 (dams, and male and female offspring), and PND21 (dams, and male and female offspring) and TSH levels measured. Thyroids were collected for histopathological evaluation from the control and 1.0 mg/kg bw/day maternal dose groups from GD20 foetuses, PND4 and PND21 male and female offspring. Other evaluations included thyroid follicular epithelial cell height and dimensions (PND4 and PND21 male and female offspring) and immunohistochemical staining (GD20 foetuses). mRNA transcripts were obtained from liver samples and the gene expression of thyroid hormones was evaluated from GD20 dams and foetuses, and PND21 male offspring with 1.0 mg/kg bw/day maternal dose.

81.             Mortality: There was no evidence of treatment-related effects on offspring survival (no data presented for dams).

82.             General toxicity and body weight: No clinical signs of general toxicity were observed in dams or offspring. Maternal body weights were significantly decreased in the 1.0 mg/kg bw/day dose group from PND4 through to PND21, due to lower mean body weight gains during gestation and PND1 to PND4. Food consumption was also significantly decreased at this dose. Maternal weight gains during the remainder of lactation and offspring body weights and body weight gain were unaffected by treatment compared with controls.

83.             Histopathology: No treatment-related histological changes, including the number or distribution of follicles, were observed in thyroid sections from GD20 foetuses or PND4 and PND21 offspring, compared with controls. There were no treatment related changes to follicular colloid area in PND4 and PND21 offspring. Follicular epithelial cell height was unaffected by treatment in PND4 male and female offspring, and PND21 female offspring. In PND21 male offspring, follicular epithelial cell height was significantly higher than controls, although this difference was considered spurious by the authors because of the low result in the male control group as compared to the female control group.

84.             Histopathology: There was a significant increase in number of thyroid follicular epithelial cells in GD20 female foetal thyroids from dams treated with 1.0 mg/kg bw/day, compared with controls. Due to the range in corresponding control values, the authors were unable to determine the toxicological significance of this finding. No treatment-related effects were observed in males. Thyroid hormone levels: Mean serum TSH levels taken from treated dams and their offspring were unchanged relative to controls on GD20, PND4 or PND21.

85.             Gene expression: mRNA transcripts with a potential relationship to thyroid status (ME, P450 oxidoreductase (Por), type 1 deiodinase (Dio1), UGT1A family, and apolipoprotein A1 (ApoA1)) were unaffected in dams and offspring following maternal treatment.

86.             The authors concluded K⁺PFOS administration up to 1.0 mg/kg bw/day to maternal rats during gestation and lactation has no clear adverse effect on thyroid status (morphology, hormone homeostasis, proliferation or liver gene expression, specifically hepatic genes mediated by thyroid hormones).

Chang et al. 2017

87.             Chang et al. (2017) investigated the effects of PFOS exposure on TH levels in monkeys. Cynomolgus monkeys (6/sex/group) were assigned to three groups: group 1 was administered vehicle by gavage on days 1, 43, 106, 288 and 358; group 2 was administered a single dose of K⁺PFOS at a dose of 9 mg/kg bw by gavage on day 106 and vehicle on days 1, 43, 288 and 358; group 3 was administered three doses of K⁺PFOS at doses of 14 mg/kg bw on day 43, 14.8 mg/kg bw (males) or 17.2 mg/kg bw (females) on day 288, 11 mg/kg bw on day 358, and vehicle on days 1 and 106, all by gavage. Blood samples for PFOS and TT4, FT4, TT3 and TSH analysis were collected at numerous time points including prior to treatment, during treatment, and on the final day of the study on day 420.

88.          Mortality: No mortalities occurred during the study.

89.             General toxicity and body weight gain: No clinical signs of general toxicity were observed. Body weight and body weight gain were unaffected by treatment in either group 2 or group 3, compared with group 1.

90.             Thyroid hormone levels: Non-significant decreases in TT4 levels were observed in males and females at 9 mg/kg bw (group 2), at 11 and 14.8 mg/kg bw in males, or 11 and 17.2 mg/kg bw in females (group 3) compared with controls. There were no significant changes to TSH, FT4 and TT3 levels in males or females from either group 2 or 3. The authors proposed that because unlike TSH and FT4, TT4 is not a clinically relevant endpoint for interpreting thyroid function, and these results do not indicate an adverse effect on thyroid function.

91.             Serum PFOS concentrations: The highest mean PFOS serum concentrations for the duration of the study from each treatment group were: 0.013 µg/mL on day 365 and 0.007 µg/mL on day 365 from males and females, respectively (group 1); 67.7 µg/mL on day 113 and 68.8 µg/mL on day 113 from males and females, respectively (group 2); and 160.8 µg/mL on day 365 and 165.0 µg/mL on day 365 from males and females, respectively (group 3).

92.             The authors concluded that, when compared with time-matched controls, the administration of a single dose or multiple single doses of PFOS to monkeys did not result in any toxicologically meaningful or clinically relevant changes in thyroid related hormones at serum PFOS concentrations up to 165 µg/mL.

Conley et al. 2022

93.             Conley et al. (2022) investigated the effects of PFOS exposure on thyroid hormones in rats. In a developmental study, pregnant SD rats (5/group) were administered K+PFOS at doses 0, 0.1, 0.3, 1, 2 or 5 mg/kg bw/day by gavage from GD8 – PND2. Dams and offspring were sacrificed on PND2 and blood was collected for various analyses: TT4, TT3, free triiodothyronine (FT3), FT4 and PFOS in dams, and TT4, TT3 and rT3 in offspring.

94.             Mortality: In dams, mortality was unaffected, whereas offspring survival was significantly reduced at 5 mg/kg bw/day, compared with controls. General toxicity and body weight: No clinical signs of general toxicity were reported. In dams, there was a significant reduction in body weight at 5 mg/kg bw/day on GD22 and PND2, and body weight gain (GD8-GD22), compared with controls, while in offspring, body weights were significantly reduced at 2 mg/kg bw/day on PND2 and at birth at 5 mg/kg bw/day when adjusted for litter size and birthdate.

95.             Thyroid hormone levels: In dams, TT4 and TT3 levels were significantly reduced at ≥ 0.1 and ≥ 0.3 mg/kg bw/day, respectively, compared with controls. FT4 levels were significantly reduced at 0.3, 2 and 5 mg/kg bw/day, whereas FT3 levels were unaffected. In offspring, TT4 levels were reduced at ≥ 0.3 mg/kg bw/day, and both TT3 and rT3 were significantly reduced at ≥1 mg/kg bw/day.

96.             Serum PFOS concentrations: In dams, mean PFOS concentrations were ND (controls), 2.2 µg/mL (0.1 mg/kg bw/day), 5.8 µg/mL (0.3 mg/kg bw/day), 31.4 µg/mL (1 mg/kg bw/day), 68.8 µg/mL (2 mg/kg bw/day), and 203.3 µg/mL (5 mg/kg bw/day).

97.             The authors concluded PFOS reduced serum TH concentrations, which occurred at nearly all doses depending on the specific hormone and life stage (maternal or neonatal). However, PFOS did not reduce maternal FT3.

Curran et al. 2008

98.             Curran et al. (2008) investigated the effects of PFOS on thyroid weight and TH levels in rats. In a repeated dose study, SD rats (15/sex/group) were administered K⁺PFOS at doses 0, 2, 20, 50 or 100 mg/kg diet (equivalent to 0, 0.14, 1.33, 3.21 or 6.34 mg/kg bw/day for males, and 0, 0.15, 1.43, 3.73 or 7.58 mg/kg bw/day for females) in the diet for 28 days. At necropsy, blood samples were collected for TT4, TT3 and PFOS analysis, and thyroids were removed.

99.             General toxicity and body weight: No clinical signs of general toxicity were reported. In males, body weights were significantly reduced at 3.21 and 6.34 mg/kg bw/day on days 14, 21 and 28, compared with controls. In females, body weights were significantly reduced at 3.73 mg/kg bw/day on days 21 and 28, and at 7.58 mg/kg bw/day on days 14, 21 and 28, compared with controls. Feed consumption was significantly reduced in both males and females at these doses, leading the authors to propose that palatability may be a factor.

100.             Gross pathology: Absolute thyroid weights in males and females were unaffected by treatment. Relative thyroid weight: body weight was significantly increased at 6.34 and 7.58 mg/kg bw/day in males and females, respectively, compared with controls.

101.             Thyroid hormone levels: In males, TT4 levels were significantly decreased at 1.33, 3.21 and 6.34 mg/kg bw/day, and in females, at 1.43 mg/kg bw/day, 3.73 and 7.58 mg/kg bw/day. In males, TT3 levels were significantly decreased at 6.34 mg/kg bw/day, and in females, at 3.73 and 7.58 mg/kg bw/day.

102.             Serum PFOS concentrations (converted from µg/g to µg/mL using serum density 1.018 g/mL): In males, mean PFOS concentrations at study termination were 0.48 µg/mL (control), 0.97 µg/mL (0.14 mg/kg bw/day), 13.69 µg/mL (1.33 mg/kg bw/day), 21.31 µg/mL (3.21 mg/kg bw/day) and 30.42 µg/mL (6.34 mg/kg bw/day). In females, mean PFOS concentrations on day 28 were 0.97 µg/mL (control), 1.53 µg/mL (0.15 mg/kg bw/day), 15.68 µg/mL (1.43 mg/kg bw/day), 32.50 µg/mL (3.73 mg/kg bw/day) and 43.98 µg/mL (7.58 mg/kg bw/day).

103.             The authors concluded that serum thyroid hormone levels were decreased in PFOS-treated rats. Decreased TT4 and TT3 occurred concurrently with hepatic changes indicative of peroxisome proliferation. Significant treatment-related changes in thyroid weight, relative to body weight, were suggestive of altered endocrine functions.

104.             The authors determined the following lowest observed effect levels (LOEL). Reduced TT4: 1.33 mg/kg bw/day (serum 13.69 µg/mL) in males and 1.43 mg/kg bw/day (serum 15.68 µg/mL) in females. Reduced TT3: 6.34 mg/kg bw/day (serum 30.42 µg/mL) in males and 3.73 mg/kg bw/day (serum 32.50 µg/mL) in females. Reduced relative thyroid weight:body weight: 6.34 mg/kg bw/day (serum 30.42 µg/mL) in males and 7.58 mg/kg bw/day (serum 43.98 µg/mL) in females.

Elcombe et al. 2012

105.             Elcombe et al. (2012) examined thyroid parameters as part of a 7-day dietary study in rats. Male SD rats (40/group) were given either 20 ppm or 100 ppm K⁺PFOS (equivalent to 1.93 and 9.65 mg/kg bw/day, respectively) in the diet for 7 days, followed by a control diet for a recovery period of either 1, 28, 56 or 84 days. Subgroups of rats (10/group) were sacrificed on the first day of recovery (recovery day 1) and subsequently on recovery days 28, 56 and 84.

106.             Upon necropsy, blood was collected, and thyroids removed from each rat. Thyroid tissue samples underwent histopathological examination and were used to determine cell proliferation rates and apoptotic index. Blood serum PFOS concentrations were determined.

107.             Mortality: All rats survived to scheduled necropsy.

108.             General toxicity and body weight: No clinical signs of general toxicity were observed. Body weights were significantly decreased at 1.93 mg/kg bw/day recovery days 21 and 28, and at 9.65 mg/kg bw/day on recovery days 16, 21, 28. Food consumption was unaffected due to treatment.

109.             Histopathology: There were no treatment-related effects observed upon microscopic examination of thyroid gland sections. Similarly, K⁺PFOS treatment had no effect on S-phase activity and hence cell proliferation. Levels of apoptosis in thyroid follicles were similar to controls for all time points.

110.             Serum PFOS concentrations: mean serum PFOS concentrations following administration of 1.93 mg/kg bw/day were 39.49 µg/mL, 15.49 µg/mL, 8.03 µg/mL and 4.38 µg/mL, on day 1, 28, 56 and 84, respectively, and 140.4 µg/mL, 53.82 µg/mL, 43.68 µg/mL and 25.79 µg/mL on day 1, 28, 56 and 84, respectively following treatment with 9.65 mg/kg bw/day. PFOS concentrations in controls not reported.

111.             The authors concluded that K⁺PFOS at up to 9.65 mg/kg bw/day via the diet for 7 days did not appear to have any effect on the thyroid parameters evaluated (histology, follicular epithelial cell proliferation and follicular epithelial apoptosis).

Lau et al. 2003

112.             Lau et al. (2003) investigated the effects of PFOS on thyroid hormones in rats and mice. As part of a developmental study in rats, pregnant SD rats (17 – 28/group) were treated with K⁺PFOS at doses of 0, 1, 2, 3, 5 or 10 mg/kg bw/day by gavage from GD2 until GD21. Four offspring from each litter were sacrificed within 2-4 hours of birth and trunk blood collected. Remaining offspring were randomised and redistributed to nursing dams within their respective dosage groups, a process that was repeated every few days to maintain litters sizes at 10 – 12 offspring each. Offspring were weaned on PND21. Male and female offspring were randomly chosen for sacrifice on PND2, 5, 9, 15, 21, 28 and 35, where blood was collected, and serum prepared.

113.             As part of a developmental study in mice, pregnant CD-1 mice (21 – 22/group) were treated with K⁺PFOS at doses of 0, 1, 5, 10, 15 or 20 mg/kg bw/day by gavage from GD1 until GD17. Offspring were weaned on PND21. Male and female offspring were randomly selected from litters and sacrificed within 2-4 hours of birth and on PND3, 7, 14, 21, 28 and 35, where blood was collected, and serum prepared. Litters were culled at intervals of several days to maintain sizes at 10–12 offspring each.

114.             Serum TT4, FT4, triiodothyronine (T3) (assumed to be both TT3 and FT3 as not specified in the paper) and TSH levels and serum PFOS concentrations were measured in offspring at sacrifice. Mortality: In rats, neonatal exposure to PFOS reduced postnatal survival in offspring at 5 and 10 mg/kg bw/day, and in mice at 15 and 20 mg/kg bw/day. Greater than 95 % of offspring in these groups died within 24 hours of birth. The authors noted that approximately 50 % rat and mice offspring died at 3 and 10 mg/kg bw/day, by PND22 and PND24, respectively.

115.             General toxicity and body weight: In surviving rat offspring, at 2 mg/kg bw/day body weight was significantly decreased between PND0 to PND3, and on PND9; at 3 mg/kg bw/day body weight was significantly decreased between PND0 to PND5; and at 5 mg/kg bw/day body weight was significantly decreased between PND0 to PND22. In surviving mice offspring, body weights were unaffected by treatment.

116.             Thyroid hormone levels: In rat offspring, TT4 and FT4 levels were significantly reduced on PND2 at either 1 or 2 mg/kg bw/day maternal doses depending on the statistical method used, compared with controls. TT4 levels in all treated groups were similar to controls on PND21 and PND35, whereas FT4 levels remained depressed from PND2 to PND35 in all treated groups. There were no significant changes in serum TT3, FT3 or TSH levels in maternally exposed offspring compared with controls at any time point. In mice offspring, there were no significant changes in TT4, TT3, FT3 or TSH levels in maternally exposed mice compared with controls from PND3 until PND28, although a non-significant decrease in levels was observed at 5 and 10 mg/kg bw/day on PND14.

117.             Serum PFOS concentrations: In rats, at birth serum PFOS concentrations increased with dose in a non-linear manner (numerical values not provided). By PND5, serum PFOS concentrations were lower than at birth. Serum PFOS concentrations for mice were not reported.

118.             The authors concluded that hypothyroxinemia was observed in PFOS- exposed rat offspring. Serum T4 levels (assumed TT4 and FT4) were suppressed in the PFOS-exposed rat offspring, although T3 (assumed to be TT3 and FT3) and TSH were not altered.

119.             Luebker et al. (2005) investigated the effects of PFOS on TH levels and histopathology in rats and their offspring. In a two-generation reproduction study, female SD rats (20/group) were administered K⁺PFOS at doses of 0, 0.4, 0.8, 1.0, 1.2, 1.6 and 2.0 mg/kg bw/day by gavage for 42 days pre- mating, through mating with untreated males, gestation (GD0 to GD21) until PND4. Dams and offspring were sacrificed on PND5 and blood was collected for TT4, FT4, TT3, FT3 and TSH analysis. Offspring were not dosed directly but were exposed in utero or via lactation. Liver ME activity was measured in livers collected from PND5 dams and offspring from the 0.0, 0.4, 1.6, and 2.0 mg/kg bw/day groups. Thyroids were collected from offspring from the 0 and 2.0 mg/kg bw/day groups for histopathological examination.

120.             Mortality and morbidity: In dams, the authors indicate deaths occurred, but these were not attributed to treatment (no further details given). In offspring, the number of dams with stillborn pups was within the historical control range. The number of dams with all offspring dying PND1 to PND5 was significantly increased at 2.0 mg/kg bw/day, compared with controls.

121.             General toxicity and body weight: No clinical signs of general toxicity in dams or surviving offspring were reported. In dams, body weights were significantly decreased at 1.6 and 2.0 mg/kg bw/day from GD0 to GD21, and at 2.0 mg/kg bw/day from PND1 to PND5, compared with controls. Body weight gain was significantly decreased at 2.0 mg/kg bw/day for day 1 to day 42 pre-mating, and at doses ≥ 0.8 mg/kg bw/day from PND1 to PND5 (with the exception of 1.2 mg/kg bw/day), compared with controls. The authors state a general trend for decreased feed consumption with increasing dose was seen during pre-mating through to PND4. In offspring, mean pup weights per litter were significantly reduced for all maternal dose groups at birth and on PND5, compared with controls. Histopathology: No microscopic changes were observed in thyroids collected from offspring from the 2.0 mg/kg bw/day group on PND5, compared with controls.

122.             Thyroid hormone levels: In dams, TT4 levels were significantly reduced at doses ≥ 0.4 mg/kg bw/day on PND5, while TT3 levels were significantly reduced at doses ≥ 1.2 mg/kg bw/day on PND5, compared with controls. Both TSH and FT4 levels were unaffected by treatment (observations for FT3 levels were not reported). In offspring, TT4 and TT3 levels were significantly reduced at 1.0 mg/kg bw/day on PND5, compared with controls. TSH levels were also significantly elevated at 1.6 mg/kg bw/day on PND5, compared with controls. No significant changes to FT4 levels were observed. The authors considered that the use of the standard reference method for FT4 (analog radioimmunoassay (RIA) kit) can lead to artificially lower FT4 levels (especially when protein bound T4 and serum binding capacity is low, and because the protein bound T4 and FT4 equilibrium can be disturbed by dilution by assay components). A method using equilibrium dialysis followed by radioimmunoassay (ED/RIA) is not prone to these biases and was used here to measure FT4.

123.             Liver ME activity measured in dam and offspring on PND5 was unaffected by treatment.

124.             The authors concluded that although there were apparent reductions in serum TT3 and TT4 in offspring, on the whole, the data from this study did not suggest a hypothyroid state in offspring. The lack of a major increase in TSH and the fact that liver ME activity, a marker for hepatic response to thyroid hormones, was comparable to controls, suggest that offspring were in a normal (euthyroid) state. In addition, there were no histopathological changes in offspring thyroids based on evaluation of tissues from control and 2.0 mg/kg bw/day groups. In dams, FT4, TSH and liver ME activity were unaltered by PFOS treatment, and serum TT3 and TT4 were reduced.

125.             NTP (2022b) investigated the effects of PFOS on thyroid weight, histopathology and TH levels in rats. In a repeated dose study, SD rats (10/sex/group) were administered PFOS at doses 0, 0.312, 0.625, 1.25, 2.5 or 5 mg/kg bw/day by gavage for 28 days. At necropsy on day 29, blood samples were collected for TT4, TT3, FT4, TSH and PFOS analysis, and thyroids were removed for histopathological evaluation.

126.             Mortality: One female rat at 5 mg/kg bw/day died, all other rats survived to scheduled necropsy.

127.             General toxicity and body weight: No clinical signs of general toxicity were observed. Terminal body weights were significantly reduced at 5 mg/kg bw/day in males and females, compared with controls.

128.             Gross pathology: Thyroid weights in males and females were unaffected by treatment.

129.             Histopathology: Histopathology in males and females was unaffected by treatment.

130.             Thyroid hormone levels: In males and females, TT4 and FT4 levels were significantly decreased at ≥ 0.312 mg/kg bw/day, compared with controls. TT3 levels were significantly decreased at ≥ 0.625 mg/kg bw/day, compared with controls. TSH levels were unaffected by treatment.

131.             Plasma PFOS concentrations: In males, mean plasma PFOS concentrations on day 29 were ND (control), 23.730 µg/mL (0.312 mg/kg bw/day), 51.560 µg/mL (0.625 mg/kg bw/day), 94.260 µg/mL (1.25 mg/kg bw/day), 173.700 µg/mL (2.5 mg/kg bw/day) and 318.200 µg/mL (5 mg/kg bw/day). In females, mean plasma PFOS concentrations on day 29 were 0.054 µg/mL (control), 30.530 µg/mL (0.312 mg/kg bw/day), 66.970 µg/mL (0.625 mg/kg bw/day), 135.100 µg/mL (1.25 mg/kg bw/day), 237.500 µg/mL (2.5 mg/kg bw/day) and 413.556 µg/mL (5 mg/kg bw/day). The authors concluded that TT4, FT4 and TT3 decreased in a dose- response manner. TSH was unaffected, nor were there any histopathologic changes in the thyroid gland (hyperplasia/hypertrophy).

Seacat et al 2002

132.             Seacat et al. (2002) investigated the effect of PFOS exposure on thyroid organ weight and TH levels in monkeys. Cynomolgus monkeys (4 — 6/sex/group) were administered K⁺PFOS at doses of 0, 0.03, 0.15 and 0.75 mg/kg bw/day by capsule for 182 days. Two monkeys from the 0, 0.15 and 0.75 mg/kg bw/day groups were monitored for a recovery period of one year following the end of treatment. Blood samples for serum PFOS and TT4, T3 (assumed TT3) and TSH analysis were collected at numerous time points prior to the commencement of treatment, during treatment and during the recovery period. FT4 and FT3 levels were measured on day 184 only. Monkeys were sacrificed on days 184 and 185 and thyroid glands removed for examination.

133.             Mortality and morbidity: A male monkey from the 0.75 mg/kg bw/day group died on day 155. A second male monkey from the 0.75 mg/kg bw/day group was in a moribund condition on day 179 and was sacrificed.

134.             General toxicity and body weight: No clinical signs of general toxicity were reported. Body weight gain from day 0 to day 184 was significantly reduced in males and females at 0.75 mg/kg bw/day, compared with controls. Terminal body weights on day 184 were unaffected by treatment. No data are provided for the recovery groups.

135.             Gross pathology: No adverse findings regarding thyroid weights were reported.

136.             Thyroid hormone levels: A significant increase in TSH levels was observed in males and females at 0.75 mg/kg bw/day on days 182 and 184, compared with controls. A significant decrease in TT3 levels was observed in males at 0.75 mg/kg bw/day on days 62, 91, 182 and 184, and in females at 0.75 mg/kg bw/day on days 91, 182 and 184. FT3 levels were also significantly reduced in males and females at 0.75 mg/kg bw/day on day 184. On day 184, FT4 levels in males and females were unchanged from control values, but a statistically significant reduction in FT3 levels was observed in both sexes but only at a dose of 0.75 mg/kg bw/day. Other changes to TT4, TSH, FT4 and FT3 levels were observed over the various time points monitored throughout the study, however, the authors state these changes were not consistent over time or consistent with a dose-response. In the recovery groups, all TH levels returned to control levels between days 33 to 61.

137.             Serum PFOS concentrations: In the 0.75 mg/kg bw/day group, mean PFOS serum concentrations were 173 µg/mL and 171 µg/mL on day 183, in males and females, respectively. In the 0.15 mg/kg bw/day group, mean PFOS serum concentrations were 82.6 µg/mL and 66.8 µg/mL on day 183, in males and females, respectively. No numerical data are presented for the recovery groups, although the authors commented that serum PFOS concentrations declined overall during the recovery period except for an initial increase in the week following cessation of treatment.

138.             The authors concluded that significant adverse effects occurred only in the 0.75 mg/kg bw/day group and included compound-related mortality in 2 of 6 male monkeys, decreased body weights and lowered TT3 concentrations (without evidence of hypothyroidism).

Thibodeaux et al 2003

139.             Thibodeaux et al. (2003) investigated the effects of PFOS on thyroid hormones in rats and mice.

140.             As part of a developmental study in rats, pregnant SD rats (25 – 50/group) were administered K⁺PFOS at doses of 0, 1, 2, 3, 5 or 10 mg/kg bw/day by gavage from GD2 until GD20. Blood samples were collected from dams on GD7, GD14 and at sacrifice on GD21 TT4, FT4, TT3 TSH levels and PFOS concentrations measured. In a separate study, adult female SD rats (6 – 8/group) were treated with K⁺PFOS at doses of 0, 3 or 5 mg/kg bw/day by gavage for 20 days. Blood samples were collected from adults on days 3, 7, 14 and at sacrifice on day 20 TT4, FT4, TT3 and TSH levels measured.

141.             As part of a developmental study in mice, pregnant CD-1 mice (60 – 80/group) were treated with K⁺PFOS at doses of 0, 1, 5, 10, 15 or 20 mg/kg bw/day by gavage from GD1 until GD17. Dams were selected for sacrifice on either GD6, GD12 or GD18. Blood samples were collected at sacrifice for analysis of TT4, FT4, T3 (assumed TT3), TSH levels and PFOS concentrations.

142.             General toxicity and body weight: In pregnant rats, PFOS treatment reduced body weight gain in a dose-dependent manner and was significantly reduced at ≥2 mg/kg bw/day compared with controls. The authors state weight gain deficits in pregnant rats corresponded to significant reductions in food consumption. No data are given for adult female rats. In pregnant mice, body weight gain was significantly reduced at 20 mg/kg bw/day during late gestation (inferred GD14 to GD18). Food consumption was unaffected.

143.             Thyroid hormone levels: In pregnant rats, both TT4 and FT4 levels were significantly reduced at ≥1 mg/kg bw/day on GD7 to GD21, compared with controls. TT3 levels were significantly reduced at 10 mg/kg bw/day on GD7 to GD21; at 3, 5 and 10 mg/kg bw/day on GD14 and GD21; and at ≥ 1 mg/kg bw/day on GD21. TSH levels were unaffected by treatment. In adult female rats, both TT4 and FT4 levels were significantly reduced at 3 and 5 mg/kg bw/day on day 3 to day 20. TT3 levels were significantly reduced at 3 and 5 mg/kg bw/day on day 7 to day 20. TSH levels were dose dependent and significantly increased at 3 mg/kg bw/day on day 7, returning to within control levels from day 14 onwards. Conversely, TSH levels were decreased (not statistically significant) at 5 mg/kg bw/day from day 3 to day 20. In pregnant mice, TT4 levels were significantly reduced at 20 mg/kg bw/day on GD6, but unchanged compared with controls on GD12 and GD18. FT4, TT3 and TSH levels were unaffected by treatment.

144.             Serum PFOS concentrations: In pregnant rats, PFOS concentrations increased with dose, reaching maximum concentrations at GD14 before reducing by PND21. Numerical values were not provided. In pregnant mice, the authors state that serum PFOS concentrations were similar to those in the rat (no further data provided). The mean PFOS concentration at 10 mg/kg bw/day on GD18 was 190 µg/mL (no further data provided).

145.             The authors concluded that serum PFOS levels increased with dosage. Serum T4 (assumed TT4 and FT4) and T3 (assumed TT3) in the PFOS-treated pregnant rats were significantly reduced as early as one week after chemical exposure, although no feedback response of TSH was observed. A similar pattern of reduction in T4 (assumed TT4) was also seen in the pregnant mice. T3 and T4 results from the study with adult female rats by and large substantiated the findings in pregnant rats, discounting potential confounding effects of pregnancy. The adverse effect of PFOS on TH was less pronounced in the mouse than in the rat.

Wang et al. 2011

146.             Wang et al. (2011) investigated the effect of PFOS on TH levels in rats. As part of a developmental study evaluating the mixture effects of PFOS and 2,2’,4,4’-tetrabromodiphenyl ether (BDE-47), a known thyroid hormone disruptor, pregnant Wistar rats (3 – 9 dams/group, 9 – 12 offspring/group) were administered K⁺PFOS in the diet at doses 0, 3.2, 32 mg/kg diet /day (converted to 0.38 and 3.8 mg/kg bw/day using conversion factor of 0.12 for rats for a subacute study (EFSA, 2012)) in the diet from GD1 to PND14. Only results from exposure to PFOS alone are reported.

147.             Dams and offspring were sacrificed on PND1, 7 and 14 (numbers not specified) and blood samples collected for measurement of TT4 and TT3 levels and serum PFOS concentrations.

148.             Mortality: No deaths were attributed to treatment in dams or offspring.

149.             General toxicity and body weight: No clinical signs of general toxicity were observed in dams or offspring. At 3.8 mg/kg bw/day, male and female offspring body weights were significantly decreased on PND1, PND7 and PND14 compared with controls. Body weights at 0.38 mg/kg bw/day were unaffected.

150.             Thyroid hormone levels: In dams, TT4 levels were significantly reduced at 0.38 and 3.8 mg/kg bw/day on PND1, PND7 and PND14 (no data available for 3.8 mg/kg bw/day on PND7), compared with controls. TT3 levels were significantly reduced at 3.8 mg/kg bw/day on PND1 and PND14 (no data for PND7 at 3.8 mg/kg bw/day). In offspring, TT4 levels were significantly reduced at 0.38 mg/kg bw/day and 3.8 mg/kg bw/day on PND1, PND7 and PND14, with the exception of PND1 (0.38 mg/kg bw/day). TT3 levels were significantly reduced at 0.38 mg/kg bw/day and 3.8 mg/kg bw/day on PND14.

151.             Serum PFOS concentrations: In dams, at 0.38 mg/kg bw/day mean concentrations in serum were 2.29 µg/mL, 4.16 µg/mL and 3.15 µg/mL for PND1, PND7 and PND14, respectively; at 3.8 mg/kg bw/day mean concentrations were 16.9, 27.3 and 28.7 µg/mL for PND1, PND7 and PND14, respectively. In offspring, at 0.38 mg/kg bw/day mean concentrations were 5.85 µg/mL, 3.65 µg/mL and 4.89 µg/mL for PND1, PND7 and PND14, respectively; at 3.8 mg/kg bw/day mean concentrations were 32.9 µg/mL, 21.3 µg/mL and 25.2 µg/mL for PND1, PND7 and PND14, respectively. Controls were all ND.

152.             The authors concluded that there were exposure- and time-dependent alterations in thyroid hormone concentrations.

Yu et al. 2009

153.             Yu et al. (2009) investigated the effect on TH levels following prenatal and/or postnatal (lactational) exposure to PFOS. In a cross-foster study, pregnant Wistar rats (20/group) were administered K⁺PFOS at 0 and 3.2 mg/kg diet/day (converted to 0.29 mg/kg bw/day using conversion factor of 0.09 for rats for a subchronic study (EFSA, 2012)) in the diet from GD0 until weaning on PND21. Two control litters and two PFOS-treated litters were used for sample collection on the day of birth (PND0). Remaining litters were cross-fostered to create the following three groups: litters from control dams fostered by other control dams (unexposed control, n = 8); litters from treated dams fostered by control dams (prenatal PFOS exposure, n = 8), litters from control dams fostered by treated dams (postnatal PFOS exposure, n = 8), and litters from treated dams fostered by treated dams (combined prenatal and postnatal PFOS exposure, n = 10). Litter sizes were adjusted to 10 offspring (5 male and 5 female where possible). Offspring were weaned on PND21.

154.             Offspring were weighed and sacrificed on PND0, 7, 14, 21 or 35 and blood and liver samples collected. Serum TT4, TT3, and rT3 levels were measured at all time points from all cross-fostered groups. Total RNA was isolated from livers from offspring from all cross-fostered groups on PND0 and PND21 to estimate transcript levels and gene expression. Genes studied included hepatic genes associated with thyroid function (transthyretin (TTR)), Dio1, UGT1A1, and uridine diphosphoglucuronosyl transferase 1A6 (UGT1A6)), and thyroid receptors in the developing liver (thyroid hormone receptor α (TRα) and thyroid hormone receptor β (TRβ)). Serum PFOS concentrations were determined for all time points from all cross-fostered groups.

155.             General toxicity and body weight: No clinical signs of general toxicity were observed in offspring. Offspring body weights for all groups were unaffected by treatment.

156.             Thyroid hormone levels: Combined prenatal and postnatal PFOS exposure resulted in significantly reduced TT4 levels in offspring on PND14, 21 and 35, compared with controls. Prenatal only exposure and postnatal only exposure significantly reduced TT4 levels on PND21 and 35. The authors noted that reduced TT4 levels in postnatally exposed offspring at PND21 and 35 did not occur at PND14, which was attributed to the cumulative effect of PFOS during the lactation and postweaning period. There were no treatment- related effects on TT3 or rT3 levels at any time point or exposure scenario.

157.             Gene expression: No alteration in mRNA expression was observed. However, the transcript level of TTR was significantly increased compared with controls at PND21 following combined prenatal and postnatal exposure. The authors proposed that this may not be indicative of TH status due to the absence of TTR up-regulation in the corresponding prenatal or postnatal exposure groups.

158.             Serum PFOS concentrations: Serum PFOS concentrations in postnatally exposed offspring increased in a dose-dependent manner. The mean concentrations on PND35 were 6.64 and 7.04 µg/mL in males and females, respectively. A similar trend was observed in offspring with combined prenatal and postnatal exposure. Mean concentrations on PND35 were10.61 and 11.53 µg/mL in males and females, respectively. Conversely, serum PFOS concentrations in prenatally exposed offspring decreased with age. On PND0 concentrations were 5.98 µg/mL from a pooled serum sample, on PND35 concentrations were 0.41 and 1.02 µg/mL, for males and females respectively. Controls were all ND. The authors proposed that these results indicate the transfer of PFOS from dams to offspring via placenta and milk.

159.             The authors concluded that prenatal only PFOS exposure and postnatal only PFOS exposure at 0.29 mg/kg bw/day induced hypothyroxinemia in rat offspring to a similar extent, which suggested that prenatal PFOS exposure and postnatal PFOS accumulation, especially though maternal milk, are matters of great concern. Of particular note is that prenatal PFOS exposure significantly suppressed serum TT4 levels on PND21 and 35. The finding suggests that exposure to PFOS during gestation results in a long-lasting adverse effect on serum TH in rat offspring. Although no alteration of mRNA expression was observed for selected genes that are important for TT4 deiodination, glucuronidation and TH receptors, this does not rule out possible changes in regulation at the translation or post translational levels.

PFHxA

In vivo toxicity data

Loveless et al. 2009

160.                Loveless et al. (2009) investigated the effects of sodium perfluorohexanoate (NaPFHxA) on thyroid weight and histopathology. In the main study, adult Crl:Cd Sprague Dawley (SD) rats (10/sex/group) were administered NaPFHxA daily by gavage at doses of 0, 20, 100, or 500 mg/kg bw/day for 92 days (males) or 93 days (females). Two further groups (10/sex/group) were designated for either a 90-day recovery (0, 20, 100, or 500 mg/kg bw/day) or 30-day recovery (0 and 500 mg/kg bw/day). This study conformed to the OECD Guideline 408 (Repeated Dose 90 Day Oral Toxicity Study in Rodents).

161.             Mortality: No deaths were attributed to treatment.

162.                General toxicity and body weight: No clinical signs of general toxicity were observed. Mean body weight was significantly decreased from day 42 to 105 in male rats treated with 500 mg/kg bw/day with a 90-day recovery period, compared with controls with the same recovery period. No treatment- related effects on either body weight or body weight gain were observed in any female group, or males from the main study or 30-day recovery. Terminal weights of males and females at the end of either a 30- or 90-day recovery (i.e, day 122-123 or 182-183, respectively) were unaffected.

163.                Gross pathology: Thyroid weights were significantly increased in female rats treated with 500 mg/kg bw/day following the 30-day recovery period, compared with controls with the same recovery period. There were no significant changes observed in females in either the main study or 90-day recovery group, or in males at any time point or dose.

164.                Histopathology: In the main study, minimal hypertrophy of the thyroid follicular epithelium was observed in both male and female rats exposed to 500 mg/kg bw/day, and one male rat exposed to 100 mg/kg bw/day. In the 30- day recovery group, minimal hypertrophy of the thyroid follicular epithelium was present in males and females exposed to 500 mg/kg bw/day. In the 90- day recovery group, minimal hypertrophy of the thyroid follicular epithelium was only observed in males exposed to 500 mg/kg bw/day.

165.                Authors noted that since hypertrophy of the thyroid follicular epithelium was present primarily in rats exposed to 500 mg/ kg bw/day in the main study and in the 30- and 90-day recovery groups, the increased thyroid weights observed in females in the 30-day recovery group may be adverse and treatment related. Thyroid follicular cell hypertrophy was also potentially adverse albeit minimal and likely to be secondary and related to liver hypertrophy and induction of metabolic liver enzymes, which were also seen at 500 mg/kg bw/day. The authors also commented this response may not be relevant to non-rodent species.

166.                The authors concluded that the administration of NaPFHxA by gavage for approximately 90 days was associated with adverse changes at 500 mg/kg bw/day that included thyroid pathology.

NTP, 2022a

167.                The National Toxicology Program (NTP, 2022a) investigated the effects of PFHxA on thyroid weight, histopathology and TH levels in rats. In a repeated dose study, SD rats (10/sex/group) were administered PFHxA at doses 0, 62.6, 125, 250, 500 or 1000 mg/kg bw/day with half the dose being administered twice daily by gavage for 28 days. At necropsy on day 29, blood samples were collected for TT4, TT3, FT4, TSH and PFHxA analysis, and thyroids were removed for histopathological evaluation.

168.             Mortality: All rats survived to scheduled necropsy.

169.             General toxicity and body weight: No clinical signs of general toxicity were observed. In males, terminal body weights were significantly reduced at 1000 mg/kg bw/day, compared with controls. In females, terminal body weights were unaffected by treatment.

170.             Gross pathology: Thyroid weights in males and females were unaffected by treatment.

171.             Histopathology: Histopathology in males and females was unaffected by treatment.

172.             Thyroid hormone levels: In males, TT4, FT4 and TT3 levels were significantly decreased at ≥ 62.6 mg/kg bw/day compared with controls. TSH was unaffected by treatment. No effects were seen in females.

173.             Plasma PFHxA concentrations: In males, mean plasma PFHxA concentrations were 0.378 µg/mL (62.6 mg/kg bw/day), 0.503 µg/mL (125 mg/kg bw/day), 1.297 µg/mL (250 mg/kg bw/day), 3.339 µg/mL (500 mg/kg bw/day) and 10.899 µg/mL (1000 mg/kg bw/day). In females, mean plasma PFHxA concentrations were 0.129 µg/mL (62.6 mg/kg bw/day), 0.292 µg/mL (125 mg/kg bw/day), 0.475 µg/mL (250 mg/kg bw/day), 1.668 µg/mL (500 mg/kg bw/day) and 6.712 µg/mL (1000 mg/kg bw/day). No PFHxA was seen in controls.

174.             Overall TT4 and FT4 were decreased (males only), while TSH was unaffected in males or females. There were no histopathologic changes in the thyroid gland.

In vivo toxicity data

Conley et al, 2022

175.                Conley et al. (2022) investigated the effects of PFOA exposure on TH in rats. In a developmental study, pregnant SD rats (5/group) were administered PFOA ammonium salt at doses 0, 10, 30, 62.5, 125 and 250 mg/kg bw/day by gavage from GD8 – PND2. Dams and offspring were sacrificed on PND2 and blood was collected for various analyses: TT4, TT3, FT3, FT4 and PFOS in dams, and TT4, TT3 and rT3 in offspring.

176.                Mortality: In dams, due to a significant reduction in body weight at 250 mg/kg bw/day within 3-4 days of treatment, animals were euthanised. In offspring, survival was significantly reduced at 125 mg/kg bw/day, compared with controls.

177.                General toxicity and body weight: No clinical signs of general toxicity were reported. In dams, significant weight loss was observed in 2 of the 5 dams at 125 mg/kg bw/day within 4 – 5 days treatment, and exposure was terminated for these animals. Body weight on GD22 and body weight gain on GD8-GD22 were significantly reduced at ≥62.5 mg/kg bw/day, while body weight on PND2 and body weight gain om GD8-PND2 were reduced at 125 mg/kg bw/day, compared with controls. In offspring, birthweights were significantly reduced at 10 and ≥ 62.5 mg/kg bw/day, while adjusted birthweights and body weights on PND2 were significantly reduced only at ≥62.5 mg/kg bw/day.

178.                Thyroid hormone levels: In dams, TT4 and TT3 levels were significantly reduced at ≥10 mg/kg bw/day, compared with controls, while FT3 and FT4 levels were significantly reduced at 10, 62.5 and 125 mg/kg bw/day. In offspring, TT3 and TT4 levels were significantly reduced at ≥10 mg/kg bw/day while rT3 was significantly reduced at 10 and 30 mg/kg bw/day but significantly increased at 125 mg/kg bw/day.

179.                Serum PFOA concentrations: In dams, mean PFOA concentrations were 0.16 µg/mL (controls), 31.8 µg/mL (10 mg/kg bw/day), 96.1 µg/mL (30 mg/kg bw/day), 168.9 µg/mL (62.5 mg/kg bw/day) and 280.0 µg/mL (125 mg/kg bw/day).

180.                The authors concluded PFOA reduced serum TH concentrations, which occurred at nearly all doses depending on the specific hormone and life stage (maternal or neonatal).

NTP, 2022a

181.                NTP (2022a) investigated the effects of PFOA on thyroid weight, histopathology and TH levels in rats. In a repeated dose study, SD rats (10/sex/group) were administered PFOA at doses 0, 0.625, 1.25, 2.5, 5 or 10 mg/kg bw/day) for males, or 0, 6.25, 12.5, 25, 50, or 100 mg/kg bw/day for females by gavage for 28 days. At necropsy on day 29, blood samples were collected for TT4, TT3, FT4, TSH and PFOA analysis, and thyroids were removed for histopathological evaluation.

182.                Mortality: One female rat at 100 mg/kg bw/day died, all other rats survived to scheduled necropsy.

183.                General toxicity and body weight: One female rat at 100 mg/kg bw/day which subsequently died was lethargic, ataxic and thin prior to death. No clinical signs of general toxicity were observed in the remaining rats. In males, terminal body weights were significantly reduced at ≥ 2.5 mg/kg bw/day, compared with controls. In females, terminal body weights were unaffected by treatment.

184.             Gross pathology: In males, thyroid weights were significantly increased at 2.5 mg/kg bw/day, compared with controls and relative thyroid weight: body weight was significantly increased at ≥1.25 mg/kg bw/day. In females, thyroid weights and relative thyroid weight: body weight was unaffected by treatment.

185.             Histopathology: In males, there was a non-significant increase in the incidence of thyroid gland follicular cell hypertrophy of minimal severity at 10 mg/kg bw/day, compared with controls. In females, there was a significant increase in the incidence of thyroid gland follicular cell hypertrophy of minimal severity at 100 mg/kg bw/day.

186.             Thyroid hormone levels: In males, TT4 and FT4 levels were significantly decreased at ≥ 0.625 mg/kg bw/day, and TT3 levels were significantly decreased at 0.625 mg/kg bw/day, 1.25 mg/kg bw/day, 2.5 mg/kg bw/day and 5 mg/kg bw/day, compared with controls. TSH levels were significantly decreased at 5 and 10 mg/kg bw/day. In females, TT4 and FT4 levels were significantly decreased at 100 mg/kg bw/day and TSH levels were significantly increased at ≥ 6.25 mg/kg bw/day. TT3 levels were unaffected by treatment.

187.             Plasma PFOA concentrations: In males, mean plasma PFOA concentrations on day 29 were 0.098 µg/mL (control), 50.690 µg/mL (0.625 mg/kg bw/day), 73.480 µg/mL (1.25 mg/kg bw/day), 95.430 µg/mL (2.5 mg/kg bw/day), 110.720 µg/mL (5 mg/kg bw/day) and 148.570 µg/mL (10 mg/kg bw/day). In females, mean plasma PFOA concentrations on day 29 were ND (control), 0.491 µg/mL (6.25 mg/kg bw/day), 1.153 µg/mL (12.5 mg/kg bw/day), 2.960 µg/mL (25 mg/kg bw/day), 9.326 µg/mL (50 mg/kg bw/day) and 23.444 µg/mL (100 mg/kg bw/day).

188.             Overall, TT4 and FT4 were decreased in males and females, while TSH was decreased in males but increased in females. Thyroid gland follicular hypertrophy was observed in males and females.

In vivo toxicity data

NTP, 2022a

189.                NTP (2022a) investigated the effects of perfluorononanoic acid (PFNA) on thyroid weight, histopathology and TH levels in rats. In a repeated dose study, SD rats (10/sex/group) were administered PFNA at doses 0, 0.625, 1.25, 2.5, 5 or 10 mg/kg bw/day) for males, or 0, 1.56, 3.12, 6.25, 12.5 or 25 mg/kg bw/day for females by gavage for 28 days. At necropsy on day 29, blood samples were collected for TT4, TT3, FT4, TSH and PFNA analysis, and thyroids were removed for histopathological evaluation.

190.                Mortality: In males, all rats died at 10 mg/kg bw/day, and eight rats died at 5 mg/kg bw/day before scheduled necropsy. In females, all rats died at 25 mg/kg bw/day, and nine rats died at 12.5 mg/kg bw/day before scheduled necropsy. General toxicity and body weight: All rats that died before scheduled necropsy were described as thin. In males, terminal body weights were significantly reduced at 1.25 and 2.5 mg/kg bw/day, compared with controls. In females, terminal body weights were significantly reduced at 3.12 and 6.25 mg/kg bw/day. Data for 5 and 10 mg/kg bw/day (males), or 12.5 mg/kg bw/day and25 mg/kg bw/day (females) are not available due to high mortality.

191.                Gross pathology: In males, thyroid weights and relative thyroid weight: body weight were significantly increased at 2.5 mg/kg bw/day, compared with controls. In females, thyroid weights and relative thyroid weight: body weight was unaffected by treatment.

192.                Histopathology: Histopathology in males and females was unaffected by treatment.

193.                Thyroid hormone levels: In males, TT4 levels were significantly decreased at 0.625 and 1.25 mg/kg bw/day, FT4 levels at ≥ 0.625 mg/kg bw/day, TT3 levels at 2.5 mg/kg bw/day; and TSH levels at 1.25 and 2.5 mg/kg bw/day, compared with controls. In females, TT4 and FT4 levels were significantly decreased at 3.12 and 6.25 mg/kg bw/day. TT3 and TSH levels were unaffected by treatment.

194.                Plasma PFNA concentrations: In males, mean plasma PFNA concentrations on day 29 were 0.055 µg/mL (control), 56.730 µg/mL (0.625 mg/kg bw/day), 161.000 µg/mL (1.25 mg/kg bw/day), 380.000 µg/mL (2.5 mg/kg bw/day) and 358.000 µg/mL (5 mg/kg bw/day). In females, mean plasma PFNA concentrations on day 29 were 0.098 µg/mL (control), 26.400 µg/mL (1.56 mg/kg bw/day), 54.360 µg/mL (3.12 mg/kg bw/day) and 112.200 µg/mL (6.25 mg/kg bw/day). Data for 10 mg/kg bw/day (males), and 12.5 or 25 mg/kg bw/day (females) are not available due to high mortality.

195.                Overall, TT4 and FT4 were decreased, while TSH was unaffected. There were no histopathologic changes in the thyroid gland.

In vivo toxicity data

 Langley and Pilcher, 1985

196.                Langley and Pilcher (1985) investigated the effect of perfluorodecanoic acid (PFDA) on TH levels in rats. Male Wistar rats (30/group) received a single intraperitoneal (i.p.) injection of 75 mg/kg bw PFDA. A control group of weight-matched rats received a single injection of vehicle 24 hours after treatment and was subsequently pair-fed with the PFDA treated group (called thereafter pair-fed controls). A further control group of eight rats were fed ad libitum (ad libitum controls). Groups of five rats (PFDA-treated or pair-fed) were sacrificed beginning 12 hours after treatment and thereafter at 1, 2, 4, 6 and 8 days. Blood was collected and TT4 and TT3 levels were measured. Control rats fed ad libitum were sacrificed on days 0, 1 and 2.

197.                Body weight: The average body weight of PFDA-treated rats reduced over 8 days of treatment (statistical significance not determined). A similar trend was observed in the pair-fed control rats.

198.                Thyroid hormone levels: TT4 levels were significantly reduced compared with pair-fed and ad libitum controls at all timepoints, reaching a minimum concentration at 2 days post-treatment. TT3 levels were significantly lower than pair-fed controls at 12 hours, 1 day and 2 days. After 2 days, serum TT3 levels were similar to pair-fed controls. The authors proposed that the TT4 findings in pair-fed controls indicate the depression in thyroid hormones in PFDA treated animals was not the result of reduced feed intake.

199.                The authors concluded that following 4 days of pair-feeding, TT3 values were at the same level as that produced by PFDA treatment; however, TT4 levels in PFDA-treated rats were significantly lower than in those pair-fed throughout the study. These data indicate that the depression of thyroid hormones levels produced by PFDA is not solely a result of starvation.

200.                NTP (2022a) investigated the effects of PFDA on thyroid weight, histopathology and TH levels in rats. In a repeated dose study, SD rats (10/sex/group) were administered PFDA at doses 0, 0.156, 0.312, 0.625, 1.25, or 2.5 mg/kg bw/day by gavage for 28 days. At necropsy on day 29, blood samples were collected for TT4, TT3, FT4, TSH and PFDA analysis, and thyroids were removed for histopathological evaluation.

201.                Mortality: All rats survived to scheduled necropsy.

202.                General toxicity and body weight: No clinical signs of general toxicity were observed. In males and females, terminal body weights were significantly reduced at 1.25 and 2.5 mg/kg bw/day, compared with controls.

203.                Gross pathology: In males, relative thyroid weight:body weight was significantly increased at 1.25 and 2.5 mg/kg bw/day, compared with controls. Thyroid weights were unaffected by treatment. In females, thyroid weights were significantly increased at 0.312 mg/kg bw/day, 0.625 mg/kg bw/day and 1.25 mg/kg bw/day, and relative thyroid weight:body weight was significantly increased at ≥0.312 mg/kg bw/day.

204.                Histopathology: Histopathology in males and females was unaffected by treatment.

205.             Thyroid hormone levels: In males, TT4 levels were significantly decreased at 0.312 mg/kg bw/day, and FT4 levels were significantly decreased at ≥ 0.312 mg/kg bw/day, compared with controls. TT3 and TSH levels were unaffected by treatment. In females, FT4 and TT3 levels were significantly decreased at 1.25 and 2.5 mg/kg bw/day, respectively, and TT4 and TSH levels were unaffected by treatment.

206.             Plasma PFDA concentrations: In males, mean plasma PFDA concentrations on day 29 were 0.022 µg/mL (control), 8.505 µg/mL (0.156 mg/kg bw/day), 23.030 µg/mL (0.312 mg/kg bw/day), 42.720 µg/mL (0.625 mg/kg bw/day), 101.580 µg/mL (1.25 mg/kg bw/day) and 259.400 µg/mL (2.5 mg/kg bw/day). In females, concentrations were 0.042 (control), 11.208 µg/mL (0.156 mg/kg bw/day), 25.700 µg/mL (0.312 mg/kg bw/day), 50.290 µg/mL (0.625 mg/kg bw/day), 117.150 µg/mL (1.25 mg/kg bw/day) and 246.875 µg/mL (2.5 mg/kg bw/day).

207.             The authors concluded that FT4 was decreased, and TT4 was decreased in males only. TSH was unaffected and there were no histopathologic changes in the thyroid gland.

Van Rafelghem et al. 1987

208.             Van Rafelghem et al. (1987) investigated the effect of PFDA on thyroid hormone levels and thyroid histology in rats. Adult male SD rats (8 – 16/group) received a single i.p. injection of 20, 40 or 80 mg/kg bw PFDA on Day 0. Two controls groups were used both receiving a single injection of the vehicle. One control group was pair-fed whilst the other was allowed to feed ad libitum. Body weights and feed intake were measured daily for 7 days following treatment.

209.             Eight rats were sacrificed from each group on Day 7 after dosing. Blood was collected for thyroid hormone level analysis and thyroid glands for histopathological assessment. Free thyroxine index (FTI) was calculated as the product of the TT4 concentration and T3 uptake.

210.             General toxicity and body weight: PFDA treatment resulted in a dose- dependent reduction in body weight and cumulative feed intake over the 7-day period. Both were significantly reduced at doses of 40 and 80 mg/kg bw when compared with ad libitum controls but were unaffected compared with pair-fed controls.

211.             Gross pathology: Thyroid gland weights were significantly reduced in the 80 mg/kg bw group compared with ad libitum controls and pair-fed controls. Pair feeding with the 80 mg/kg bw treated group also significantly reduced thyroid weights, compared with ad libitum controls, leading the authors to propose that the reduced thyroid weight in treated rats was partly attributed to hypophagia (reduced ingestion of food).

212.          Histopathology: There were no histological changes attributed to treatment.

213.             Thyroid hormone levels: Treatment with PFDA significantly decreased TT4 levels in a dose-dependent manner from 20 mg/kg bw, compared with both pair-fed and ad libitum controls. Controls that were pair-fed with the 80 mg/kg bw treated group also showed a significantly reduced TT4 level compared with ad libitum controls. TT3 levels in PFDA-treated rats were unaffected compared with ad libitum controls at 80 mg/kg bw, whereas controls that were pair-fed with the 80 mg/kg bw treated group had significantly reduced TT3 levels. T3 uptake was significantly reduced at 80 mg/kg bw, compared with ad libitum controls, however no effects were seen in pair-fed controls. The authors state the treatment-related effects on FTI were similar to those for TT4 (data not provided).

214.             The authors concluded that reductions in TT4 concentration and FTI at a low dose of PFDA (20 mg/kg bw) indicate that PFDA-induced hypothyroxinemia can be dissociated from its overtly toxic effects (i.e., severe hypophagia and body weight loss) observed at higher doses. Although PFDA caused a dose-dependent decrease in thyroid gland weight (not completely paralleled by pair feeding), thyroid histology was unremarkable. These results suggest that despite alterations in plasma thyroid hormone levels there is no consistent pattern of effects on functional thyroid status which could explain the overt toxicity of PFDA.

Table 1 In vivo thyroid toxicity effects following acute exposure to PFSAs

*Derived by contractor; NR – not reported; NA – not applicable.

Substance	Species / sex / number	Dose / route of administration / duration (mg/kg bw)	Serum concentration (µg/mL)	Observed effects at LOAEL	Published NOAEL / LOAEL (mg/kg bw)	Reference
PFOS (potassium salt).	SD rats / female / 5 – 15/group.	15 / gavage / single dose.	At 15 mg/kg bw 37.28 ± 8.49 at 2 hours 66.90 ± 9.00 at 6 hours 61.58 ± 8.81 at 24 hours.	Serum ↑ FT4 at 2 – 6 hrs but not at 24hrs ↓ TSH at 2 – 6 hrs ↓ TT4 at 2 – 24 hrs. Liver ↑ ME mRNA at 2 hrs ↑ ME activity at 24 hrs ↑ UGT1A mRNA at 2 – 6 hrs.	NA / 15.	Chang et al. (2008).
PFOS (potassium salt).	SD rats / male and female / 4/group (male), 5/group (female).	15 / gavage / single dose.	NR.	↓ TT4 at 24 hrs) ↓ 125I in serum and liver) ↑ 125I in urine and faeces).	NA / 15*.	Chang et al. (2008).
PFOS (potassium salt).	Cynomolgus monkeys / male and female / 6/group.	Group 1: 0 or 9 / gavage / single dose on day	At 9 mg/kg bw on day 113.  67.7 ± 7.5 in males 68.8 ± 2.5 in Females.	No adverse effects on thyroid status.	9 / NA*.	Chang et al. (2017).

PFOS (potassium salt).

Cynomolgus monkeys / male and female / 4- 6/sex/dose.

0, 14, 14.8 / 17.2 (male/female) and 11 / gavage / single doses on day 43, day 288 and day 358 followed by 62 days recovery.

At 14 mg/kg bw/day on day 50 104.8 ± 502 in males 96.5 ± 6.2 in Females. At 14.8/17.2 bw/day on day 288 141.0 ± 13.1 in males 147.6 ± 17.5 in Females. At 11 mg/kg bw on day 365 160.8 ± 14.2 in Males  165.0 ± 6.7 in Females.

No adverse effects on thyroid status ↓ TT4 but values within normal Range.

Males: NA / 13.3 (average dose) Females: NA / 14.

Chang et al. (2017).

Table 2 In vivo thyroid toxicity effects following acute exposure to PFCAs

*Derived by contractor; NR – not reported; NA – not applicable.

Substance	Species / sex / number	Dose / route of administration / duration (mg/kg bw)	Serum concentration (µg/mL)	Observed effects at LOAEL	Published NOAEL / LOAEL (mg/kg bw)	Reference
PFDA	Wistar rats / male / 30/group.	75 / i.p. / single dose.	NR.	↓ TT4 ↓ TT3 (transient ≤ 2 days) ↓ Body weight (BW).	NA / 75*.	Langley and Pilcher (1985).
PFDA	SD rats / male / 8 – 16/group.	0, 20, 40 or 80 /  i.p. / single dose.	NR.	↓ TT4 ↓ FTI.	NA / 20*.	Van Rafelghem et al. (1987).

Table 3 In vivo thyroid toxicity effects following repeated exposure to PFSAs

*Derived by contractor; ** calculated according to EFSA. (2012); NR – not reported; NA – not applicable.

Substance	Species / sex / number / study type	Dose / route of administration / duration (mg/kg bw)	Serum concentration (µg/mL)	Observed effects at LOAEL	Published NOAEL / LOAEL (mg/kg bw)	Reference
PFBS (potassium salt).	ICR mice / female / 10/group / developmental study.	0, 50, 200 or 500 / gavage / GD1 – 20.	At 50 mg/kg bw/day Maternal serum: 0.074 ± 0.022. At 200 mg/kg bw/day Maternal serum: 0.332 ± 0.053.	Maternal: ↓ TT4, TT3 and FT4 ↑ TSH. Offspring: ↓ BW in females at all ages ↓ TT4 and TT3 in all ages ↑ TSH on PND30 ↑ Trh mRNA in hypothalamus on PND30.	Maternal: 50 / 200. Offspring: 50 / 200.	Feng et al. (2017).
PFBS	SD rats / male and female / 10/group / repeated dose study.	0, 62.6, 125, 250,500 or 1000 / gavage / 28 days.	At 62.6 mg/kg bw/day Plasma:2.222 ± 0.477 in males 0.154 ± 0.048 in Females.	↓ TT4, FT4 and TT3.	NA / 62.6.	NTP (2022b).
PFHxS (potassium salt).	SD rat / male and female / 15/sex/group / developmental study.	0, 0.3, 1, 3 or 10 / gavage / day 1 – day 43 (males); day 1 – PND21 or GD25 (females).	At 1 mg/kg bw/day on day 42 Serum: 89.1 ±0.80 in males. At 3 mg/kg bw/day on day 42 Serum: 128.67 ± 10.30 in males.	↑ Hyperplasia of thyroid follicular cells in males.	1 / 3.	Butenhoff et al. (2009).
PFHxS (potassium salt).	Long-Evans rats / female / 6 – 9/group / developmental study.	0 or 50 / gavage / GD6 - PND21.	NR	Maternal: ↓ TT4, TT3 and FT4. Offspring: ↓ TT4, TT3 and FT4↓ TT4 in brain tissue (PN 0 only).	Maternal: NA / 50*.- Offspring: NA / 50*.	Gilbert et al. (2021).
PFHxS (potassium salt).	SD rats / male and female / 10/group / repeated dose study.	0, 0.625, 1.25, 2.5, 5 or 10 (males), 0, 3.12, 6.25, 12.5, 25 or 50 (females) / gavage / 28 days.	At 0.625 mg/kg bw/day Plasma: 66.76 ± 3.518 in Males.	↓ TT4, FT4 and TT3 in males.	NA / 0.625*.	NTP (2022b).
PFHxS (potassium salt).	Wistar rats / female / 8/group / developmental study.	0, 25 or 45 / gavage / GD7 – PND22.	At 25 mg/kg bw/day Maternal serum: 139 on PND22 (SD not given).	Maternal and offspring: ↓TT4.	Maternal: ND / 25. Offspring: ND / 25.	Ramhøj et al. (2018).
PFHxS (potassium salt).	Wistar rats / female / 16 – 20/group / developmental Study.	0, 0.05, 5 or 25 / gavage / GD7 – PND22.	NR	Maternal and offspring: ↓TT4.	0.05 / 5.	Ramhøj et al. (2018).
PFHxS (potassium salt).	Wistar rats / female / 16 – 20/group / developmental study.	0, 0.05, 5 or 25 /gavage / GD7 – PND22.	NR	Maternal ↓TT3. Offspring: ↓ Thyroid weight in females.	Maternal: 5* / 25*. Offspring: 0.05* / 5*.	Ramhøj et al. (2020).
PFOS (potassium salt).	SD rats / male and female / 60 – 70/dose / repeated dose study.	0, 0.5, 2, 5 or 20 ppm equivalent to 0, 0.024, 0.098, 0.242, 0.984 or 1.144 (recovery group) (males) or 0, 0.029, 0.120, 0.299, 1.251 or 1.385 (recovery group) (females) / diet / 104 Weeks.	At 0.984 mg/kg bw/day Serum: 69.3 ± 0.351 in Males.	No adverse effects on thyroid status.	0.984* / NA.	Butenhoff et al. (2012).
PFOS (potassium salt).	SD rats / male / 6/group / repeated dose Study.	0 or 3 / gavage / 7 days.	NR	↓ TT4 and TT3.	NA / 3*.	Chang et al. (2008).
PFOS (potassium salt).	SD rats / female / 25/group / developmental study.	0, 0.1, 0.3 and 1.0 / gavage / GD0 - PND20.	NR	Maternal: No adverse effects on thyroid status ↓ BW. Offspring: Possible effect on thyroid epithelial cells in females on GD20.	Maternal: 1.0* / NA. Offspring: NA / 1.0.	Chang et al. (2009).
PFOS (potassium salt).	SD rats / female / 5/dose / repeated dose study.	0, 0.1, 0.3, 1, 2 / gavage / GD8 – PND2.	At 0.1 mg/kg bw/day on PND2 Serum: 2.2 ± 0.1.	Maternal: ↓ TT4 on PND2. Offspring: ↓ TT4 on PND2.	Maternal: NA / 0.1*. Offspring: NA/ 0.3*.	Conley et al. (2022).
PFOS	SD rats / male and female /	0, 2, 20, 50 or 100 ppm	At 0.14 mg/kg bw/day in males	↓ TT4.	Males: 0.14 / 1.33.	Curran et al. (2008).
(potassium salt).	20/group / repeated dose study.	equivalent to 0,0.14, 1.33, 3.21 or 6.3 (males) and 0, 0.15, 1.43, 3.73 or 7.58 (females) / diet / 28 days.	Serum: 0.95 ± 0.13. At 1.33 mg/kg bw/day in males Serum: 13.45 ± 1. 48. At 0.15 mg/kg bw/day in females Serum: 1.50 ± 0.23. At 1.43 mg/kg bw/day in females Serum: 15.40 ± 0.56.	N/A	Females: 0.15 / 1.43.	N/A
PFOS (potassium salt).	SD rats / male / 40/group / repeated dose study.	0, 20 or 100 ppm equivalent to 1.93 or 9.65 / diet / 7 days.	At 1.93 mg/kg bw/day Serum: 39.49 ± 7.76 on day 1 15.49 ± 1.60 on day 28 8.03 ± 1.14 on day 56 4.38 ± 0.72 on day 84.	No adverse effects on thyroid status ↓ BW.	NA / 1.93*.	Elcombe et al. (2012).
PFOS (potassium salt).	SD rats / female / 17 – 28/group / developmental Study.	0, 1, 2, 3, 5 or 10 / gavage / GD2- GD21.	NR	Offspring: ↓ TT4 and FT4.	Offspring NA / 1*.	Lau et al. (2003).
PFOS (potassium salt).	CD-1 mice / female / 21 – 22/group / developmental Study.	0, 1, 5, 10, 15 or 20 / gavage / GD1 – GD17.	NR	Offspring: No adverse effects on thyroid hormones Mortality.	Offspring: NA / 15*.	Lau et al. (2003).
PFOS (potassium salt).	SD rats / female / 20/group / two generation reproductive Study.	0, 0.4, 0.8, 1.0, 1.2, 1.6 or 2.0 / gavage / 42 days prior to mating through to PND4.	NR	Maternal: ↓ TT4. Offspring: ↓ TT4 and TT3.	Maternal: NA / 0.4. Offspring: 0.8* / 1.0*.	Luebker et al. (2005).
PFOS	SD rats / male and female / 10/group / repeated dose study.	0, 0.312, 0.625, 1.25, 2.5 or 5 / gavage / 28 days.	At 0.312 mg/kg bw/day. Mean plasma: 23.73 ± 1.114 in Males. 30.53 ± 0.918 in Females.	↓ TT4 and FT4.	NA / 0.312.	NTP (2022b).
PFOS (potassium salt).	Cynomolgus monkeys / male and female / 4- 6/group / repeated dose study.	0, 0.03, 0.15 or 0.75 / gavage / 182 days.	At 0.75 mg/kg bw/day on day 183.  Serum: 173 ± 37 in males 171 ± 22 in Females. At 0.15 mg/kg bw/day on day 183. Serum: 82.6 ± 25.2 in males 66.8 ± 10.8 in Females.	↓ TT3 and FT3 ↑ TSH ↓ BW gain. ↑ Mortality.	0.15 / 0.75.	Seacat et al. (2002).
PFOS (potassium salt).	SD rats / female / 25 – 50/group / developmental Study.	0, 1, 2, 3, 5 or 10 / gavage / GD2 – GD20.	ND	↓ TT4 and FT4 on GD7 – 21 ↓ T3 on GD21.	NA / 1.	Thibodeaux et al. (2003).
PFOS (potassium salt).	SD rats / female / 6 – 8 /group / repeated dose.	0, 3, 5 / gavage / 20 days.	ND	↓ TT4 and FT4 on day 3 – 20 ↓ T3 on day 7 – 20 ↑ TSH on day 7.	NA / 3*.	Thibodeaux et al. (2003).
PFOS (potassium salt).	CD mice / female / 60 – 80/group / developmental study.	0, 1, 5, 10, 15 or 20 / gavage / GD1 – GD17.	At 10 mg/kg bw/day on GD18 Maternal serum: 190 ± 7.	↓ TT4 on GD6.	15 / 20.	Thibodeaux et al. (2003).
PFOS (potassium salt).	Wistar rats / female / 3 – 9/group / developmental study.	0, 3.2 and 32 mg/kg diet equivalent to 0.38 and 3.8** / diet / GD1 – PND14.	At 0.38 mg/kg bw/day Maternal serum: 2.29 ± 0.15 on PND1 4.16 ± 0.04 on PND7 3.15 ± 0.21 on PND14. Offspring serum: 5.85 ± 0.33 on PND1 3.65 ±0.23 on PND7 4.89 ± 0.29 on PND14.	Maternal and offspring: ↓ TT4 and TT3.	NA / 0.38*.	Wang et al. (2011).
PFOS (potassium salt).	Wistar rats / female / 20/group / developmental study.	0 or 3.2 mg/kg diet equivalent to 0.29** / diet / Prenatal exposure GD0 – PND0; Postnatal exposure PND1 – 35; Combined prenatal and	At 0.29 mg/kg bw/day in offspring (Prenatal exposure) on PND35 Serum: 0.41 ± 0.02 in males.	Offspring: ↓ TT4 in all exposure groups.	NA / 0.29*.	Yu et al. (2009).
		postnatal group GD0 – PND35.	1.02 ± 0.08 in Females.  At 0.29 mg/kg bw/day in offspring (postnatal exposure) on PND35 Serum: 7.04 ± 0.59 in females. At 0.29 mg/kg bw/day in offspring (Combined prenatal and postnatal exposure) on PND35 Serum: 11.53 ± 0.28 in females on PND35.

Table 4 In vivo thyroid toxicity effects following repeated exposure to PFCAs

*Derived by contractor; NR – not reported; NA – not applicable.

Substance	Species / sex / number / study type	Dose / route of administration / duration (mg/kg bw/day)	Serum / plasma concentration (µg/mL)	Observed effects at LOAEL	Published NOAEL / LOAEL (mg/kg bw/day)	Reference
PFHxA (sodium salt).	Crl:Cd SD rats / male and female / 10/sex/group / repeated dose study.	0, 20, 100 or 500/ gavage / 92/93 days (males/females.	NR.	↑ Thyroid follicular epithelial hypertrophy (females) ↑ thyroid weight (males and females) ↓ BW (males).	100 / 500.	Loveless et al. (2009).
PFHxA	SD rats / male and female / 10/group / repeated dose study.	0, 62.6, 125, 250, 500 or 1000 / gavage / 28 days.	At 62.6 mg/kg bw/day Plasma:0.378 ± 0.178 in Males.	↓ TT4, FT4 and TT3 in males.	NA / 62.6.	NTP (2022a).
PFOA (ammonia salt).	SD rats / female / 5/dose / repeated dose study.	0, 10, 30, 62.5, 125 or 250 / gavage / GD8 – PND2.	At 10 mg/kg bw/day on PND2 Maternal serum: 31.8 ± 1.1.	Maternal: ↓ TT4, TT3, FT4 and FT3 on PND2. Offspring:↓ birthweights↓ TT3, TT4 and↓ rT3 on PND2.	Maternal: NA / 10*. Offspring: NA/ 10*.	Conley et al. (2022).
PFOA	SD rats / male and female / 10/group / repeated dose study.	0, 0.625, 1.25, 2.5, 5 or 10 (males), 0, 6.25, 12.5, 25, 50, or 100 (females) / gavage / 28 days.	At 0.625 mg/kg bw/day. Plasma: 50.690 ± 2.207 in Males.	↓ TT4, FT4 and TT3 in males.	NA / 0.625.	NTP (2022a).
PFNA	SD rats / male and female / 10/group / repeated dose study.	0, 0.625, 1.25, 2.5, 5 or 10 (males), 0, 1.56,3.12, 6.25, 12.5 or 25 (females) / gavage / 28 days.	At 0.625 mg/kg bw/day Plasma:56.730 ± 1.878 in Males.	↓ TT4 and FT4 in males.	NA / 0.625.	NTP (2022a).
PFDA	SD rats / male and female / 10/group / repeated dose study.	0, 0.156, 0.312, 0.625, 1.25, or 2.5 / gavage / 28 days.	At 0.156 mg/kg bw/day Plasma:8.505 ± 0.578 in males. 11.208 ± 0.436 in Females. At 0.312 mg/kg bw/day Plasma:23.030 ± 1.771 in males 25.700 ± 1.048 in Females.	↓ TT4 and FT4 in males. ↑ thyroid weight and relative thyroid weight: body weight in females.	Males: 0.156* / 0.312*. Females: 0.156 / 0.312.	NTP (2022a).

Summary

215.                This paper presents an overview of the in vivo data for thyroid toxicity. It does not contain an overall summary and conclusions as these will be provided once the review of in vitro and epidemiological data has taken place. A review of authoritative body opinions and evaluation of adversity will also be undertaken. Nevertheless, some key points to note are discussed below.

216.                All seven PFAS showed decreases in TH levels (including in offspring) but generally without an associated rise in TSH levels, and there were sex differences in response. There is disagreement between researchers about the biological significance of these findings.

217.                Thyroid morphology was examined in 18 of the 27 studies on the seven PFAS, but adverse findings (follicular epithelial cell hyperplasia and hypertrophy) were only found in four studies on four different PFAS, of which only one study measured TH and showed it to be reduced. The significance of these morphological changes in these studies is uncertain as it is suggested that they could be secondary to liver hypertrophy and the induction of metabolic liver enzymes leading to an increase in T4 and TSH. Moreover, as T4 in rodents has a short half-life, they are more sensitive, hence this effect may not be relevant to non-rodent species.

218.                Authoritative bodies such as the Agency for Toxic Substances and Disease Registry (ATSDR) based their point of departure (POD) on effects on thyroid pathology, while the United States Environmental Protection Agency (US EPA) consider reduced TH levels to be an indication of adverse effects. These opinions will be explored in future papers.

219.                Overall, the in vivo evidence indicates that low doses of PFAS can produce adverse effects on levels of thyroid hormones (without affecting TSH levels), and at higher doses, can produce morphological alterations in the thyroid. However, some of the findings are inconsistent, sex-specific and difficult to interpret in terms of adversity and human relevance.

Questions on which the views of the Committee are sought

220.             Members are invited to consider the following questions:

i).     Are there any specific papers that the subgroup would like to review in more detail?

iii)     Would a discussion of adversity and human relevance be useful at this stage, or should this await a consideration of epidemiological data?

IEH Consulting under contract supporting the UKHSA COT Secretariat

August 2023 List of Abbreviations and Technical terms

ApoA1	Apolipoprotein A1
BDE-47	2,2’,4,4’-tetrabromodiphenyl ether
Dio1	Type 1 deiodinase, iodothyronine deiodinase type 1
EFSA	European Food Safety Authority
FT3	Free triiodothyronine
FT4	Free thyroxine
FTI	Free thyroxine index
GD	Gestational day
HBGV	Health-based guidance value
HPT	Hypothalamic–pituitary–thyroid
i.p.	Intraperitoneal
K+PFBS	Potassium perfluorobutanesulfonate
K+PFHxS	Potassium perfluorohexanesulfonate
K+PFOS	Potassium perfluorooctane sulfonate
LOEL	Lowest observed effect level
Mdra1	Multidrug resistance 1
ME	Malic enzyme
mRNA	Messenger ribonucleic acid
NA	Not applicable
NAM	New approach methodology
NaPFHxA	Sodium perfluorohexanoate
ND	Not detected
nis	Sodium-iodide symporter
Nkx2.1	NK2 homeobox 1 (TTF-1)
NOAEL	No observed adverse effect level
NR	Not reported
NTP	National Toxicology Program
Pax8	Paired box 8
PFBS	Perfluorobutane sulfonate
PFCA	Perfluoroalkyl carboxylic acid
PFDA	Perfluorodecanoic acid
PFHxA	Perfluorohexanoate / Perfluorohexanoic acid
PFHxS	Perfluorohexanesulfonate/Perfluorohexanesulfonic acid
PFNA	Perfluorononanoic acid
PFOA	Perfluorooctanoic acid
PFOS	Perfluorooctane sulfonate
PFSA	Perfluorosulfonic acids
PND	Postnatal day
Por	P450 oxidoreductase
RNA	Ribonucleic acid
rT3	Reverse triiodothyronine
SD	Sprague Dawley
Slc5a5 (NIS)	Solute carrier family 5
Spot14	Thyroid hormone-inducible hepatic protein, or THRSP
T3	Triiodothyronine
T4	Thyroxine
TT3	Total triiodothyronine
TT4	Total thyroxine
TH	Thyroid hormone
Tpo	Thyroid peroxidase (also threoperoxidase)
TR	Thyroid hormone receptor
Trh	Thyrotropin releasing hormone
TRα	Thyroid hormone receptor α
TRα-LBD	Thyroid hormone receptor α ligand-binding domain
TRβ	Thyroid hormone receptor β
TSH	Thyrotropin also thyroid stimulating hormone
Tshr	Thyroid stimulating hormone receptor
ttf-1	Thyroid transcription factor 1
TTR	Transthyretin
TWI	Tolerable weekly intake
UGT1A	UDP‑glucuronsyltransferase 1A
UGT1A1	Uridine diphosphoglucuronosyl transferase 1A1
UGT1A6	Uridine diphosphoglucuronosyl transferase 1A6