Comments on "In vitro and in vivo estrogenicity of UV screens".

Schlumpf et al. (1) reported on the in vitro and in vivo “estrogenicity” of six ultraviolet (UV) filters: benzophenone-3 (Bp-3), homosalate (HMS), 4-methyl-benzylidene camphor (4-MBC), octyl-methoxycinnamate (OMC), octyl-dimethyl PABA (OD-PABA), and butyl-methoxydibenzoylmethane (BMDM). The authors concluded that “UV screens should be tested for endocrine activity, in view of possible long-term effects in humans and wildlife.” There is international consensus that in vitro data should serve only for screening purposes and that they are not suited for conclusions regarding risk assessment. The interpretation of the in vivo data presented is very much hampered by the fact that Schlumpf et al. (1) used nonstandard and non-GLP protocols, although official guidelines have been issued (2). Specifically, we refer to Schlumpf et al.’s choice of unusual rat strains (Long-Evans and Nu rats) for the uterotrophic assay and to the mode of dermal administration (pups were totally immersed in oily solutions of the test compound). Because of the administration protocol used by Schlumpf et al. (1), the calculation of the absorbed dose after dermal exposure remains obscure. Also, the time of administration of the test compounds (postnatal day 26) was very close to or at the onset of puberty in most rat species. Following established protocols and GLP procedures, a uterotrophic assay was performed in Sprague-Dawley rats (the standard strain) using three daily doses of 10, 100, or 1,000 mg/kg 4-MBC subcutaneously (3); no uterotrophic response was observed. In another uterotrophic assay (4), Bp-3 and OMC were tested in female immature Wistar rats. Bp-3 was administered in four oral doses of 500 and 1,000 mg/kg/day, and OMC was applied in three oral doses of 500 and 1,000 mg/kg/day; no uterotrophic effect was observed (4). Strain variations such as these are not entirely unusual. According to Table 3 of Schlumpf et al. (1), effective oral doses (uterotrophic effect) were 0.342 μg/kg/day ethinylestradiol, 119 mg/kg/day 4-MBC, 1,035 mg/kg/day OMC, and 1,525 mg/kg/day Bp-3. The lower doses tested, (i.e., 0.085 μg/kg/day ethinylestradiol, 66 mg/kg/day 4-MBC, 522 mg/kg/day OMC, and 937 mg/kg/day Bp-3) must be regarded as no-hormonaleffect levels (NHELs), based on the data of Schlumpf et al. (1). The effect of Bp-3 (called “weak” by the authors) appears in a range above the “limit dose,” according to current Organisation for Economic Cooperation and Development guidelines (2). A very weak effect of Bp-3 is not considered contradictory with negative findings in other studies (5), and it appears consistent with an estrogenic effect of the minor Bp-3metabolite p-hydroxy-benzophenone (6), which comprises ≈1% of a benzophenone dose in rats (7). Although the data of Schlumpf et al. (1) are in contrast to findings of others and have technical shortcomings, they can nevertheless be incorporated into risk assessment scenarios leading to worst-case views. We used the data presented by Schlumpf et al. in their Table 3 (1) as the basis of two assessments: a) we calculated a traditional margin of safety (MOS) based on the NHEL observations of Schlumpf et al., and b) we compared the estrogenic load that might be imposed on the human organism by the UV filter compounds under consideration with the estrogenic load imposed by phytoestrogens in the normal diet [hygienebased margin of safety (HBMOS)] (8). Official exposure scenarios for 4-MBC and OMC have been described by the Scientific Committee of Cosmetic Products and Non-Food Products (SCCNFP) of the European Union as a basis of associated risk assessments (9,10). The effects of Bp-3 in the study by Schlumpf et al. (1) are very much borderline if one considers that they are observed only at doses above the limit dose. Hence, the subsequent assessments are restricted to the two compounds (4-MBC and OMC) for which uterotrophic effects at lower doses have been reported by Schlumpf et al. (1). With regard to human toxicity, experimentally based no-observed-adverse-effect levels (NOAEL) are the toxicologic key element. In contrast to other approaches, the MOS methodology of the European Union does not make use of numerically fixed assessment factors; the MOS is calculated by comparing the level of human exposure (estimated to a large extent by modeling) with the NOAEL from animal experiments. Application of this concept to hormonally active compounds (endocrine modulators) is easily possible if the hormonal effect is considered the critical toxicity; this would mean that NHELs could serve as specific substitutes of the NOAEL (11). In principle, this avenue of thinking has been advanced from the scientific side in discussions concerning regulations of hormonally active growth promoters in meat (12). An MOS can be derived by comparing the NHEL data of the two substances 4MBC and OMC from Table 3 of Schlumpf et al. (1) with official exposure scenarios (systemic exposure doses) of the SCCNFP (Table 1) (9,10). Bolt et al. (8) developed a supplementary route of comparative risk calculation using the concept of HBMOS. Basically, they compared exposure scenarios for individual industrial compounds with those of endocrine modulators of natural origin, under consideration of the respective relative potency ratios in vivo. The dietary intake figures of estrogenic isoflavones have been assessed in our laboratory (8); data in the published literature are in general support of the scenario of the Senate Commission on the Evaluation of Food Safety of the Deutsche Forschungsgemeinschaft (SKLM) (13), which arrived at a human daily intake of isoflavones in the order of 1 mg/kg body weight. Relative estrogenic potency assessment figures based on in vivo studies of 4-MBC and OMC, in relation to isoflavones (e.g., daidzein), can be derived from a synopsis of the results of the uterotrophic assays by Schlumpf et al. (1) (shown in their Table 3; the potencies of 4-MBC and OMC compared to that of ethinylestradiol) and by Bolt et al. (8). The latter data refer to uterotrophic assays by Diel et al. (14) that compare the potencies of the phytoestrogen daidzein and the reference compound ethinylestradiol, as well as other compounds. Based on the concept of “dose additivity” for combinations of similarly acting compounds (15), Schlumpf et al. (1) provide data in their Table 3 of equally effective doses of ethinylestradiol, 4-MBC, and