Potential endocrine disrupting properties of toys for babies and infants

Plastic toys mouthed by children may be a source of exposure to endocrine active substances. The purpose of this study was to measure hormonal activity of substances leaching from toys and to identify potential endocrine disruptors causing that activity. For this purpose, migration experiments of toys were conducted in saliva simulants. The CALUX® assays were used to detect (anti-) estrogenic and (anti-) androgenic activity of 18 toys. Chemical trace analysis–namely, GC-MS and HPLC-MS- was used to identify which compounds may be responsible for endocrine activity in the sample migrates. Nine out of 18 tested toys showed significant estrogenic activity. For two samples, the detected estrogenic activity could be well explained by detecting the known endocrine active substance bisphenol A (BPA). For all identified substances, including BPA, a risk assessment for human health was performed by comparing the exposure dose, calculated based on the determined substance concentration, to toxicological reference values. Using worst-case scenarios, the exposure to BPA by mouthing of the two estrogen active, BPA-containing toys could be above the temporary TDI that EFSA has calculated. This demonstrates that some toys could significantly contribute to the total exposure to BPA of babies and infants. For seven out of nine estrogen active samples, the source of the estrogen activity could not be explained by analysis for 41 known or suspected endocrine active substances in plastic, indicating that the estrogen activities were caused by currently unknown endocrine active substances, or by endocrine active substances that would currently not be suspected in toys.

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Unfunded studies
Enter: The author(s) received no specific funding for this work.   Children, in particular, those below the age of 36 months, are considered particularly vulnerable to 11 chemical substance exposure, since the ratio between exposure and body weight is different 12 between children and adults [1]. Furthermore, developing periods constitute windows of 13 susceptibility to some compounds showing endocrine activity. This may explain the increased 14 incidence of certain diseases such as neurodevelopmental disorders or effects on the reproductive 15 tract [2]. 16 Children are exposed in their daily life to multiple chemical compounds, via food, dust, personal 17 care products and other consumer products [3,4]. Several studies based on observations on the 18 mouthing behaviour of children from 0 to 36 months confirm that children put a great diversity of 19 objects into their mouths, including toys [1]. Plastic toys account for the majority of toys purchased 20 in France. However, only few studies have assessed the risks associated with chemical 21 substances present in plastic toys and children's equipment intended for infants and children up to 22 three years [5]. Plastic toys are often made with complex mixtures of one or more polymers 23 combined with multiple additives such as plasticizers, flame retardants, antioxidants …. As some of 24 these constituents are not covalently bound to the polymers, plastics have been shown to release 25 chemicals such as phthalates or UV filters that are known endocrine disrupters [4]. 26 Toys are regulated in the European Union by a Directive which stipulates that toy manufacturers 27 shall carry out an analysis of the chemical, physical, mechanical, electrical, flammability, hygiene 28 and radioactivity hazards that the toy may present and assess the potential exposure to these 29 hazards. In terms of chemical safety, the directive prohibits the use of carcinogenic, mutagenic and 30 reprotoxic (CMR) substances in categories 1A, 1B or 2 in toys or structurally separate parts, except 31 if the substance is inaccessible or present in concentrations below a certain threshold. Plastic 32 materials have been suggested as a relevant source of human exposure to endocrine disrupting 33 Today, even if a common definition of what is an endocrine disrupter has been adopted at the 35 European level, there is no regulatory obligations for those placing toys on the market to address 36 this concern for endocrine effects yet. On the contrary, in the recently published Medical Device 37 Regulation 2017/745, it is stated that substances having endocrine-disrupting properties with 38 scientific evidence of probably serious effects on humans, shall only be present in a medical device 39 above 0.1% by weight (w/w) when justified. 40 Phthalates are a group of substances, which have been widely used in toys as plasticizers to 41 increase their flexibility. Several phthalates have been classified as reprotoxicants 1B and 42 therefore been banned in toys or other articles which may be put into the children mouth. For 43 example, the use of di(2-ethylhexyl) phthalate (DEHP), Di-n-butylphthalate (DnBP), Di-iso-44 butylphthalate (DiBP) and Butylbenzyl-phthalate (BBP) is restricted to a maximum level of 0.1% in 45 all toy plastic parts following entry 51 of Annex XVII to REACH [9]. In addition, the same maximum 46 threshold has to be applied to Di-iso-nonyl-phthalate (DiNP), di-n-octyl phthalate (DnOP), di-iso-47 decyl phthalate (DiDP) when children can place toys in their mouth (entry 52 of Annex XVII to REACH). Some phthalates have also been recently identified as endocrine disrupters under the 49 REACH regulation [10]. 50 Actually, the described low regulatory limit of 0.1 % is equivalent to a ban of phthalates in plastics, 51 as a minimal level 10% phthalates is required to achieve the softening effect on PVC. As a result, 52 efforts have been made by industry to substitute many of the reprotoxic phthalates with less potent 53 substances, such as Bis-2-ethylhexyl terephtalate (DEHTP) or Acetyl tributyl citrate (ATBC), in 54 toys. However, for many substitutes endocrine activity data is not available. Other compounds 55 used in plastics such as Bisphenol A (BPA) have also been recently identified as endocrine 56 disrupters under the REACH regulation [10]. 57 In addition to chemicals with known endocrine disrupting potential, toys may contain substances 58 that have never been evaluated for their endocrine activity. Especially for non-intentionally-added 59 substances, such as degradation products or contaminants, little is known on their potential 60 endocrine effects. To be able to detect not only known or suspected endocrine disruptors, but also 61 currently unknown substances with an endocrine activity, biodetection systems can be used 62 [11,12]. Berger et al. Thus, the purpose of this study was to analyse selected toys for leaching of endocrine active 68 substances potentially absorbed by toy-mouthing children. Several studies have already been 69 carried out to assess the health risks associated with substance exposure originating from toys 70 [18][19][20][21][22]. Most of these studies, however, assessed exposure scenarios based on the composition 71 as well as migration or emission tests to identify hazardous chemicals that need to be avoid or 72 reduce in the material composition,.
Hence, in the present study, toys and children's equipment intended for children up to three years 74 were screened for EDCs using effect-directed analysis [23]. The CALUX ® assays (Chemically 75 Activated Luciferase gene expression) were applied to detect substances (i.e. ligands) interacting 76 with the human estrogen or androgen receptors and thereby modifying the subsequent 77 transcriptional response [24,15]. 78 In a previous study, composition and migration tests were already conducted on these items, in  Table 2 shows the samples that were selected for analysis and ordered online. 115

Characterization of materials 116
Toy samples were characterized following a 3-step approach. First, the toy itself, its packaging and 117 the producers' web sites were examined for available product information (e.g. recycling codes). If 118 not enough information could be obtained, a formal request asking for the used raw materials was 119 directly sent to the toy manufacturer as a second step. Several toy producers agreed to share this 120 information (see table 2). In a third step the remaining samples were analysed by Fourier-transform 121 infrared (FT-IR) spectroscopy analysis using a FT-IR-spectrometer type Perkin-Elmer, model 122 Spectrum One to identify the materials. As a general approach the samples were measured on the 123 surface as well as in the internal part. Recordings were made using a universal ATR unit (equipped 124 with Diamond/ZnSe crystal) in a spectral range from 4000-600 cm -1 . 125

Migration experiments 126
To simulate the process of toy mouthing by children, the toy subparts were incubated with saliva  Table 1

Selected toys and characterization of materials 251
In a 3-step approach (research, contact to the manufacturer and FT-IR analysis) the material of the 252 tested toys was characterized. Table 2 gives an overview on all selected toys and sampling areas 253 and lists the determined material of all tested sample parts. 254 The results of the bioassay screening are listed in Table 3.   In comparison to estrogen activities taken up by food, the detected estrogen activities were low. 302 Many food products naturally contain estrogens, for instance 23 ng/L of the natural female sex 303 hormone 17β estradiol were analytically determined in cow milk by Courant et al. [27]. By orders of 304 magnitude higher estrogen activities than in cow milk, can be found in soy bean rich food, that 305 contains phytoestrogens. The maximum total daily uptake of estrogen activity by food is estimated 306 to be up to 10,000 ng estradiol equivalents per day for babies who are fed by milk on soy bean 307 basis [28]. The activities detected in the migrates of the saliva solvent were also 308 significantly lower than estrogen activities previously reported for mineral water [7, 29, 30], 309 where no estrogen activity would be expected. The two parts of toy "T05", in which BPA could be detected, consist of neither of these materials, 350 but of flexible PVC. BPA has previously been a common additive in PVC as a production aid to 351 stabilize vinyl chloride monomer, but the use of BPA as a stabilizer for vinyl chloride monomer was 352 stopped in Europe since 2001. BPA has also been restricted in food contact plastic in Europe. 353 Nevertheless, BPA still seems to be used in the PVC production for toys [37]  Besides "T05", BPA could be detected in one out of three independently prepared migrates of 360 sample T07b. However, the detected concentration was too low to be quantified. 361 Benzophenone was identified in five different tested sample parts (T02, T08a, T09a, T09b and 362 T11a). Four of these are built from flexible PVC, one sample (T11a) is made of Acrylonitrile 363 butadiene styrene (ABS) with a sticker of printed paper on it. In the latter one printing inks from the 364 paper sticker seem to be the most plausible source for the detected benzophenone, as 365 benzophenone is a common photo initiator in UV printing colors (IARC 2013). In PVC 366 benzophenone is often us as a UV-stabilizer. 367 The 41 target compounds in the analysis include seven phthalates, previously widely used in database (see Table 4). 381

Correlation between bioassays results and chemical trace analysis 416
In order to identify the source of the hormonal activity in positively tested samples in the ER 417 CALUX ® , results from chemical analysis and bioassays were compared. For this purpose, all 418 detected substances were tested as pure chemicals in the estrogen receptor CALUX ® , if they were 419 available. The response curves for the positively tested substances in the bioassay are shown in 420 figure 2. From that the half-maximal effective concentration (EC50) was calculated for all 421 compounds showing estrogenic effects in the ER-CALUX ® (see table 5). 422 The determined estrogen activities of BPA, benzophenone and DEP were to be expected based on 423 previously published results [2, 34, 35]. The phthalate alternatives TXIB, ATBC, DINCH and 424 DEHTP were tested negative in the ER-CALUX ® . Unexpectedly, the plasticizer TBC (Merck) 425 showed a low, but significant estrogen activity at high concentrations, although the structure of the 426 molecule would not suggest binding affinity to the estrogen receptor. It is possible, that this low 427 estrogen activity, was caused by contaminants of this substance, rather than the substance itself, 428 as the purity of the tested substance was below 97 %. 429 For substances, that could not be obtained as pure chemicals a literature survey was conducted. 430 1-Propene-1,2,3,-tricarboxilic acid, tributyl ester, propanoic acid and 2-methyl,3-hydroxy-431 2,4,4,trimethylpentyl ester are not registered yet under the REACH regulation. No data on the 432 endocrine activity of these substances is available. 433  Tributyl citrate (TBC) 77-94-1 yes 5*10 -5