# Melamine regulatory assessment for endocrine disruption

Isabelle Charron, Brigitte Le Magueresse-Battistoni, René Habert, Marie-Chantal Canivenc-Lavier, Sakina Mhaouty-Kodja, Cécile Michel-Caillet

► To cite this version:

Isabelle Charron, Brigitte Le Magueresse-Battistoni, René Habert, Marie-Chantal Canivenc-Lavier, Sakina Mhaouty-Kodja, et al. Melamine regulatory assessment for endocrine disruption. Environment International, 2024, 194, pp.109188. 10.1016/j.envint.2024.109188. anses-04830539

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Distributed under a Creative Commons Attribution 4.0 International License
Review article

Melamine regulatory assessment for endocrine disruption

Isabelle Charron<sup>a, *, ID</sup>, Brigitte Le Magueresse-Battistoni<sup>b</sup>, René Habert<sup>c</sup>, Marie-Chantal Canivenc-Lavier<sup>d</sup>, Sakina Mhaouty-Kodja<sup>e</sup>, Cécile Michel-Caillet<sup>a</sup>

<figure><img src="image_2.png" /></figure>

ARTICLE INFO

Keywords:

Melamine (CAS No 108-78-1)

Endocrine disruption

Risk assessment

Reproductive toxicity

Human health

A B S T R A C T

Melamine has several domestic and industrial uses as a flame retardant or in the manufacture of melami-
ne-formaldehyde resins. Based on available scientific literature data, the French Agency for Food, Environmental
and Occupational Health & Safety (ANSES) included this substance in the list of “chemicals that may present
endocrine disruptor (ED) properties”, and the substance was prioritized to assess whether it should be classified
as an ED in European Union (EU) regulations for hazard identification. This review reports the assessment of
melamine based on relevant studies from the registration dossier under REACH, and peer-reviewed literature.
Among the various adverse effects, reproductive, neurodevelopmental, and thyroid effects were analyzed in
particular, because they could be the consequence of an endocrine disruption. The different modes of action
(endocrine or non-endocrine) potentially leading to these effects were scrutinized to understand whether the
WHO definition for ED and the criteria for hazard identification were met. It was concluded that the reproductive
effect on spermatogenesis was not a consequence of endocrine activity. A biologically plausible link between this
effect and endocrine activity was not established, and other modes of action (oxidative stress or altered energy
metabolism) could be involved. Similarly, thyroid and neurodevelopmental effects appeared at higher doses than
those leading to renal toxicity. Our assessment confirms that melamine is a reprotoxic substance but does not
support ED classification. This assessment illustrates the scientific and regulatory challenges in differentiating
specific endocrine disruption from an indirect endocrine effect resulting from non-ED mediated systemic toxicity.

1. Introduction

Melamine (CAS no. 108-78-1; EC no. 203-615-4) is used as a flame retardant in polyurethane foam-containing products such as mattresses or furniture. It is also used in the manufacture of melamine-formaldehyde resins for application in laminate flooring and panels, plastic kitchenware, paints, and coatings. Melamine is reportedly used in agriculture as a fertilizer and is a metabolite of the pesticide cyromazine (EFSA, 2007; Health Canada 2020, WHO, 2009). Standards for melamine in food and feed have been developed internationally to ensure the safety of consumers and to differentiate between the presence of background levels of melamine in food and intentional adulteration (EFSA 2010, Codex Alimentarius 2019). These measures were adopted following the incidents associated with melamine-adulterated pet food in the United States in 2007 and, above all, melamine-adulterated milk,

infant formula, and other milk-derived products in China in 2008 (EFSA 2010). The main consequences were urinary stone formation in children with associated renal damage, and child mortality in the worst cases (EFSA 2010).

Toxicokinetic studies carried out in rats exposed to melamine showed that oral absorption is very high (73 to 98 %), and distribution to the tissues is rapid (distribution/absorption half-time of 15 min.) (Mast et al., 1983). The highest concentrations of melamine were detected in the kidney and urinary bladder, although melamine was also distributed to other tissues (including the liver, stomach, spleen, heart, uterus, ovaries, testes, brain, and lungs). The authors reported a high clearance rate from plasma, and urinary excretion was found to be the sole route of elimination (elimination half-life of 3 h). No metabolism of melamine was reported, and the substance was therefore considered to be excreted in the native form. Based on the literature, humans display a

<footnote><sup>a</sup> ANSES, Risk Assessment Department, 14 Rue Pierre et Marie Curie, Maisons-Alfort 94701, France</footnote>

<footnote><sup>b</sup> Univ-Lyon, CarMeN Laboratory, INSERM U1060, INRAE U1397, Université Claude Bernard Lyon 1, Pierre-Bénite 69310, France</footnote>

<footnote><sup>c</sup> Laboratory of Development of the Gonads, UMR-008 Genetic Stability Stem Cells and Radiations, Université Paris-Cité, Fontenay-aux-Roses 92265, France</footnote>

<footnote><sup>d</sup> Centre des Sciences du Goût et de l'Alimentation, CNRS, INRAE, Institut Agro, Université de Bourgogne, Dijon 21000, France</footnote>

<footnote><sup>e</sup> Sorbonne Université, CNRS UMR 8246, INSERM U1130, Neuroscience Paris Seine-Institut de Biologie Paris Seine, Paris 75005, France</footnote>

<footnote>* Corresponding author at: ANSES, Risk Assessment Department, 14 Rue Pierre et Marie Curie, Maisons-Alfort 94701, France.
E-mail address: isabelle.charron@anses.fr (I. Charron).</footnote>
similar toxicokinetic profile. Urinary melamine excretion after low-dose exposure (migration from melamine resin plastic bowls) was investigated in a randomized crossover human study (Wu et al. 2013). The half-life of urinary melamine elimination was approximately 6 h. Melamine was detected in the native form in the urine of pediatric patients exposed to melamine-tainted milk products (Cheng et al. 2009, Kong et al. 2011, Lam et al. 2009, Zhang et al. 2010). Importantly, melamine can cross the blood-brain barrier (Wu et al. 2009) and the placental barrier in rats and humans (Chu et al. 2010, Partanen et al. 2012), implying that melamine exposure can occur during the critical early periods of development in both animals and humans.

Based on concerns related to its toxicological profile, melamine was included by the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) in the list of “chemicals that may present endocrine disruptor (ED) properties” (ANSES 2021a). Its inclusion in this list originated from the work performed by DEDUCT v.1.0 (Database of Endocrine Disrupting Chemicals and their Toxicity Profiles). DEDUCT is based on an analysis of existing scientific literature containing experimental evidence for endocrine-specific disruption in humans and rodents, and the methodology is described in Karthikeyan et al. (2019). The DEDUCT database mentioned 4 articles related to the reproductive effects of melamine involving endocrine-mediated perturbations (DEDUCT 2024). Three of them described adverse effects in the reproductive system of male mice (Chang et al. 2014, Sun et al. 2016a, and Yin et al. 2013), while one study showed adverse effects related to toxicity in the reproductive system of female rats (Sun et al. 2016b). In addition to the toxicological concerns, more than 100,000 tons of melamine are produced in or imported to Europe each year, excluding the volume directly exported and intermediate registrations (ECHA 2024a). Therefore, considering the hazard concern for ED properties and widespread exposure of the population, melamine was prioritized for formal assessment.

The ANSES group of experts on endocrine disruptors was mandated to assess the ED properties of melamine based on the ED definition of the WHO (2002), and the recent amendment to the Classification, Labelling and Packaging (CLP) Regulation (EC) No 1272/2008 introducing new hazard classes and criteria, among which classification for endocrine disruption for human health (EC 2022). This classification is based on evidence from human, animal, and/or predictive data that the substance meets the criteria for endocrine activity, adverse effects in an intact organism or its offspring or future generations, and a biologically plausible link between the endocrine activity and the adverse effect.

Under the CLP Regulation, assessment of the scientific evidence should consider all relevant scientific data in order to establish whether the criteria for ED properties of the substance are fulfilled (EC 2022). As such, the assessment of melamine's potential ED properties on human health was performed following the weight-of-evidence analysis methodology previously reported by ANSES (2017, 2021b) and according to the guidance for the identification of EDs in the context of EU regulations (ECHA and EFSA 2018). The conclusion of this assessment and the need for follow-up regulatory action at the EU level were reported on the European Chemicals Agency (ECHA) website (ECHA 2024b).

This paper aims to report results of the literature review and analysis performed by the ANSES group of experts for the assessment of melamine ED properties. It illustrates the assessment strategy to perform hazard identification for ED properties in the context of European regulations, namely REACH (EC no 1907/2006) and CLP (EC no 1272/2008). Finally, this article also reports the scientific challenges encountered by regulators performing the ED assessment, especially in establishing the biological plausibility of an ED mode of action based on the WHO definition.

## 2. Methods

Evidence to assess the potential ED properties of melamine included the relevant studies available from the registration dossier for melamine

under the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) Regulation, as well as a literature review to identify relevant *in vitro*, *in vivo*, and human studies. Reports by national agencies and cross-references of peer-reviewed research articles were also included in the literature search and identification of studies. In addition, the ToxCast and Endocrine Disruptor Screening Program (EDSP) databases were queried for melamine bioactivity results using the Chemical Abstracts Service (CAS) registry number to identify high-throughput *in vitro* screening assays that measured endocrine activity (US EPA 2024).

A targeted search strategy was applied in the PubMed and Scopus databases to retrieve documents published until December 2023 by using terms related to the substance melamine (CAS number, IUPAC name, or chemical name synonyms) and by adding specific terms related to the relevant endpoint for ED assessment. Given the previous findings from DEDUCT, the investigation of adverse effects following melamine exposure focused primarily on male reproductive function, with supporting evidence on female reproductive function. Additionally, due to the concerns reported above regarding infant exposure to melamine, neurodevelopment was assessed together with the thyroid hormone system, a key regulator of cellular processes in the developing brain (Bernal 2005). Effects on renal function (identified at the EU level and included in the Harmonised Classification and Labelling report on melamine) were also considered in the scope of the analysis, to compare adverse effect doses and to assess whether the mechanism leading to renal toxicity could explain spermatotoxicity (ECHA 2024c).

Keywords and equations used for each endpoint are detailed in Supplementary Materials S1 and S2. The studies retrieved from both databases were imported into the web tool Rayyan to create, share, and organize the review (Ouzzani et al. 2016). Reference duplicates were removed to obtain the preliminary bibliography. Inclusion and exclusion criteria were determined and used to select the relevant publications (Supplementary Material S3). A Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) flow chart diagram summarizes the methodology used to identify the studies targeting the male reproduction endpoint (Fig. 1). Pilot testing of the searching, screening, and extraction stages was not conducted. Five people were involved in article screening. For each of the endpoints, the first screening was performed by one screener on title and abstract. The second screening of publications related to reproduction was performed by three screeners on the full text, while the second screening of publications related to neurodevelopmental and thyroid effects was performed by two screeners on the full text. For reproductive and thyroid effects, the search terms did not target a specific period of exposure. However, analysis of the neural effects of melamine focused on critical development periods, including the prenatal and postnatal periods, known to be important in the organization of the nervous system and highly sensitive to thyroid disruption.

The available data on the different endocrine activities were reviewed to identify if one of the adverse effects could be a potential consequence of the ED mode of action (MoA). Data relating to non-ED MoA, potentially triggering these adverse effects, were also identified and described. The strength of evidence of the different MoAs was then weighted and discussed to see which MoA is more likely to lead to the adverse effects observed.

## 3. Results

The following sections describe the experimental data selected based on the abovementioned criteria used to scope the problem formulation: is melamine an ED? A summary of the studies on the adverse effects of melamine is presented in Table S4.
<figure><img src="image_3.png" /><figcaption>**Fig. 1.** PRISMA flow chart diagram for studies on melamine targeting the male reproduction endpoint. * See Table S3 for literature search inclusion and exclusion criteria. Adapted from Page et al. (2021).</figcaption></figure>

## 3.1. Evidence of adverse effects related to repeated exposure to melamine

### 3.1.1. Nephrotoxicity induced by repeated exposure to melamine

Repeated melamine exposure led to adverse effects on the urinary tract system, comprising urolithiasis and signs of nephrotoxicity such as chronic inflammation and renal injuries (WHO 2009, EFSA 2010, Health Canada 2020). These adverse effects were reproducibly observed in repeated toxicity studies, including in a 13-week study showing melamine-induced urolithiasis in male rats at 72 mg/kg body weight (bw)/day (d) and calcareous renal deposits in female rats at 84 mg/kg bw/d (National Toxicology Program, 1983). Similar effects were also reported in the Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG) 443: Extended One-Generation Reproductive Toxicity (EOGRT) Study from the registration dossier used to evaluate the fertility and developmental effects of melamine (Study Report, 2020). The kidney was identified as a target organ for melamine in parent animals and their progeny. Dietary exposure to melamine showed nephrotoxic effects in both males and females rats, at mid-dose (4,000 ppm eq. to 268/355 mg/kg bw/d in males/females) and high-dose (12,500 ppm eq. to 833/1,124 mg/kg bw/d in males/females). Reduced white blood cell counts were found at the high dose in

parent females, which could be a consequence of renal impairment, as described in Heinzelmann et al. (1999).

The proposed mechanism of melamine-induced nephrotoxicity involves an induction of oxidative stress and an inflammatory response triggered by renal crystals (EFSA 2010, Chu and Wang 2013, and Rai et al. 2014 in Health Canada 2020). Based on the nephrotoxicity observed in the National Toxicology Program (NTP) study (1983), a tolerable daily intake (TDI) dose of 0.2 mg/kg bw was established for melamine, relying on a 10 % increase in the incidence of urinary bladder crystals occurring at the benchmark dose of 41 mg/kg bw/d (BMD₁₀) and its lower confidence limit (BMDL₁₀) of 19 mg/kg bw/d (EFSA 2010).

### 3.1.2. Melamine-induced effects on male reproductive function

In the EOGRT Study from the registration dossier, adverse effects on male reproductive function were described as follows (Study Report, 2020): effects on sperm quality were observed at high-dose (12,500 ppm) in both parent (eq. to 833 mg/kg bw/d) and offspring (F1) generations (eq. to 1,200 mg/kg bw/d) and concerned a significant reduction in motile sperm (−17 % in parent and −21 % in F1), and an alteration of morphology. Testicular histology revealed degeneration/atrophy of germ cells in the testes and cell debris in the lumen of the
epididymis at mid- and high-doses in the parent generation and at high-dose in F1 animals. Except for these effects, epididymal sperm count and spermatogenic profiling were not modified. These effects of tubular degeneration/atrophy in the testis and sperm (detached heads) appeared at doses also inducing nephrotoxicity. In this study, male reproductive function of parent and F1 generations was not affected at the lowest dose (1,000 ppm eq. to 65 mg/kg bw/d), for which no nephrotoxicity was observed.

The other test guideline studies available in the registration dossier did not provide further data on male reproductive function and fertility. Five reliable, good quality studies from the literature review provided additional information on male reproductive function, as presented below.

Testicular acute toxicity of melamine was investigated by Chang et al. (2014), where male mice (n = 12/group) received melamine once a day by oral gavage for three consecutive days at doses of 30, 140, and 700 mg/kg bw/d. No general toxicity (normal general state, body weight, and behavior) was detected, but white precipitates were observed in the bladders of a few animals exposed to low- and mid-doses and in half of the mice 1 day after exposure to the high dose. It should be mentioned that "hard crystalline solids" in the bladder were already observed in 80% to 100% of male rats at doses of 790 mg/kg bw/d and above in a sub-acute toxicity study in which animals received melamine orally for 14 days (NTP, 1983). Together with effects on the bladder, the percentage of abnormal spermatozoa in the epididymis was elevated (between + 10 and + 35 %) at all doses, 1 and 5 days after treatment. Destruction of the blood-testis barrier was evidenced by electronic microscopy and by immunostaining of vimentin filaments, an important component of the cytoplasmic skeleton, in Sertoli cells. Since Sertoli cells are the supporting and nurturing cells for germ cells, injury to vimentin filaments could severely reduce the supportive capacity of Sertoli cells, and could potentially affect spermatogenesis.

Chen et al. (2021) studied male reproductive dysfunction in mice. A total of 32 male Kunming mice (8/group) were administered 0, 12.5, 25, and 50 mg/L melamine in drinking water (equivalent to 0, 2, 4, and 8 mg/kg bw/d) for 13 weeks. The age of the mice at the beginning of the experiment was not given by the authors, but based on the reported body weight (18 g), they were in the prepubertal period. No effect on body weight was observed at 2 and 4 mg/kg bw/d, but body weight was significantly lower compared to controls in the 8 mg/kg bw/d melamine-treated group. Strong adverse effects were observed on sperm motility (−10 to −25% in a non-dose dependent manner) and on sperm count (reduced by half at the highest doses) starting at 2 and 4 mg/kg bw/d, respectively compared to controls. Changes in testicular histology (number of seminiferous epithelium cell layers, thickness of seminiferous tubules, and diameter of seminiferous tubules) were also observed, starting at the lowest dose of 2 mg/kg bw/d.

In Khalil et al. (2017), the authors demonstrated that oral exposure to 50 mg/kg bw/d of melamine (the single dose tested) for 65 consecutive days induced reproductive toxic effects in adult mice, together with general toxicity manifesting as lethargy, rough hair coat, and anorexia, as well as a trend for decreased body weight. The treated group presented marked vacuolation of Leydig cells (the cells that synthesize and secrete testosterone), cystic dilatation, and histological lesions of the seminiferous tubules, with a marked reduction in the percentage of spermatocytes in most seminiferous tubules. The sperm analysis showed large reductions in sperm number and motility, and an increase in sperm abnormalities.

In Sun et al. (2016a), no general toxicity related to body weight or food intake was reported at the tested doses (2, 10, and 50 mg/kg bw/d) given orally for 28 days to 5-week-old male mice. However, although testicular weight did not change, the sperm count was dose-dependently reduced in all the treated groups (from −20% at 2 mg/kg bw/d to −50% at the highest dose), and the rate of abnormal sperm was dose-dependently increased in all the treated groups (from ~ 4-fold at 2 mg/kg bw/d to more than 5-fold at the highest dose), compared to the

control group. Looseness of the seminiferous tubule structure was observed at 10 mg/kg bw/d. Disruption of the seminiferous tubule structure, reduced spermatogenic cell series, nuclei pyknosis, and a low sperm number were found at 50 mg/kg bw/d. Quantitative analysis showed that the number of Leydig cells was significantly lower in animals exposed to 10 and 50 mg/kg bw/d of melamine compared to controls, but no significant difference was observed at 2 mg/kg bw/d.

Finally, Yin et al. (2013) exposed mice to 2, 10 and 50 mg/kg bw/d of melamine for 14 days. Testicular histopathology examination showed irregular nucleus shape in some spermatogenic cells at 2 mg/kg bw/d of melamine. At the exposure dose of 10 mg/kg bw/d, seminiferous tubules had indistinct basement membranes, and spermatogenic cells at all stages of the seminiferous epithelium exhibited a slightly loosened organization with decreased cell layers. At 50 mg/kg bw/d of melamine, damage to the basement membrane of seminiferous tubules was more severe compared to the effects observed at lower doses, with highly reduced layers of epithelial cells and structural damage found in many spermatogenic cells at all stages (from spermatogonia to spermatids towards the lumen of the tubules). In addition, swelling and lysis of the nucleus occurred in primary spermatocytes, secondary spermatocytes, and sperm cells with uneven size and staining of varying intensity. Also, a reduction in the number of sperm, even with no mature sperm, was observed in some seminiferous tubules. These changes in the testes were observed in a dose-dependent manner with no apparent general toxicity. In a second experiment, high doses of melamine (412, 824, and 1,648 mg/kg bw/d) were administered for 5 days. No mortality was recorded, but a trend for body weight decrease was observed at the highest dose. The percentage of spermatozoa with morphological alteration increased dose-dependently from ~ 2% in controls to ~ 6% at the highest melamine dose.

An additional study by Huang et al. (2018) described toxic effects of melamine on normal formation and maturation of mouse sperm, and damaged testicular structures after 35 days of high-dose exposure (400, 800 or 1,600 mg/kg bw/d). This study is considered to be of limited quality because of shortcomings in the methodology description.

In conclusion, three studies showed that administration of a low dose of melamine (2 mg/kg bw/d) altered sperm and/or spermatogenesis in mice (Chen et al. 2021, Sun et al. 2016a, Yin et al. 2013). Additional studies demonstrated effects at higher doses (Chang et al. 2014, Khalil et al. 2017). The only study performed in rats is a guideline study, and effects on sperm or spermatogenesis appeared at melamine doses two orders of magnitude higher, and in the presence of nephrotoxicity (Study Report, 2020). Potential factors that could explain the discrepancy in the doses that led to adverse effects in regulatory vs academic studies have not been analyzed. No epidemiologic data were available regarding reproductive function.

### 3.1.3. Neurotoxicity induced by exposure to melamine

Analysis of the cohort for developmental neurotoxicity in the OECD TG 443 Study did not show a significant effect on brain weight in rats from the F1 generation exposed to 1,000, 4,000, or 12,500 ppm of melamine (Study Report, 2020). There were no significant changes in brain dimensions and no concordance between cohorts or sexes. The thickness of the corpus callosum was reduced by less than 10% in male rats from the F1 generation at both postnatal day (PND) 21-22 and PND 76-90 and was increased by less than 10% in F1 females at both ages (PND 21-22 and PND 76-90) compared to respective controls at the only dose tested for this parameter (12,500 ppm equivalent to 1,200 and 1,227 mg/kg bw/d in males and females, respectively). These sex-dependent effects induced by melamine exposure may be related to the previously reported sex-dependent neuroanatomical differences in the adult rat corpus callosum, such as myelinated and unmyelinated axon densities or the ratio of myelinated/total axons (Al Qattan et al. 2019).

At the behavioral level, there was no treatment effect on locomotor activity tested for 1 h using the Kinder Scientific Motor Monitor System
in the F1 generation (PND 69–75). Acoustic startle response tests performed once between PND 23–25 for the F1 generation showed that the average response amplitude was significantly lower for males in the mid-dose group (4,000 ppm) in 1 of the 5 tested blocks. However, the data from this test and the Biel maze used to assess the F1 generation at PND 62–68 were considered inconclusive because of insufficient reporting on the methodology and lack of specific description of the statistics. Therefore, except for the thickness of the corpus callosum, no other neurodevelopmental effects based on this OECD guidance were considered relevant for an endocrine disruption assessment.

The effects of melamine exposure on the brain and behavior were analyzed in several academic studies carried out by a single group. Exposure of female rats to 400 mg/kg bw/d of melamine during pregnancy impaired spatial memory of their offspring, assessed using the Morris water maze (An and Sun 2017a, An and Sun 2017b, An et al. 2019, An and Zhang 2014). Reduced litter size, differences in the sex ratio, as well as diminished hippocampal/brain weight ratio (although no difference in the mean body weights for the offspring) were observed in these studies. In additional studies analyzing the effects of postnatal exposure, 3-week-old rats were orally exposed to 300 mg/kg bw/d of melamine for 4 weeks (An et al. 2012, An et al. 2013, Sun et al. 2021a, Sun et al. 2021b, Sun et al. 2021c, Xu et al. 2013, Yang et al. 2011). The obtained data showed impaired learning and memory, assessed using the Morris water maze or the Y maze. In contrast, no significant effect was found on locomotor activity. Reduced body weight and weight gain were also observed at this dose (An et al. 2012, Sun et al. 2021a, Yang et al. 2011).

In a study conducted by another group, relatively low doses of melamine were used (Yang et al. 2012). In this study, acute oral treatment of prepubertal rats (21–28 days of age) with a single dose of melamine at 5 to 25 mg/kg/d, 1 h before the behavioral tests, had no effect on immobility time in the forced swim test or on locomotor activity assessed in the open field test. No analysis of learning and memory was performed in this study.

Based on the available literature data, chronic prenatal or postnatal exposure to melamine impaired learning and memory at 300 and 400 mg/kg bw/d.

### 3.1.4. Melamine effects on thyroid function

Effects on the thyroid were investigated in the OECD TG 443 Study (Study Report, 2020). Significant reductions in T4 levels have been observed at high melamine doses in parent males (833 mg/kg bw/d) and F1 offspring at PND 4 (1,200 and 1,227 mg/kg bw/d of males and females, respectively). A significant increase in relative thyroid weight was observed in F1 animals (+22 % in F1A males at PND 89–95, +15 % in F1B males at PND 140–155, and + 18 % in F1A females at PND 89–95) at the highest dose. This could be partly related to reduced body weights (−8.1 % in F1A males, −7.3 % in F1B males, and −8.3 % in females) as absolute thyroid weights increased (+10 %) in each group. However, although a decrease in T4 levels constitutes an alert *per se*, no histopathological changes were observed in the thyroid gland analyzed by tissue staining with hematoxylin and eosin (HE).

A single paper from the literature search mentioning the adverse effects of melamine on thyroid function was included for analysis (Son et al. 2014). In this study, absolute and relative weights of the thyroid gland were significantly higher compared to controls in male rats exposed to 63 mg/kd bw/d of melamine given orally for 50 days. Unfortunately, neither histopathological analysis of the thyroid gland nor thyroid hormone levels were available for this study.

Therefore, in the two available studies, elevated thyroid weight, which is considered a T-mediated parameter (ECHA and EFSA 2018), was observed and was accompanied by reduced T4 levels in male rats and in pups of the F1 generation at the high-dose of melamine in the EOGRT study.

## 3.2. Data available on ED and non-ED modes of action leading to reproductive and neurodevelopmental adverse effects

### 3.2.1. Overview of the *in vitro* / *in silico* data available on the endocrine activity of melamine

All the 234 assays with melamine listed in the ToxCast database were indicated as inactive (US EPA 2024). ToxCast Model Predictions indicated no androgenic nor estrogenic activity. Endocrine disruptome predicted a low probability of binding to androgen and estrogen receptors (Endocrine Disruptome 2024). No additional ED-related *in silico* nor *in vitro* mechanistic studies were available in the literature at the time of the review.

### 3.2.2. Studies investigating the mechanisms of male reproductive adverse effects

The OECD TG 443 Study from the registration dossier in rats (Study Report, 2020) and the Yin *et al.* study (2013) in mice presented in section 3.1.2 did not measure testosterone levels.

In the study by Chang *et al.* (2014), the elevated percentage of abnormal spermatozoa in the epididymis at all doses of melamine (30, 140, and 700 mg/kg bw/d for 3 consecutive days) was not accompanied by changes in serum testosterone levels, with the exception of a significant increase in serum testosterone levels observed 5 days after treatment at 140 mg/kg bw/d (3.65 ± 2.09 ng/mL vs 0.47 ± 0.44 ng/mL for the control). This showed that melamine altered spermatogenesis and the blood-testis barrier, without a related dose-dependent variation in testosterone levels and in the presence of urinary adverse effects.

In Chen *et al.* (2021), expression of the *p38* gene (related to cell apoptosis) in mouse testes was significantly reduced in all treated groups (2, 4, and 8 mg/kg bw/d). Expression of its downstream regulated genes *MAX* (myc-associated factor X) and the ternary complex factor *Sap1a* (two transcription factors regulated by the mitogen-activated protein kinases (MAPK) signaling pathways) were also diminished at the highest dose. Phosphorylation of p38 protein was also reduced at the highest dose. In addition, the authors observed significant reductions in the expression of extracellular signal-regulated kinases (ERK1 and ERK 2), known to be involved in cell growth, proliferation, and differentiation. The results indicated that p38 and the ERK1-2 MAPK pathways may be involved in melamine-induced reproductive dysfunction. Hormone measurements were not performed in this study.

Abnormal formation and maturation of spermatozooids, damaged testicular tissue structures, and reduced sperm quality and quantity were reported in mice by Huang *et al.* (2018), 35 days after exposure to high-doses of melamine (400, 800 or 1,600 mg/kg bw/d). These effects were accompanied by impairment of antioxidant capacity, namely lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH) enzymatic activities (involved in glucose metabolism and promoting the apoptosis of spermatogenic cells). At these doses (400, 800 or 1,600 mg/kg bw/d), serum testosterone levels were increased to reach 3.9 ± 0.9 nmol/L (1.1 ng/mL) compared to controls 2.2 ± 0.5 nmol/L (0.6 ng/mL). However, this increase was not dose-dependent and was in the low range of serum testosterone levels usually found in adult male mice (Chen *et al.* 2024, Lin *et al.* 2020, Tong *et al.* 2004). As mentioned above (section 3.1.2), this study is considered to be of limited quality.

A general toxicity with severe reproductive toxic effects including structural and functional alterations of Leydig cells through cystic dilatation of the seminiferous tubules with a marked reduction in the spermatocyte percentage of most seminiferous tubules was reported in Khalil *et al.* (2017). After melamine administration at a concentration of 50 mg/kg bw/d orally for 65 consecutive days, the adverse effects were accompanied by a reduction in testicular enzyme activities involved in energy metabolism (by three-fold) compared to the control group. The levels of testosterone and luteinizing hormones were significantly reduced (from 2.5 ng/mL to 0.5 ng/mL for testosterone) in the melamine-exposed group, and this reduction correlated with the decreases in mRNA transcription levels of some key steroidogenic actors,
including StAR, P450scc, and 3β-HSD-1.

Sun et al. (2016a) reported a dose-dependent reduction in sperm count (down to around -20 % at 2 mg/kg bw/d) and in Leydig cell number, without any changes in testis weight and without any indication of general toxicity. Significant dose-dependent decreases in serum testosterone levels were observed in the 10 and 50 mg/kg bw/d groups (from 15 ng/mL in the control group to less than 5 ng/mL). Importantly, no significant changes in serum testosterone levels were observed at 2 mg/kg bw/d, whereas spermatogenesis was altered at this dose. The mechanisms involved in melamine toxicity on the reproductive system therefore target the germ cells at doses lower than those affecting Leydig cells. The decline in the number of Leydig cells observed at higher doses indicated that these cells were secondary targets of melamine and were less sensitive than germ cells.

Although female reproductive function was not prioritized in our analysis of the possible ED-mediated adverse effects, three studies indicated a similar mode of action possibly involved in both sexes (see Supplementary Material S4). Duan et al. (2015) investigated melamine-induced adverse effects on oocyte quality in 3-week-old Female ICR mice exposed to 0, 10, and 50 mg/kg bw/d of melamine in drinking water for 8 weeks. Melamine had toxic effects at 50 mg/kg bw/d on oocyte quality and fertility due to its effects on the oocyte cytoskeleton, apoptosis and autophagy induction, and epigenetic modifications. The early apoptosis rate of oocytes at the germinal vesicle stage and caspase 9 mRNA levels were significantly higher at 50 mg/kg bw/d when compared to controls. Melamine also significantly affected histone methylation in mouse oocytes at both doses. Erisgin et al. (2023) reported a significant decrease in the corpus luteum and a significant increase in the number of atretic follicles at doses of 50 and 75 mg/kg bw/d in weaning female rats treated with melamine for three weeks. Apoptosis increased in both melamine groups, and anti-Mullerian hormone plasma levels decreased significantly in the 75 mg/kg bw/d group. In another study performed in female rats, Sun et al. (2016b) reported that melamine caused ovarian lesions, including elevated numbers of atretic follicles and necrosis of granulosa cells and oocytes at 40 mg/kg bw/d. Moreover, the ovaries of melamine-treated rats showed apoptosis of granulosa cells, oxidative damage, and reduced expression of genes associated with steroidogenesis, indicating that melamine is toxic to rat ovaries. The apoptosis and oxidative stress contributed to ovarian lesions and reduced expression of steroidogenic enzymes, but did not lead to changes in serum estradiol and progesterone levels.

### 3.2.3. Studies investigating the mechanisms of neurodevelopmental effects induced by exposure to melamine

Reduced T4 levels were observed at PND 4 in the EOGRT Study that reported effects on corpus callosum thickness in the postnatal F1 generation. There is therefore a concern that these reduced plasma T4 levels might have contributed to the brain morphometric changes. Development is a critical sensitive window for thyroid hormone regulation of neural processes (Batista and Hensch 2019). However, it must be pointed out that these neural and thyroid effects in offspring were observed at comparable high doses (1,200 and 1,227 mg/kg bw/d in males and females, respectively), while renal effects were observed at lower doses.

In academic studies, the impairment of learning and memory induced by exposure to melamine at 300 and 400 mg/kg bw/d (Section 3.1.3) was associated with alterations in the hippocampal CA1 area, including a reduced number of neurons (An et al. 2012), reduced expression of the glutamate receptor (GluR) 2/3 subunit of the AMPA (α-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid) receptor (Sun et al. 2021a, Sun et al. 2021c), disruption of acetylcholine and acetylcholine esterase expression (An et al. 2013, Sun et al. 2021b), and impaired synaptic activity (An et al. 2019, An and Sun 2017a, An and Sun 2017b, An and Zhang 2014, Xu et al. 2013, Yang et al. 2011). Alteration of electrophysiological properties was confirmed using hippocampal slices of 10–14 day- and 3-week-old rats perfused with 5 ×

$10^{-4}$ and $5 \times 10^{-5}$ g/mL, but not $5 \times 10^{-6}$ g/mL of melamine, displaying a possible threshold of effect (Yang et al. 2010a, Yang et al. 2011, Yang et al. 2012). Neuroinflammation, elevated apoptotic index, and disruption of the blood–brain barrier were also observed following oral gavage of prepubertal rats with 50 and 75 mg/kg bw/d of melamine for three weeks (Erisgin 2021). Under this experimental condition, body and brain weights were comparable between the treatment groups.

In addition, *in vitro* studies using PC12 cells reported reduced cell viability after 36 h of treatment with $5 \times 10^{-6}$ g/mL of melamine and 12 h of treatment at a melamine concentration of $5 \times 10^{-4}$ g/mL (Yang et al. 2010b). It was suggested that this effect involved alteration of neuronal excitability. Similarly, Han et al. (2011) observed a dose- and time-dependent inhibition effect on cell growth. Melamine induced apoptosis from concentrations of $3.3 \times 10^{-5}$ to $3.3 \times 10^{-3}$ µg/mL in a dose-dependent manner. Oxidative damage was mentioned as a factor increasing the neurotoxicity potential of melamine in SH-SY5Y neuroblastoma and PC12 cell lines (Goyal et al. 2023, Han et al. 2011).

## 4. Discussion

The present review highlights the adverse effects induced by melamine exposure, in particular on the nervous system, thyroid function, and male reproductive function. The plausibility, or not, that these effects could have been induced by an endocrine mode of action is discussed below for each endpoint.

Evidence of neurodevelopmental effects of melamine was illustrated by brain morphometric changes reported in the EOGRT Study (Study Report, 2020). As explained in section 3.1.3, the behavioral analyses conducted in this study were inconclusive due to methodological and data reporting issues. Impairment of learning and memory following exposure to melamine was described in several academic studies (An and Sun 2017a, An and Sun 2017b, An et al. 2019, An and Zhang 2014, An et al. 2012, An et al. 2013, Sun et al. 2021a, Sun et al. 2021b, Sun et al. 2021c, Xu et al. 2013, Yang et al. 2011). The mechanisms underlying learning and memory impairment were related to reduced cellular viability and increased apoptosis (Han et al. 2011), disrupted neuronal activity and synaptic plasticity and transmission (An et al. 2019, An and Sun 2017a, An and Sun 2017b, An and Zhang 2014, Xu et al. 2013, Yang et al. 2011), as well as generation of oxidative stress (An et al. 2012, Goyal et al. 2023, Han et al. 2011). For the brain morphometric changes reported in the EOGRT Study, despite the associated reduced T4 levels observed in the F1 generation, the possibility that melamine acted through disruption of the thyroid hormone system is poorly supported.

First, neural and thyroid effects in offspring were observed at comparable high doses (1,200 and 1,227 mg/kg bw/d in males and females, respectively). In contrast, nephrotoxicity occurred at lower doses (circa 250 mg/kg bw/d) as mentioned in section 3.1.1. In the EORGT Study, the no-observed adverse effect level (NOAEL) for nephrotoxicity was set at the lowest dose tested, i.e., 1,000 ppm (65 and 87 mg/kg bw/d in males and females, respectively) for the parent generation and 1,000 ppm (89 and 93 mg/kg bw/d in males and females, respectively) for F1 animals. Biochemical parameters (urea and creatinine concentrations) were not modified in melamine-treated animals, but the results showed histological changes with retrograde nephropathy at the mid- and high-doses. As a result, renal function appears to be a more sensitive endpoint than the thyroid or neurodevelopment. Reduced body weight was also reported in some of these studies, supporting the idea that the neural effects induced by melamine were due to general toxicity, possibly related at least in part to renal failure. Second, previous studies have highlighted bidirectional communication between the kidney and the thyroid gland on the one hand, and the kidney and the brain on the other (Echterdiek et al. 2022, Yan et al. 2024, Malek, 2018, Pépin et al. 2024). In particular, it has been shown that kidney dysfunction induces thyroid and cognitive impairments, possibly through disruption of transport, release of inflammatory molecules, or retention of toxins (Malek, 2018). It is plausible that the renal dysfunction induced by melamine was the
initiating event that further impacted physiological systems including the thyroid hormone profile and cognitive processes. Finally, according to the specificity criterion of the European guidance for the identification of endocrine disrupting compounds (see Section 3.3.1 of ECHA and EFSA 2018), the adverse effects should not be the “non-specific secondary consequences of other toxic effects”. Therefore, based on these observations and the specificity criterion of the European guidance (ECHA and EFSA, 2018), the criterion to identify melamine as a thyroid hormone disruptor, thereby inducing neurotoxicity, is not met.

The present set of data clearly showed that melamine was able to alter male reproductive function by impairing spermatogenesis. In the EOGRT Study, reduced sperm quality and quantity (–42 % of epididymal sperm count in cohort 1A) did not trigger changes in rat fertility. In rodents, a reduction in spermatozoa production must be higher than 55 % to affect fertility (Forand et al., 2019). This is not the case in the human population since even a small decrease in spermatozoa concentrations in semen could lead to low couple fecundability and increased demand for assisted reproduction (Leridon and Slama 2008, Slama et al. 2004, Skakkebæk et al. 2022). Therefore, the conclusion drawn for melamine about the absence of effects on rodent fertility should be transposed with caution regarding human fertility.

The potential endocrine mechanisms underlying the effects on spermatogenesis were addressed in the EOGRT Study (Study Report, 2020) and in 4 academic studies (Chang et al. 2014, Sun et al. 2016a, Khalil et al. 2017, Huang et al. 2018). They all focused on testosterone-controlled spermatogenesis although they were performed using different study protocols, doses, routes, durations and periods of exposure. Two studies described impaired spermatogenesis in the absence of reductions in serum testosterone levels (Chang et al. 2014 at 30, 140, and 700 mg/kg bw/d, Huang et al. 2018 at 400, 800, and 1,600 mg/kg bw). In Sun et al. (2016a), no significant changes in serum testosterone levels were observed at 2 mg/kg bw/d, whereas spermatogenesis was altered at this dose. In this study, plasma testosterone levels decreased at higher doses (10 and 50 mg/kg bw/d). The EOGRT Study showed altered spermatogenesis while the weights of androgen-dependent organs were unchanged following exposure from 65 to 1,200 mg/kg bw/ d of melamine (Study Report 2020). One study described a clear concomitance between spermatogenesis alterations and testosterone level decrease (Khalil et al. 2017 at 50 mg/kg bw/d). This study described a clear and marked reduction in general cellular metabolism in the testis, which appears as the direct cause of spermatogenic defects. Similarly, in females, melamine effects consisted of reduced oocyte development related to meiotic maturation, and oocyte abnormalities indicative of comparable oxidative effects of melamine on gametogenesis (ovogenesis and spermatogenesis). Considering the presence of other toxic effects observed after melamine exposure in these studies, spermatogenesis alteration occurred primarily in the absence of a demonstrated endocrine MoA and could involve general cellular biology alterations.

As discussed above for the thyroid gland and brain, an indirect MoA related to nephrotoxicity could also have induced adverse effects on the reproductive system. Based on evidence of impaired fertility in women and men with chronic kidney disease reported in Kuczera et al. (2022), we hypothesize that renal toxicity induced by melamine could have triggered indirect reprotoxic effects. Indeed, in Sun et al. (2016a), impaired spermatogenesis at 2 mg/kg bw/d in male rats occurred in the absence of testosterone variations, showing that other modes of action were involved. In addition, in female rats, Sun et al. (2016b) observed adverse effects on ovarian function and oogenesis (elevated numbers of atretic follicles and necrosis of granulosa cells and oocytes) in the absence of significant variations in estrogen levels (40 mg/kg bw/d). These results are consistent with the study of Dai et al. (2015) in female mice, linking reprotoxic effects to oxidative stress but not to an ED mechanism. Furthermore, an increase in oxidative stress and a reduction in the activities of enzymes involved in energy metabolism in the testis were observed in all the studies that investigated these parameters,

which paralleled the observation of melamine-induced general toxicity (Chen et al. 2021, Khalil et al. 2017, Huang et al. 2018). The non-sex-specific effects reported could therefore support a non-specific secondary consequence of the renal toxicity. In Chang et al. (2014), 1 day after exposure by oral gavage, white precipitates were found in the bladders of one mouse in each of the two low- and mid-dose groups (30 and 140 mg/kg bw/d; n = 12/group) and in half of the mice in the highest melamine dose group (700 mg/kg bw/d), while plasma urea and creatinine levels were not affected. Simultaneously, the percentage of abnormal spermatozoa in the epididymis was increased in all dose groups, while serum testosterone levels were unchanged. Further histological or biochemical data regarding renal damage are needed to confirm the link between the reprotoxic effects and the upstream nephrotoxic activity induced by melamine exposure.

Taken as a whole, adverse effects of melamine on spermatogenesis do not appear to be the consequence of reduced testicular testosterone production, but rather a consequence of cellular toxicity affecting gametogenesis. The female reproductive studies support this analysis. A causal relationship between testosterone reduction and spermatogenesis alteration was not demonstrated. The effects observed on spermatogenesis were probably mainly due to melamine-induced alterations in the physiology of testicular cells, and specifically that of germ cells, which are highly sensitive. Later impairment of Leydig cells may occur, inducing changes in testosterone production.

## 5. Conclusion

Concerning the effects reported on neurodevelopment and the thyroid hormone system, the available information showed that both occurred concomitantly, at doses of melamine higher than those inducing nephrotoxicity. Given the vulnerability of the thyroid gland and the brain to renal dysfunction, both neurodevelopmental and thyroid effects are possibly due to general toxicity related at least partly to renal failure. The specificity criterion of the European guidance for the identification of endocrine disrupting chemicals (ECHA and EFSA, 2018) is not met and, according to the guidance, these effects are considered non-ED related.

Based on the present analysis, we conclude that the effects inducing germ cell damage occurred at doses below those related to toxicity of Leydig cells, and thus leading to changes in testosterone levels. Importantly, melamine impaired spermatogenesis, but the spermatotoxicity was possibly primarily induced by modes of actions (such as oxidative stress and reduction in energy metabolism) other than changes in testosterone levels. The publication of La Merill describes the characteristics of endocrine-disrupting chemicals, as a basis for hazard identification (Merrill et al., 2020). One of these characteristics is that the substance "Alters the fate of hormone-producing or hormone-responsive cells", which could indeed apply to the direct cytotoxicity described in this paper, targeting first germ cells and later Leydig cells. However, in the framework of the CLP regulation, the effect on germ cells is consistent with a reprotoxic rather than an endocrine-disrupting hazard.

Therefore, we conclude that potential ED properties of melamine are not confirmed for human health, but that melamine should be considered a reprotoxicant and classified as such under the CLP regulation.

This assessment illustrates the scientific challenges faced by regulators in differentiating specific endocrine disruption from an indirect endocrine effect resulting from non-ED mediated systemic toxicity. It emphasizes the amount of data necessary to properly identify endocrine disruptors and distinguish them from other hazard classes.

## CRediT authorship contribution statement

**Isabelle Charron:** Writing – review & editing, Writing – original draft, Project administration, Methodology, Investigation.
**Brigitte Le Magueresse-Battistoni:** Writing – review & editing, Validation, Formal analysis, Writing – original draft.
**René Habert:** Writing – review &
editing, Validation, Formal analysis, Writing – original draft. Marie- Chantal Canivenc-Lavier: Writing – review & editing, Validation, Formal analysis, Writing – original draft. Sakina Mhaouty-Kodja: Writing – review & editing, Validation, Formal analysis, Writing – original draft. Cécile Michel-Caillet: Writing – review & editing, Writing – original draft, Supervision, Conceptualization.

## Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.envint.2024.109188.

## Data availability

Data used for assessment come from published scientific literature

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