# Limits of the regulatory evaluation of resorcinol as a thyroid disruptor: When limited experimental data challenge established effects in humans

Elodie Pasquier, Catherine Viguié, Jean-Baptiste Fini, Sakina Mhaouty-Kodja, Cécile Michel-Caillet

Mhaouty-Kodja, Cécile Michel-Caillet

► To cite this version:

Elodie Pasquier, Catherine Viguié, Jean-Baptiste Fini, Sakina Mhaouty-Kodja, Cécile Michel-Caillet. Limits of the regulatory evaluation of resorcinol as a thyroid disruptor: When limited experimental data challenge established effects in humans. Environmental Research, 2023, 222, pp.115330. 10.1016/j.envres.2023.115330. hal-04749039

HAL Id: hal-04749039

https://hal.science/hal-04749039v1

Submitted on 22 Oct 2024

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

Public Domain
1 **Title:**

2 Limits of the regulatory evaluation of resorcinol as a thyroid disruptor: when limited experimental data

3 challenge established effects in humans

4 **Authors:**

5 Elodie Pasquier¹, Catherine Viguié², Jean-Baptiste Fini⁴, Sakina Mhaouty-Kodja³, Cécile Michel-Caillet¹.

6 ¹ANSES, Risk Assessment Department, Maisons-Alfort, France

7 ²Toxalim (Research Centre in Food Toxicology), INRAE, ENVT, INP-Purpan, UPS, Toulouse, France

8 (corresponding author)

9 ³Sorbonne Université, CNRS, INSERM, Neuroscience Paris Seine - Institut de Biologie Paris Seine, Paris, France

10 ⁴UMR 7221, CNRS, MNHN, Laboratoire PHYMA Paris, France

11

12

13

14

15 **Corresponding author:** Catherine Viguié

16 TOXALIM UMR 1331, INRA INP UPS

17 180 chemin de Tournefeuille, BP 93173

18 31027 Toulouse cedex3, France

19 Phone: +33 582 06 64 14

20 Email: catherine.viguie@inrae.fr

21

22 All authors declare they have nothing to disclose.

23

24
25

26 **Abstract**

27

28 Severe hypothyroidism has been reported in humans during resorcinol therapeutic use. However, available
29 data highlight differences in the severity of resorcinol-induced thyroid effects between humans and rodents,
30 leading to a debate on the relevance of human data for its classification as a thyroid disruptor.

31 The aim of this review is to illustrate some of the limitations of the evaluation framework for thyroid
disrupters using resorcinol as a case study of a chemical with clear thyroid-disrupting properties in humans
that could not have been identified solely from regulatory studies on animals.

34 The reliability of human data has been called into question due to the specific exposure patterns in humans
35 and the paucity of robust toxicokinetic data. In humans, therapeutic use of resorcinol induces severe
36 hypothyroidism, but in rodents, thyroid disruption is limited to decreased thyroxine concentrations and
37 histological changes in the thyroid. The adverse effects of thyroid disruption, such as impaired
38 neurodevelopment, have not been sufficiently investigated, and experimental neurobehavioral data for
39 resorcinol remain scarce and inconclusive.

40 Although regulatory toxicological evaluations have not included in-depth investigations of thyroid regulation
41 and related adverse effects, they have been used to challenge the relevance of human data. Resorcinol is an
42 emblematic example of how the framework for regulatory evaluations of thyroid disruptors relies almost
43 exclusively on animal studies which may not be suitable for assessing thyroid disruption. This review
44 highlights the need to revise regulatory guidelines and to adopt strategies based on up-to-date, scientifically
45 sound approaches to identify thyroid disruptors. The limits of the current regulatory framework for
46 identifying thyroid disruptors can lead to opposing positions between regulatory bodies. The French Agency
47 for Food, Environmental and Occupational Health & Safety (ANSES)' proposal to identify resorcinol as a
48 "substance of very high concern" due to its ED properties has not been adopted by the European instances.

49

50 **Keywords:** Endocrine disruptors- Resorcinol- Thyroid- neurodevelopmental toxicity -Risk and public health –
regulatory toxicological evaluation -

52
53

54 Funding: the work reported in this study has been conducted by the "endocrine disruptor" working group supported by ANSES, the French National agency for food, environment and occupational safety. This work did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors.

58

59

60
## 61 Introduction

## 62

63 Risk assessments of endocrine-disrupting chemicals (EDCs) constitute a huge challenge because the 64 endocrine system regulates nearly all physiological processes in a timely and often non-monotonic manner. 65 Regulatory bodies throughout the world are therefore continuously looking for new strategies for the 66 assessment of these compounds (Barton-Maclaren et al., 2022; Browne et al., 2020). Although the World 67 Health Organization (WHO)'s definition of EDCs is consensually recognized (WHO, 2002), the existing 68 institutional framework for chemical evaluation specifically addressing endocrine disruption — whether 69 based on the Environmental Protection Agency (EPA)'s tiered approach in the United States or on the 70 Organization for Economic Co-operation and Development (OECD)'s five-level rating approach in the 71 European Union — remain incomplete and limited to very few guidelines, some of which are obsolete and/or 72 inappropriate for EDCs. Historically, the focus has been placed on the male and female reproductive 73 functions. The thyroid endocrine system came to the forefront much later, highlighting major gaps in the 74 tools available for the hazard assessment of thyroid-disrupting chemicals (TDCs). The thyroid function is 75 critical for both the regulation of key physiological processes in adult and adequate development. The 76 amphibian metamorphosis assay, based on the thyroid-dependent metamorphosis process, is used for low- 77 level (OECD level 2 and 3) or tier (US EPA tier 1) tests to investigate thyroid-specific endpoints (Table 1). 78 However, even in the amphibian test guidelines, investigation of parameters characterizing the thyroid 79 function remains extremely limited. In higher vertebrates, there is no specific endpoint such as 80 metamorphosis that can be considered as diagnostic of thyroid disruption. Further, many adverse effects 81 potentially associated with exposure to TDCs can result from other modes of action (MoA). Thus, disruption 82 of such a complex and pluripotent endocrine function is difficult to test with available traditional regulatory 83 schemes elaborated for highly specialized function such as reproduction. The robust identification of a cause- 84 effect relationship between a thyroid-related MoA and an adverse effect, which is mandatory for a substance 85 to be recognized as an EDC targeting the thyroid function, may be very challenging. To overcome such 86 difficulties, regulatory bodies around the world have encouraged the use of adverse outcome pathway (AOP) 87 approaches, which link molecular events to adverse effects via a plausible MoA (Browne et al., 2020, 2017), 88 and/or other approaches based on a broad, holistic view of the mechanistic evidence (La Merrill et al., 2020) 89 for the identification of EDCs including TDCs (Noyes et al., 2019). Although these approaches have proved 90 helpful in improving the efficiency of TDC evaluation by allowing the integration of academic data into the 91 evaluation process, there are only a few reviewed and published AOPs available for TDCs, and these are 92 limited to neurodevelopmental toxicity endpoints. Despite this clear data gaps, until 2013, no EU-funded, 93 large-scale project had directly addressed TDC issues. It was only in 2020 that three large-scale EU projects
94 dedicated to the development of innovative and specific methods to detect TDCs were finally launched (the
95 ATHENA (Kortenkamp et al. 2020), SCREENED and ERGO projects).

96 Despite the peculiarities of the thyroid function and the fact that existing guidelines are clearly insufficient
97 when considering thyroid disruption, there is currently no guideline exclusively dedicated to TDC evaluation.
98 For example, evaluating thyroid disruption is barely mentioned in the different levels/tiers of either OECD or
99 US-EPA frameworks (Noyes et al., 2019). When measurements of thyroid-related parameters are mentioned,
100 they are rarely mandatory and usually very perfunctory. They are generally limited to a merely descriptive
101 evaluation of thyroid gland histology and/or thyroid hormone (TH) measurements (Table 1). Guidance has
102 been produced at the EU level (ECHA/EFSA 2018), emphasizing what should be considered an adverse effect
103 and how to handle notable differences in the systemic regulation of TH levels between commonly used
104 experimental animal models and humans. Although there is undoubtedly a desire to improve the framework
105 of TDC evaluation, major gaps have not been addressed at all, leaving the door wide open to debates on the
106 relevance of the analysis. There is to date no comprehensive testing strategy or integrated conceptual
107 framework that can integrate all available data across various fields — from molecular testing to human
108 clinical observations — or propose decision strategies when some type of data is lacking or insufficiently
109 documented. An integrated strategy is vital for TDC, because animal models recommended for high level/tier
110 mammal trials of regulatory evaluation, i.e. frequently rodents, can be easily — and are — challenged with
111 regard to their relevance for human thyroid system regulation. Finally, one of the major caveats of TDC
112 evaluation arises from the fact that none of the guidelines are designed to establish the link between thyroid
113 disruption and apical adverse effects, one of the most worrying being the alteration of neurodevelopment.
114 From this standpoint, identifying and including neurodevelopmental endpoints that could potentially be
115 more sensitive and/or specific of thyroid disruption e.g. periventricular cortical heterotopia (Gilbert et al.,
116 2014) could reveal itself to be very helpful. An optimized protocol for thyroid hormone measurements in
117 dams and pups allowing a better coverage of the window of susceptibility in terms of the thyroid axis
118 development as well as thyroid-dependent neurodevelopmental processes would undoubtedly constitute a
119 huge progress.

120 In this context, the evaluation of resorcinol is a striking example of the gaps in TDC evaluation and, in
121 particular, of how complying strictly and solely with the few recommendations for TDCs in the ED evaluation
122 conceptual framework can be misleading if the well-established endocrine toxicity in humans is not
123 considered. Resorcinol (CAS 108-46-3) is a small molecule with a wide range of applications (WHO, 2006):
124 ultraviolet absorber, antioxidant, antiseptic, etc. In the context of its use as a medicinal product in humans
125 in particular to treat skin ulcers, resorcinol has long been described to interfere with thyroid function
126 (Fawcett and Kirkwood, 1953). There is a unique set of experimental and human studies describing its
127 thyroid-disrupting properties (Supplemental Material, Table1). Human data show severe clinical
128 hypothyroidism with associated goiter. Other than goiter, no other potential apical adverse effect related to
129 thyroid system disruption has ever been properly investigated. Resorcinol is a prime example of a situation
130 where there is a clear demonstration of thyroid disruption in humans, but for which *in vivo* experimental data
131 lead to mitigated conclusions. The evaluation of thyroid function in guideline studies, although complying
132 with official guidelines, is incomplete and limited.

133

134 Available data on resorcinol, whether from academic or regulatory studies, were analyzed in the context of
135 the French Agency for Food, Environmental and Occupational Health & Safety (ANSES)'s proposal (ANSES,
136 2020) to identify resorcinol as a "substance of very high concern" due to its TD properties under the European
137 Regulation on the Registration, Evaluation, Authorization and Restriction of Chemicals (REACH). The main
138 objective of this commentary is to use discrepancies between different types of studies, more particularly
139 human clinical and animal experimental studies, regarding the thyroid disrupting properties of resorcinol, to
140 highlight some of the limitations of the thyroid disruptor evaluation framework. More specifically,

141 we aimed to shed light on why and how a clear AOP linking a thyroid-related MoA (i.e. thyroid peroxidase
142 (TPO) inhibition) to adverse effects in humans (clinical hypothyroidism) was not considered reliable enough
143 to identify resorcinol as a "substance of very high concern" due to its TDC properties (ECHA, 2020). We set
144 out to gain an understanding of how the limitations of available regulatory data can lead to the failure to
145 identify TDCs in the absence of human data. Ultimately, our goal is to provide insights for further
146 improvements to the regulatory framework for TDC evaluation and testing.

147

148 Methods

149 Our analysis focused on demonstrating the ability of resorcinol to disrupt thyroid function based on the EU
150 Joint Research Center (JRC)'s application of the WHO definition (WHO, 2002; JRC, 2013): inducing an adverse
151 effect, having an endocrine MoA, and a plausible link between the adverse effect and the MoA based on
152 general knowledge or existing and validated AOPs. Here, we considered regulatory studies submitted in the
153 context of the European REACH regulation. In addition, studies from an exhaustive literature search
154 conducted until 12 November 2019 were selected for their relevance and adequacy for the assessment of
155 thyroid disruption, and their reliability was assessed using ToxRtool (Segal et al., 2015). A bibliographic search
156 regarding the thyroid effects of resorcinol was updated until 26 October 2021. All studies were qualitatively
157 weighted, based on expert judgment, to analyze the thyroid-disrupting properties of resorcinol. Only data
158 appropriate for the demonstration of the limitations and pitfalls of the framework for the regulatory
159 identification of EDs are presented here. Comprehensive detailed data are available in the ANSES's proposal
160 to identify resorcinol as a substance of very high concern due to its TD properties (ANSES, 2020) that has
161 been published on the European Chemicals Agency (ECHA, 2020) website.

162 Summary tables of human and experimental data are available as Supplemental Material.

163

164 **<u>Discussion</u>**

165 <u>Do animal and human data lead to conflicting conclusions?</u>

166 Ten human cases published between 1950 and 1977 (Supplemental Material, Tables 2–6) reported severe
167 clinical hypothyroidism as an adverse effect of long-term chronic exposure to resorcinol via the application
168 of a cutaneous ointment for the treatment of cutaneous ulcers (for review see Lynch et al. 2002). Biological
169 parameters and thyroid histology, when available, were consistent with a blockage of TH synthesis.
170 Regression of the goiter and other symptoms after cessation of exposure to resorcinol in 8 of the 10 reported
171 cases demonstrated the causal relationship between adverse effects of resorcinol in adults and the thyroid
172 MoA. This reversibility attenuates the perception of adversity. However, this seeming reversibility is quite
173 misleading, because more subtle subclinical thyroid alterations during developmental stages can lead to
174 irreversible damages, particularly in terms of neurobehavioral development (Korevaar et al., 2018; Zoeller
175 and Rovet, 2004). In rodent studies, results have been called into question due to the poor quality of the data
176 and/or major differences in the experimental designs. The available regulatory studies indeed provide
177 measurements of all the mandatory thyroid parameters for regulatory toxicology. However, these studies
178 were too limited for a proper investigation of thyroid function and often did not include the most relevant
179 period of measurements (no measurement of THs during gestation or concomitant to neurobehavioral tests)
180 with regard to the only thyroid-relevant apical adverse effect investigated, i.e. neurodevelopment.

181 Results obtained via the oral route in rodents differ markedly between gavage and diet or drinking water
182 administration. Most often, no effect could be demonstrated with gavage, including at very high doses. By
183 contrast, thyroid parameters were often modified in a sense suggesting decreased thyroid activity following
184 more "physiological" oral routes, such as the diet or drinking water, including at much lower doses (for detail,
185 see Supplemental Material, Tables 2–6). Interestingly, thyroid histopathological lesions and enlargement,
186 two phenomena considered as diagnostic of thyroid disruption by standard-setting organizations such as the
187 OECD or US EPA (Table 1), have been observed in rats with subcutaneous administration of aqueous
188 (Cheymol et al., 1951) or oil solutions at doses within the range of the therapeutic dose for humans, and long
189 durations of exposure (Doniach and Logothetopoulos, 1953; Samuel, 1955). The results of these old pre-
190 Guideline studies are not precisely described, and it is difficult to determine the significance of these
191 observations. Similar effects have been observed following dermal applications of ointment at a much higher
192 concentrations for 4 weeks (Samuel, 1955), but not after shorter applications for 3 weeks (Doniach and 193 Logothetopoulos, 1953).

194 Developmental exposure to resorcinol through drinking water (Unpublished Study report, 2003; Welsch et 195 al., 2008) was associated with sex, dose and age-dependent changes for some thyroid parameters. Overall, 196 except for an increased T3 concentration in F0 females and F1 and F2 pups at weaning, all observed 197 modifications argue in favor of decreased thyroid function: decreased colloid content in F0 males, increased 198 thyroid-stimulating hormone (TSH) concentration in F1 male offspring at weaning at higher doses, increased 199 thyroid weight jointly with decreased T4 levels in female F2 pups at weaning. Interestingly, similar patterns 200 of thyroid alterations have been observed in mildly hypothyroid children with TPO gene mutations, with 201 mildly elevated serum TSH levels, normal or slightly low serum T4 levels, and a high T3:T4 molar ratio (Narumi 202 et al., 2017).

203 Overall, the data available for rodents suggest that resorcinol can affect the thyroid function when given 204 through "physiological" oral or parenteral routes, but not through gavage. Therefore, there are no major 205 discrepancies between animal and human studies regarding the plausibility of a thyroid MoA of resorcinol. 206 The possibility remains however that ointment application on damaged skin can lead to overexposure in 207 human subjects. This potential overexposure has not been documented by measuring resorcinol and/or its 208 metabolite concentrations in patients. The question remains of whether the route of administration and 209 associated internal exposure can be determining for the effect of resorcinol on thyroid function in humans.

## 210 Is the human route of exposure prohibitive?

211 Two toxicokinetic (TK) lines of evidence were used to challenge the regulatory identification of resorcinol as 212 an endocrine disruptor during European debates on the proposal to identify resorcinol as a substance of very 213 high concern due to its TD properties (ECHA, 2020). Firstly, gavage data showing no effect of resorcinol on 214 the thyroid function in rats was considered as strong evidence, assuming that this route is fully representative 215 of oral exposure, with no further consideration of resorcinol TK properties. Secondly, it was assumed, again 216 with no further consideration of resorcinol TK properties, that the therapeutic use of resorcinol was 217 associated with a high level of internal exposure.

218 TK studies conducted in rats and rabbits by gavage showed highly efficient digestive absorption of resorcinol 219 (from 77% to 95% within 24 h of gavage; Garton and Williams 1949; Kim and Matthews 1987), and extensive 220 metabolism before urinary excretion. About 90% of the administered dose is recovered after 24 h in urine, 221 mainly as a glucuronide conjugate (Garton and Williams 1949; Kim and Matthews 1987). *In vitro* results 222 obtained in human hepatic S9 fractions and primary human hepatocytes (Eilstein et al., 2020) suggest that 223 the hepatic conjugation of resorcinol is very efficient in humans as well. The high and rapid metabolism
224 rates are consistent with a high hepatic first-pass effect, which would thus limit internal exposure following 225 gavage. In the case of "physiological" oral routes, the buccal and sublingual mucosae are directly exposed to 226 the compound. Depending on the physicochemical properties of the compound, this may result in partial 227 direct absorption from these mucosae, bypassing first-pass metabolic effects similarly to parenteral routes. 228 Therefore, gavage data may not be ideal for the evaluation of the effect of exposure via the oral route, in 229 particular for substances that are quickly metabolized in the liver. In addition, the stressful conditions related 230 to this route might hinder clear conclusions on biological effects including endocrine effects. Indeed, gavage 231 by itself can induce neuroendocrine modifications that might be stress-related including in the context of 232 developmental exposure (Cao et al., 2013; McDonnell-Dowling et al., 2017).

233 Thus, the relevance of the effects observed in humans exposed through parenteral route cannot be 234 challenged solely on the basis of the absence of effect with gavage. Furthermore, more generally, these 235 considerations regarding administration should prompt regulatory bodies to modify guidelines, making 236 "physiological" routes the reference administration route in regulatory studies, unless justified by technical 237 or physicochemical limitations.

238 The main line of evidence for the thyroid-disrupting activity of resorcinol comes from human data in 239 which resorcinol was applied as an ointment on damaged skin (for review, see ANSES 2020). The relevance 240 of these data was considered debatable based on the argument that the breakdown of the cutaneous barrier 241 can lead to increased bioavailability and overexposure in patients. However, no measurements of blood 242 concentration in these patients are available to test this hypothesis. For intact skin, dermal absorption seems 243 rather low as shown in human volunteers following discrete, repeated applications of hydroalcoholic 244 solutions (Yeung et al., 1983), hair dye formulations (Roberts et al., 1977) or ex vivo on skin explants. In all 245 cases, no more than 5% of the daily dose was absorbed after 24 h. These data however contradict more 246 recent studies on pig and human skin explants, indicating transcutaneous transfer of resorcinol and/or its 247 metabolites of about 50% of the administered dose after 24 h, 4.8% of which being parent resorcinol (Géniès 248 et al., 2019; Hewitt et al., 2020).

249 Although a case can be made that all the conditions might have been met to favor internal exposure in the 250 medical cases, too many uncertainties remain regarding cutaneous absorption and metabolization of 251 resorcinol to unambiguously validate this hypothesis. Furthermore, of note, the therapeutic use of resorcinol 252 on damaged skin continues to be prescribed — particularly for the treatment of hidradenitis suppurativa 253 (Docampo-Simón et al., 2022) — at concentrations of 15%.

254 The paucity of TK data precludes the evaluation of the significance of the mode of administration as a 255 determining factor of the differences in thyroid changes between humans and rodents. In conclusion, the 256 relevance of the effects observed in human cases under specific circumstances cannot be excluded for other
257 exposure conditions. Regardless of the interpretation of the few available TK studies, thyroid effects have 258 nonetheless been observed in rodents — although not as severe as in human patients — and, in some 259 instances, for much lower doses and likely lower bioavailability than on damaged skin. Therefore, the 260 singularity of human exposure is not in itself enough to consider that TDC effects of resorcinol are not of 261 concern.

262 **Rat vs. human sensitivity to thyroid disruption**

263 The MoA of resorcinol on thyroid function clearly relies on inhibition of TPO activity. Ultimately, long-term 264 TPO inhibition, as for high prolonged exposure to resorcinol, can lead to hypothyroidism and goiter formation 265 in humans, as shown in Hashimoto thyroiditis (Caturegli et al., 2014) and in a condition caused by a TPO 266 mutation resulting in moderate residual TPO activity resulting in the apparition of goiter only after more 267 than 8 years of age (Narumi et al., 2017). The TPO-targeting MoA of resorcinol has been demonstrated in 268 several *in vitro* studies using purified porcine thyroid TPO, human thyroid cell lines, rat thyroid microsomal 269 fractions, or various cell lines transfected with human TPO gene and with different substrates (Cooksey et al. 270 1985; Dong et al. 2020; Lindsay et al. 1992; Paul et al. 2014; Paul Friedman et al. 2016). All but one study 271 (Paul et al., 2014) reported very high potency (low half maximal inhibitory concentration, IC₅₀) of resorcinol 272 to inhibit TPO. In three studies, resorcinol potency was much higher than that of the reference TPO inhibitors 273 methylmercaptoimidazole and propylthiouracil (Cooksey et al., 1985; Dong et al., 2020; Lindsay et al., 1992). 274 Rodents are usually considered more sensitive to thyroid disruption than humans due to lower thyroglobulin 275 stocks, higher responsiveness to TSH, and/or lack of thyroxine binding globulin (TBG) expression in adult 276 animals (Bartsch et al., 2018). It is therefore likely that the apparent higher sensitivity of humans to resorcinol 277 is related to different pharmacodynamic properties of TPO. Unfortunately, no reports of interspecies 278 differences in TPO pharmacodynamic parameters are currently available in comparable experimental 279 designs. Further studies are therefore needed to determine whether interspecies differences in TPO 280 sensitivity to resorcinol can contribute to the difference between rodents and humans.

281 Human relevance of the rat model is still a matter of debate. To address this issue, a workshop on thyroid 282 disruption was held in 2017 with support from the European Commission and ANSES (European Commission, 283 2017). Some of the conclusions of this workshop were included in the ECHA/EFSA revision of the guidance 284 for the identification of endocrine disruptors (ECHA/EFSA et al., 2018). This guidance stated that "in the 285 absence of substance-specific data which provide proof of the contrary, humans and rodents are considered 286 to be equally sensitive to thyroid-disruption." The resorcinol database illustrates the fact that rodents should 287 not be considered by default as more sensitive than humans.

288 Despite a limited number of published cases, human clinical studies unambiguously show that resorcinol can 289 dramatically alter thyroid function. In contrast, there is a complete lack of good quality epidemiological
290 studies to conclude on an effect of resorcinol in the context of an exposure pattern more likely to occur in
291 the general population (lower dose, oral route, etc.). Given the effects of resorcinol on TPO, subclinical
292 hypothyroidism can be expected. These types of thyroid changes are generally largely underdiagnosed
293 (Andersen, 2019) and may have long-lasting deleterious effects on health, in particular during critical periods
294 of development.

295 Are thyroid-related adverse effects properly documented in animals?

296 The only potentially thyroid-related adverse effect of resorcinol that have been investigated in animal
297 experimental studies involve neurodevelopmental outcomes (Unpublished Study report, 2003). In rats, after
298 developmental exposure through drinking water (0-10-40-120-360 mg/l i.e. mean doses of
299 0-0.8-3.9-13.1-36.9 mg/kg/d in males and 0-0.8-5.1-15.60 and 46.6 mg/kg/d in females) from 28 days prior to
300 mating until weaning at postnatal day (PND) 21, no signs of altered neurodevelopment are demonstrated in
301 juveniles (Unpublished Study report, 2003). The only signs of altered neurodevelopment have been noted at
302 PND60 in F1 males and characterized by increased locomotor activity from 40 mg/L of water, i.e. about 4
303 mg/d/kg bw. This could not be demonstrated in females likely due to an improper schedule of testing ignoring
304 the stage of the estrous cycle. Estrogens are indeed well-known regulators of locomotor activity (Rosenfeld,
305 2017; Scimonelli et al., 1999). Similarly, effects of resorcinol exposure could not be demonstrated on juvenile
306 or adult F1 rats based on the acoustic startle response or spatial memory tests. It was concluded from these
307 behavioral tests and from brain histology that resorcinol has no effects on the brain. However, these
308 neurodevelopmental investigations clearly suffer from at least two major limitations. First, although the
309 behaviors analyzed in these studies are regulated by different neural structures controlling different cellular
310 processes and signaling pathways, the observations of the brain were global and not detailed according to
311 their structures and functions. Second, the results obtained for spatial memory should have been corrected
312 for increased motor activity, which can compensate for a slight memory deficit (and thus influence
313 performance in the Biel maze swimming test). Finally, although in line with neurodevelopmental parameters
314 that can be required in guideline studies, the histological analyses were not sufficient or appropriate for the
315 detection of any cellular or molecular changes induced by resorcinol exposure and potential underlying TH-
316 regulated mechanisms.

317
318 The current design of rodent studies are clearly not sufficiently sensitive to detect adverse
319 neurodevelopmental effects related to maternal and perinatal hypothyroxinemia, as previously reported
320 (Ramhøj et al., 2020). In this context, approaches such as AOPs (OECD, 2018, AOP 42
321 (https://aopwiki.org/aops/42, AOP54 https://aopwiki.org/aops/54) can help in the process of hazard
322 assessment. Two recently developed AOPs addressing the impact of decreased T4 on brain development
323 have been published. AOP 42 reports the inhibition of TPO, which is the target of resorcinol, and subsequent
324 adverse neurodevelopmental outcomes in mammals (Crofton et al., 2019). AOP 54 describes the inhibition
325 of Na+/I- symporter (NIS) leading to learning and memory impairment (Rolaki et al., 2019). Through different
326 molecular initiating events, both AOPs lead to a decrease in T4 levels with neurodevelopmental
327 consequences and establish a relationship between a decrease in T4 serum concentrations and a decrease in
328 cognitive function, with a high weight of evidence and with high relevance during brain development (fetal
329 and perinatal stages).

330

331 Conclusions

332 In conclusion, available human data (even though obtained in disease conditions and concomitantly to
333 pharmaceutical use) unambiguously show that resorcinol can act as a TDC. These observations are consistent
334 with *in vitro* data showing the high potency of resorcinol to inhibit TPO activity. Rodent data, although
335 showing some degree of thyroid disruption and related adverse effects (particularly in the context of
336 developmental exposure), are not fully consistent with human clinical and *in vitro* mechanistic data showing
337 clear thyroid disrupting properties of resorcinol. In addition, these experimental animal studies are too
338 limited and contain too many methodological deficiencies to be fully conclusive on their own on TDC
339 properties of resorcinol. However, the comprehensive dataset is highly consistent in terms of biology (Figure
340 1, Table2). Many criticisms have been raised regarding the potential impact of the routes and modes of
341 administration. Our analysis shows that neither the route of exposure in the human cases, nor the near
342 absence of a thyroid effect with gavage in rodents are in themselves sufficient to reject the possibility that
343 resorcinol acts as a TDC. In particular, TK data can predict internal exposure from the different routes, but no
344 TK studies have been conducted.

345 Our analysis examined considerations relative to interspecies differences in the regulation of thyroid
346 function. In contrast to the general assumption that rodents are more sensitive to TDC than humans, the set
347 of available data suggests the opposite. This highlights the need to weigh in other considerations e.g.
348 pharmacodynamic difference, rather than just interspecies sensitivity comparison. Although TPO-dependent
349 iodine organification is one of the most highly conserved events of TH biosynthesis among vertebrates,
350 rodents may be nonetheless less sensitive to the inhibition of TPO than humans. Given the high level of
351 uncertainty on this aspect, human data should not be dismissed on the basis of findings in rodents.

352 Clinical hypothyroidism and associated symptoms are reversible in adults. Accordingly, the reversibility of
353 these effects when resorcinol treatment is interrupted can be taken as tangible proof of the cause-effect
354 relationship between resorcinol exposure and adverse effects on health (goiter, mood disorders and
355 tiredness) related to thyroid disruption. Within the regulatory arena, this reversibility can lead to questioning
356 whether such effects are indeed adverse. In the case of resorcinol, the effects reported in human case studies
357 were sufficiently serious and invalidating to be considered as fully adverse, even though they are reversible.
358 In addition, even transient, developmental thyroid disruption can induce long-term changes in neural
359 functions and behavior. Therefore, although thyroid markers can return to normal after exposure in adults,
360 some adverse effects may not be reversible in the context of developmental exposure.

361 Finally, although developmental exposure showed some adverse neural effects on offspring that may be
362 related to thyroid disruption, the data are too scarce to conclude on a link between these two processes. In
363 addition, there are no available epidemiological data in humans that investigate this hypothesis. In-depth
364 neurobehavioral investigations in rodents and a better consideration of other biomarkers of developmental
365 thyroid disruption could help reach an undisputed conclusion.

366 Our analysis showed that, based on human data, resorcinol can fulfill the WHO definition for ED (table2),
367 even in the presence of discrepancies with animal experimental data. It is noteworthy that in the case of
368 resorcinol, animal studies were limited and that more often the experimental procedures were not in nature
369 of bringing clear conclusions on TD effects and possibly related adverse effects in particular on
370 neurodevelopmental toxicity. Nevertheless, they have been used to challenge human data. This also
371 illustrates that the available regulatory toxicological data/guidelines clearly need in depth improvements to
372 accurately assess thyroid disruption. Such limits are a matter of complexity for regulatory toxicological
373 evaluation that can lead to opposite opinions among regulatory bodies, as was the case for resorcinol in the
374 EU: ANSES's proposal to identify resorcinol as a "substance of very high concern" due to its TD properties
375 was not adopted by EU instances two years after its submission (ANSES, 2020; ECHA, 2020). Finally, our
376 analysis raises the questions of how many chemicals for which human data do not exist have been or will be
377 erroneously considered as non TDCs based solely on the perfunctory evaluation of the thyroid function
378 required in the regulatory framework for EDC evaluation.

379

380 Acknowledgments

381 We would like to thank the ANSES Working Group on endocrine disruption, and its scientific coordinators
382 François Pouzaud and Sandrine Charles for their involvement and support. We would like to acknowledge
383 the added value of the members of the ANSES Specialized Expert Committee on REACH and CLP regulations
384 on the issues addressed in this paper.

385
388 Andersen, S.L., 2019. Frequency and outcomes of maternal thyroid function abnormalities in early pregnancy. Scand J Clin Lab Invest 79, 99–107. https://doi.org/10.1080/00365513.2018.1555858

390 ANSES, 2020. Proposal for identification of a substance of very high concern on the basis of the criteria set out in reach article 57. Substance Name: resorcinol. EC Number: 203-585-2. CAS Number: 108-46-3.b.

393 Barton-Maclaren, T.S., Wade, M., Basu, N., Bayen, S., Grundy, J., Marlatt, V., Moore, R., Parent, L., Parrott, J., Grigorova, P., Pinsonnault-Cooper, J., Langlois, V.S., 2022. Innovation in regulatory approaches for endocrine disrupting chemicals: The journey to risk assessment modernization in Canada. Environmental Research 204, 112225. https://doi.org/10.1016/j.envres.2021.112225

397 Bartsch, R., Brinkmann, B., Jahnke, G., Laube, B., Lohmann, R., Michaelsen, S., Neumann, I., Greim, H., 2018. Human relevance of follicular thyroid tumors in rodents caused by non-genotoxic substances. Regul Toxicol Pharmacol 98, 199–208. https://doi.org/10.1016/j.yrtph.2018.07.025

400 Browne, P., Noyes, P.D., Casey, W.M., Dix, D.J., 2017. Application of Adverse Outcome Pathways to U.S. EPA's Endocrine Disruptor Screening Program. Environ Health Perspect 125, 096001. https://doi.org/10.1289/EHP1304

403 Browne, P., Van Der Wal, L., Gourmelon, A., 2020. OECD approaches and considerations for regulatory evaluation of endocrine disruptors. Mol Cell Endocrinol 504, 110675. https://doi.org/10.1016/j.mce.2019.110675

406 Cao, J., Rebuli, M.E., Rogers, J., Todd, K.L., Leyrer, S.M., Ferguson, S.A., Patisaul, H.B., 2013. Prenatal Bisphenol A Exposure Alters Sex-Specific Estrogen Receptor Expression in the Neonatal Rat Hypothalamus and Amygdala. Toxicological Sciences 133, 157–173. https://doi.org/10.1093/toxsci/kft035

410 Caturegli, P., De Remigis, A., Rose, N.R., 2014. Hashimoto thyroiditis: clinical and diagnostic criteria. Autoimmun Rev 13, 391–397. https://doi.org/10.1016/j.autrev.2014.01.007

412 Cheymol, J., Gay, Y., Lavedan, J.P., 1951. [Antithyroid action of diphenols]. C R Seances Soc Biol Fil 145, 999–1001.

414 Cooksey, R.C., Gaitan, E., Lindsay, R.H., Hill, J.B., Kelly, K., 1985. Humic substances, a possible source of environmental goitrogens. Organic Geochemistry 8, 77–80. https://doi.org/10.1016/0146-6380(85)90054-3

417 Crofton, K., Gilbert, M., Paul Friedman, K., Demeneix, B., Marty, M.S., Zoeller, R.T., 2019. Adverse Outcome Pathway on inhibition of Thyroperoxidase and subsequent adverse neurodevelopmental outcomes in mammals, OECD Series on Adverse Outcome Pathways.

420 Docampo-Simón, A., Beltrá-Picó, I., Sánchez-Pujol, M.J., Fuster-Ruiz-de-Apodaca, R., Selva-Otaolaurruchi, J., Betlloch, I., Pascual, J.C., 2022. Topical 15% Resorcinol Is Associated with High Treatment Satisfaction in Patients with Mild to Moderate Hidradenitis Suppurativa. Dermatology 238, 82–85. https://doi.org/10.1159/000515450
424
425

Dong, H., Godlewska, M., Wade, M.G., 2020. A rapid assay of human thyroid peroxidase activity. *Toxicol In Vitro* 62, 104662. https://doi.org/10.1016/j.tiv.2019.104662

426
427

Doniach, I., Logothetopoulos, J., 1953. The goitrogenic action of resorcinol in rats. *Br J Exp Pathol* 34, 146-151.

428

ECHA, 2020. Resorcinol not identified as a substance of very high concern.

429
430
431
432

ECHA/EFSA, E.C.A.F.S.A., Andersson, N., Arena, M., Auteri, D., Barmaz, S., Grignard, E., Kienzler, A., Lepper, P., Lostia, A.M., Munn, S., Parra Morte, J.M., Pellizzato, F., Tarazona, J., Terron, A., Van der Linden, S., 2018. Guidance for the identification of endocrine disruptors in the context of Regulations (EU) No 528/2012 and (EC) No 1107/2009. *EFSA Journal* 8. https://doi.org/10.2903/j.efsa.2018.5311

433
434
435
436
437

Eilstein, J., Grégoire, S., Fabre, A., Arbey, E., Géniès, C., Duplan, H., Rothe, H., Ellison, C., Cubberley, R., Schepky, A., Lange, D., Klaric, M., Hewitt, N.J., Jacques-Jamin, C., 2020. Use of human liver and EpiSkin™ S9 subcellular fractions as a screening assays to compare the in vitro hepatic and dermal metabolism of 47 cosmetics-relevant chemicals. *J Appl Toxicol* 40, 416-433. https://doi.org/10.1002/jat.3914

438
439

Fawcett, D.M., Kirkwood, S., 1953. The mechanism of the antithyroid action of iodide ion and of the aromatic thyroid inhibitors. *J Biol Chem* 204, 787-796.

440
441

Garton, G.A., Williams, R.T., 1949. Studies in detoxication; the fates of quinol and resorcinol in the rabbit in relation to the metabolism of benzene. *Biochem J* 44, 234-238.

442
443
444
445

Géniès, C., Jamin, E.L., Debrauwer, L., Zalko, D., Person, E.N., Eilstein, J., Grégoire, S., Schepky, A., Lange, D., Ellison, C., Roe, A., Salhi, S., Cubberley, R., Hewitt, N.J., Rothe, H., Klaric, M., Duplan, H., Jacques-Jamin, C., 2019. Comparison of the metabolism of 10 chemicals in human and pig skin explants. *J Appl Toxicol* 39, 385-397. https://doi.org/10.1002/jat.3730

446
447
448

Gilbert, M.E., Ramos, R.L., McCloskey, D.P., Goodman, J.H., 2014. Subcortical Band Heterotopia in Rat Offspring Following Maternal Hypothyroxinaemia: Structural and Functional Characteristics. *Journal of Neuroendocrinology* 26, 528-541. https://doi.org/10.1111/jne.12169

449
450
451
452

Hewitt, N.J., Grégoire, S., Cubberley, R., Duplan, H., Eilstein, J., Ellison, C., Lester, C., Fabian, E., Fernandez, J., Géniès, C., Jacques-Jamin, C., Klaric, M., Rothe, H., Sorrell, I., Lange, D., Schepky, A., 2020. Measurement of the penetration of 56 cosmetic relevant chemicals into and through human skin using a standardized protocol. *J Appl Toxicol* 40, 403-415. https://doi.org/10.1002/jat.3913

453
454

Kim, Y.C., Matthews, H.B., 1987. Comparative metabolism and excretion of resorcinol in male and female F344 rats. *Fundam Appl Toxicol* 9, 409-414. https://doi.org/10.1016/0272-0590(87)90023-6

455
456
457

Korevaar, T.I.M., Tiemeier, H., Peeters, R.P., 2018. Clinical associations of maternal thyroid function with foetal brain development: Epidemiological interpretation and overview of available evidence. *Clin Endocrinol* 89, 129-138. https://doi.org/10.1111/cen.13724

458
459
460
461
462
463
464

Kortenkamp, A., Axelstad, M., Baig, A.H., Bergman, Å., Bornehag, C.-G., Cenijn, P., Christiansen, S., Demeneix, B., Derakhshan, A., Fini, J.-B., Frädrich, C., Hamers, T., Hellwig, L., Köhrle, J., Korevaar, T.I.M., Lindberg, J., Martin, O., Meima, M.E., Mergenthaler, P., Nikolov, N., Du Pasquier, D., Peeters, R.P., Platzack, B., Ramhøj, L., Remaud, S., Renko, K., Scholze, M., Stachelscheid, H., Svingen, T., Wagenaars, F., Wedebye, E.B., Zoeller, R.T., 2020. Removing Critical Gaps in Chemical Test Methods by Developing New Assays for the Identification of Thyroid Hormone System-Disrupting Chemicals- The ATHENA Project. *Int J Mol Sci* 21, E3123. https://doi.org/10.3390/ijms21093123
465 La Merrill, M.A., Vandenberg, L.N., Smith, M.T., Goodson, W., Browne, P., Patisaul, H.B., Guyton, K.Z.,
466 Kortenkamp, A., Cogliano, V.J., Woodruff, T.J., Rieswijk, L., Sone, H., Korach, K.S., Gore, A.C., Zeise, L.,
467 Zoeller, R.T., 2020. Consensus on the key characteristics of endocrine-disrupting chemicals as a basis
468 for hazard identification. Nat Rev Endocrinol 16, 45–57. https://doi.org/10.1038/s41574-019-0273-8

469 Lindsay, R.H., Hill, J.B., Gaitan, E., Cooksey, R.C., Jolley, R.L., 1992. Antithyroid effects of coal-derived
470 pollutants. J Toxicol Environ Health 37, 467–481. https://doi.org/10.1080/15287399209531686

471 Lynch, B.S., Delzell, E.S., Bechtel, D.H., 2002. Toxicology Review and Risk Assessment of Resorcinol:
472 Thyroid Effects. Regulatory Toxicology and Pharmacology 36, 198–210.
473 https://doi.org/10.1006/rtph.2002.1585

474 McDonnell-Dowling, K., Kleefeld, S., Kelly, J.P., 2017. Consequences of Oral Gavage during Gestation
475 and Lactation on Rat Dams and the Neurodevelopment and Behavior of Their Offspring. J Am Assoc
476 Lab Anim Sci 56, 79–83.

477 Narumi, S., Fox, L.A., Fukudome, K., Sakaguchi, Z., Sugisawa, C., Abe, K., Kameyama, K., Hasegawa, T.,
478 2017. Mild thyroid peroxidase deficiency caused by TPO mutations with residual activity: Correlation
479 between clinical phenotypes and enzymatic activity. Endocr J 64, 1087–1097.
480 https://doi.org/10.1507/endocrj.EJ17-0194

481 Noyes, P.D., Friedman, K.P., Browne, P., Haselman, J.T., Gilbert, M.E., Hornung, M.W., Barone, S.,
482 Crofton, K.M., Laws, S.C., Stoker, T.E., Simmons, S.O., Tietge, J.E., Degitz, S.J., 2019. Evaluating
483 Chemicals for Thyroid Disruption: Opportunities and Challenges with in Vitro Testing and Adverse
484 Outcome Pathway Approaches. Environ Health Perspect 127, 95001.
485 https://doi.org/10.1289/EHP5297

486 Organization for Economic Co-operation and development, 2018. User's handbook supplement to
487 the Guidance Document for developing and assessing Adverse Outcome Pathways. OECD Series on
488 Adverse Outcome Pathways.

489 Paul, C., Rhind, S.M., Kyle, C.E., Scott, H., McKinnell, C., Sharpe, R.M., 2005. Cellular and hormonal
disruption of fetal testis development in sheep reared on pasture treated with sewage sludge.
490 Environ. Health Perspect. 113, 1580–1587. https://doi.org/10.1289/ehp.8028

492 Paul Friedman, K., Watt, E.D., Hornung, M.W., Hedge, J.M., Judson, R.S., Crofton, K.M., Houck, K.A.,
493 Simmons, S.O., 2016. Tiered High-Throughput Screening Approach to Identify Thyroperoxidase
494 Inhibitors Within the ToxCast Phase I and II Chemical Libraries. Toxicol Sci 151, 160–180.
495 https://doi.org/10.1093/toxsci/kfw034

496 Paul, K.B., Hedge, J.M., Rotroff, D.M., Hornung, M.W., Crofton, K.M., Simmons, S.O., 2014.
497 Development of a thyroperoxidase inhibition assay for high-throughput screening. Chem Res Toxicol
498 27, 387–399. https://doi.org/10.1021/tx400310w

499 Ramhøj, L., Hass, U., Gilbert, M.E., Wood, C., Svingen, T., Usai, D., Vinggaard, A.M., Mandrup, K.,
500 Axelstad, M., 2020. Evaluating thyroid hormone disruption: investigations of long-term
501 neurodevelopmental effects in rats after perinatal exposure to perfluorohexane sulfonate (PFHxS).
502 Sci Rep 10, 2672. https://doi.org/10.1038/s41598-020-59354-z

503 Roberts, M.S., Anderson, R.A., Swarbrick, J., 1977. Permeability of human epidermis to phenolic
504 compounds. J Pharm Pharmacol 29, 677–683. https://doi.org/10.1111/j.2042-7158.1977.tb11434.x
505

506

507

Rolaki, A., Pistollato, F., Munn, S., Bal-Price, A., 2019. Adverse Outcome Pathway on inhibition of Na+/I- symporter (NIS) leads to learning and memory impairment, in: OECD Series on Adverse Outcome Pathways, OECD Series on Adverse Outcome Pathways. p. 212.

508

509

Rosenfeld, C.S., 2017. Sex-dependent differences in voluntary physical activity. J Neurosci Res 95, 279-290. https://doi.org/10.1002/jnr.23896

510

511

Samuel, K.C., 1955. Experimental production of goiter in rats: with resorcinol and its derivatives. Lab Invest 4, 90-105.

512

513

Scimonelli, T., Marucco, M., Celis, M.E., 1999. Age-related changes in grooming behavior and motor activity in female rats. Physiol Behav 66, 481-484. https://doi.org/10.1016/s0031-9384(98)00314-x

514

515

516

517

Segal, D., Makris, S.L., Kraft, A.D., Bale, A.S., Fox, J., Gilbert, M., Bergfelt, D.R., Raffaele, K.C., Blain, R.B., Fedak, K.M., Selgrade, M.K., Crofton, K.M., 2015. Evaluation of the ToxRTool's ability to rate the reliability of toxicological data for human health hazard assessments. Regul Toxicol Pharmacol 72, 94-101. https://doi.org/10.1016/j.yrtph.2015.03.005

518

519

Unpublished Study report, 2003. A drinking water dose range finding reproductive toxicity study of resorcinol in rats.

520

521

522

Welsch, F., Nemec, M.D., Lawrence, W.B., 2008. Two-generation reproductive toxicity study of resorcinol administered via drinking water to Crl:CD(SD) Rats. Int J Toxicol 27, 43-57. https://doi.org/10.1080/10915810701876679

523

WHO, W.H.O., 2006. Concise International Chemical Assessment Document 71. RESORCINOL.

524

525

WHO, (World Health Organization), 2002. Global assessment of the state of the science of endocrine disruptors. International Programme on Chemical Safety.

526

527

528

Yeung, D., Kantor, S., Nacht, S., Gans, E.H., 1983. Percutaneous absorption, blood levels, and urinary excretion of resorcinol applied topically in humans. Int J Dermatol 22, 321-324. https://doi.org/10.1111/j.1365-4362.1983.tb02149.x

529

530

531

Zoeller, R.T., Rovet, J., 2004. Timing of thyroid hormone action in the developing brain: clinical observations and experimental findings. J Neuroendocrinol 16, 809-818. https://doi.org/10.1111/j.1365-2826.2004.01243.x

532

533
534

535

536

537

<table><caption>**Table 1.** Summary of the regulatory tests designed by the Organization for Economic Co-operation and development (OECD) and the American Environmental Protection Agency (US EPA) that include endpoints responsive to and/or diagnostic of thyroid disruption.</caption><thead><tr><th>Test</th><th>OECD level GL reference</th><th>US EPA Tier GL reference</th><th>Last updated (OECD TG/OPPTS)</th><th>Endpoints for thyroid-related activity (from OECD TG 150)*</th></tr></thead><tbody><tr><td>Hershberger Bioassay in Rats</td><td>Level 3 TG 441</td><td>Tier 1 OPPTS 890.1400</td><td>September 2009 / October 2009</td><td>Optional:<ul><li>Possible liver weight increase (in combination with other thyroid-related endpoints)</li><li>Reduction in serum T4 and T3 (anti-thyroid)</li></ul></td></tr><tr><td>Amphibian Metamorphosis Assay</td><td>Level 3 TG 231</td><td>Tier 1 OPPTS 890.1100</td><td>Septembre 2009 / October 2009</td><td><ul><li>Developmental stage</li><li>Hind limb length</li><li>Snout-vent length</li><li>Thyroid gland histopathology</li><li>Time to metamorphosis</li></ul></td></tr><tr><td>XETA: Xenopus Embryonic Thyroid Signalling Assay</td><td>Level 4 TG 248</td><td>-</td><td>June 2019</td><td><ul><li>Increase or decrease in GFP, depending on precise mode of thyroid activity</li></ul></td></tr><tr><td>Repeated Dose 28-Day Oral Toxicity Study in Rodents</td><td>Level 4 TG 407</td><td>- OPPTS 870.3050</td><td>October 2008 / July 2000</td><td><ul><li>Possible liver weight increase (in combination with other thyroid-related endpoints)</li><li>Histopathologic changes in thyroid (follicular cell height increase and colloid area decrease)</li><li>Other endpoints: serum T3 and T4 decreased, TSH increased; increased thyroid weight (anti-thyroid)</li></ul></td></tr><tr><td>Repeated Dose 90-Day Oral Toxicity Study</td><td>Level 4 TG 408</td><td>- OPPTS 870.3100</td><td>June 2018 / August 1998</td><td><ul><li>Possible liver weight increase (in combination with other thyroid-related endpoints)</li><li>Serum T4, T3 decreased, TSH increased</li><li>Histopathologic changes in thyroid gland</li><li>Changes to HDL/LDL ratio (in combination with other thyroid-related endpoints)</li></ul></td></tr><tr><td>Combined Chronic Toxicity/Carcinogenicity Studies</td><td>Level 4 TG 451-3</td><td>- OPPTS 870.4300</td><td>September 2018 / August 1998</td><td><ul><li>Increased thyroid weight.</li><li>Possible liver weight increase (in combination with other thyroid related endpoints)</li><li>Histopathologic changes in thyroid gland</li></ul></td></tr><tr><td>Combined 28-Day Reproductive Screening Tests</td><td>Level 4 TG 421 TG 422</td><td>- OPPTS 870.3550 OPPTS 870.3550</td><td>July 2016 / July 2020 March 1996 / July 2020</td><td><ul><li>Increased thyroid weight.</li><li>Histopathologic changes in thyroid gland</li><li>Serum T4, decreased, TSH increased</li></ul></td></tr><tr><td>Prenatal Developmental Toxicity Study</td><td>Level 4 TG 414</td><td>- OPPTS 870.3700</td><td>June 2018 / August 1998</td><td><ul><li>Increased thyroid weight</li><li>Histopathologic changes in thyroid gland</li><li>Serum T4, decreased, TSH increased in dams</li></ul></td></tr><tr><td>Developmental Neurotoxicity Study</td><td>Level 4 TG 426</td><td>- OPPTS 870.6300</td><td>October 2007 / August 1998</td><td>Nil referenced as specific to thyroid in TG 150</td></tr><tr><td>Subchronic Dermal Toxicity: 90-Day Study</td><td>Level 4 TG 411</td><td>- OPPTS 870.3250</td><td>May 1981 / August 1998</td><td><ul><li>Histopathologic changes in thyroid gland</li></ul></td></tr><tr><td>28-Day (Subacute) Inhalation Toxicity Study</td><td>Level 4 TG 412</td><td>-</td><td>June 2018</td><td><ul><li>Increased thyroid weight</li><li>Histopathologic changes in thyroid gland</li></ul></td></tr><tr><td>Subchronic Inhalation Toxicity: 90-Day Study</td><td>Level 4 TG 413</td><td>- OPPTS 870.3465</td><td>June 2018 / August 1998</td><td><ul><li>Increased thyroid weight</li><li>Histopathologic changes in thyroid gland</li></ul></td></tr><tr><td>Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents</td><td>Level 4 TG 409</td><td>- OPPTS 870.3150</td><td>September 1998 / August 1998</td><td><ul><li>Increased thyroid weight</li><li>Histopathologic changes in thyroid gland</li></ul></td></tr><tr><td>Pubertal Development and Thyroid Function Assay in Peripubertal Male Rats</td><td>Level 4</td><td>Tier 1 OPPTS 890.1500</td><td>October 2009</td><td><ul><li>Increased thyroid weight</li><li>Possible liver weight increase (in combination with other thyroid-related endpoints)</li><li>Histopathologic changes in thyroid (follicular cell height increase and colloid area decrease)</li><li>Serum T4 decreased, TSH increased</li></ul></td></tr><tr><td>Pubertal Development and Thyroid Function Assay in Peripubertal female Rats</td><td>Level 4</td><td>Tier 1 OPPTS 890.1450</td><td>October 2009</td><td><ul><li>Increased thyroid weight</li><li>Possible liver weight increase (in combination with other thyroid-related endpoints)</li><li>Histopathologic changes in thyroid (follicular cell height increase and colloid area decrease)</li><li>Serum T4 decreased, TSH increased</li></ul></td></tr><tr><td>Larval Amphibian Growth and Development Assay</td><td>Level 4 TG 241</td><td>Tier 2 OPPTS 890.2300</td><td>July 2015 / July 2015</td><td><ul><li>Thyroid histopathology</li><li>Time to metamorphosis</li></ul></td></tr><tr><td>Two-Generation Reproduction Toxicity Study</td><td>Level 5 TG 416</td><td>- OPPTS 870.3800</td><td>January 2001 / August 1998</td><td><ul><li>Increased thyroid weight</li><li>Possible liver weight increase (in combination with other thyroid-related endpoints)</li><li>Histopathologic changes in thyroid (follicular cell height increase and colloid area decrease)</li></ul></td></tr><tr><td>Extended One-Generation Reproductive Toxicity Study</td><td>Level 5 TG 443</td><td>-</td><td>June 2018</td><td><ul><li>Increased thyroid weight</li><li>Possible liver weight increase (in combination with other thyroid-related endpoints)</li><li>Histopathologic changes in thyroid</li><li>Serum T4, decreased, TSH increased</li></ul></td></tr><tr><td>ATGT: Avian Two-Generation Toxicity Test in the Japanese Quail</td><td>Level 5</td><td>Tier 2 OCSPP 890.2100</td><td>July 2015</td><td><ul><li>T3, T4</li></ul></td></tr></tbody></table>

5

539

540

541

542
<caption>Table2 : summary of the main lines of evidence and biological plausibility for resorcinol to fullfill the criteria of the definition of an endocrine disruptor as recommended by JRC</caption>

Details of the reference articles are given in the supplementary material

<table><thead><tr><th>Adverse effects</th><th>Plausible ED MoA/ key events:</th><th>Human relevance</th></tr></thead><tbody><tr><td><strong>Goitre and severe hypothyroidism in humans</strong><br>Bull & Fraser 1950; Hobson 1951; Berthezene 1973; Katin 1977</td><td><strong>Molecular initiating event= Inhibition of TPO</strong><br>(key enzyme of TH synthesis)<br>Taurog 1970; Lindsay 1992; Paul 2014; Jomaa 2015; Divi & Doerge 1994; Paul Friedman 2016.</td><td><strong>Effects and MoA identified in humans</strong><br><em>in vitro</em> data showing TPO inhibition in human cell line</td></tr><tr><td><strong>Decreased T4 in humans</strong><br>Berthezene 1973; Katin 1977</td><td><strong>Decreased iodine organification in vitro</strong><br>Fawcett & Kirkwood 1953; Lindsay 1992; Coval & Taurog 1970.</td><td rowspan="3"><strong>ECHA/EFSA (2018)</strong> : humans and rodents considered equally sensitive to thyroid-disruption</td></tr><tr><td><strong>Decreased T4 in rodents with exposure with diet or drinking water.</strong><br>Cooksey 1985; Main two-generation study (Unpub. 2005a); Berthezene 1979</td><td><strong>Decreased iodine organification in vivo</strong><br>Cooksey 1985; Berthezene 1979</td></tr><tr><td><strong>Histological thyroid alterations and/or increased thyroid weight in experimental animals</strong><br>Doniach 1953; Seffner 1995; Main two-generation study (Unpub. 2005a); Cheymol 1951; Samuel 1955; Cooksey 1985; Berthezene 1979.</td><td><strong>Biological plausibility of a causal link</strong> highly supported by current knowledge on neurodevelompental effects (AOP 42)</td></tr><tr><td><strong>Histological thyroid alterations and/or increased thyroid weight in human</strong><br>Bull & Fraser 1950</td><td></td><td></td></tr><tr><td><strong>Neural effects</strong><br>Indications of motor dysfunctions (low level of evidence): Increased landing footsplay in 90-day study (Unpub. 2004) and increased motor activity in the preliminary two-generation study (Unpub. 2003).</td><td></td><td></td></tr></tbody></table>

543

544

545

546

547

548
<figure><img src="image_1.png" /></figure>