Comments on "in vitro and in vivo estrogenicity of UV screens". (Correspondence).

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 [micro]g/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 [micro]g/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-hormonal-effect 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-3-metabolite p-hydroxy-benzophenone (6), which comprises [approximately equal to] 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 [hygiene-based 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 4-MBC 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 Forschungs-gemeinschaft (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 OMC: A daily (4-day) dose of 0.342 [micro]g/kg ethinylestradiol produced a mean uterine weight of 37.02 mg. By interpolation between experimental data points, it appears that the same uterine weight (37.02 mg) is elicited by 275 mg/kg 4-MBC or by 1,243 mg/kg OMC. It follows that ethinylestradiol is more potent than 4-MBC by a factor of 8 x [10.sup.5], and is more potent than OMC by a factor of 3.6 x [10.sup.6]. Using uterotrophic assay data in rats (14), Bolt et al. (8) concluded that ethinylestradiol was 40,000 times more potent than the typical isoflavone phytoestrogen daidzein. It follows that the relative potencies related to daidzein (set to 1) are 0.05 for 4-MBC and [approximately equal to] 0.01 for OMC. The potency of daidzein is 20-fold higher than that of 4-MBC and 100-fold higher than that of OMC.

The official exposure scenario for isoflavone phytoestrogens (e.g., daidzein) in European diets by the SKLM was 1 mg/kg daily. Official exposure scenarios for UV filter substances in cosmetic products have been issued by the SCCNFP (9,10). The HBMOS figures, derived from official scenarios, are 87 for 4-MBC (20 mg/kg:0.23 mg/kg) and 167 for OMC (100 mg/kg:0.6 mg/kg).

Three approximations to safety margins of the UV filters 4-MBC and OMC have been deduced:

1. Calculations of MOSs based on the experimental NHELs of Schlumpf et al. (1)

2. The official MOS data of the SCCNFP based on NOAEL figures from animal studies under repeated dosage

3. Application of the novel concept of HBMOS, which basically compares the estrogenic load by phytoestrogens in the diet to that of the compound in question under application conditions.

Table 2 provides a summary of these calculations. The calculations 1 and 3 are based on the data set of Schlumpf et al. (1), and calculation 2 is the official assessment of the SCCNFP (9, 10).

The risk assessment data seem to be consistent with each other. Thus, it must reasonably be concluded that, considering the data provided by Schlumpf et al. (1), even with their technical shortcomings, the resulting margins of safety for the compounds in question clearly provide sufficient safety under the conditions of use.

Table 1. Comparison of NHEL data used by Schlumpf et al. (1) and NOAEL
data.

Compound  NHEL                      NOAEL

4-MBC     NHEL = 66 mg/kg/day (1)   NOAEL (subchronic oral rat
                                     study) = 25 mg/kg/day (10)
          SED = 0.23 mg/kg (10)     SED = 0.23 mg/kg (10)
          MOS = NHEL/SED = 290      MOS = NOAEL/SED = 110
OMC       NHEL = 522 mg/kg/day (1)  NOAEL (13-week rat oral
                                     study) = 450 mg/kg/day (9)
          SED = 0.6 mg/kg (9)       SED = 0.6 mg/kg (9)
          MOS = NHEL/SED = 870      MOS = NOAEL/SED = 750

SED, systemic exposure dose.
Table 2. Calculations of MOSs by three different methods.

              1            2         3
Compound  MOS [NHEL]  MOS [NOAEL]  HBMOS

4-MBC        290          110        87
OMC          870          750       167

REFERENCES AND NOTES

(1.) Schlumpf M, Cotton B, Conscience M, Haller V, Steinmann B, Lichtensteiger W. In vitro and in vivo estrogenicity of UV screens. Environ Health Perspect 109:239-244 (2001).

(2.) Inoue T. Protocol for the Conduct of the OECD Rodent Uterotrophic Assay, Draft Protocol A. Tokyo:National Institute of Health Sciences, 2000.

(3.) Comotto L, Bussi R. Unpublished data.

(4.) Bachmann S, Hellwig J. Unpublished data.

(5.) Baker VA, Hepburn PA, Kennedy SJ, Jones PA, Lea LJ, Sumpter JP, Ashby J. Safety evaluation of phytosterol esters. Assessment of oestrogenicity using a combination of in vivo and in vitro assays. Food Chem Toxicol 37:13-22 (1999).

(6.) Nakagawa Y, Tayama S. Estrogenic potency of benzophenone and its metabolites in juvenile female rats. Arch Toxicol 75:74-79 (2001).

(7.) Stocklinski AW, Ware OB, Oberst TH. Benzophenone metabolism. I. Isolation of p-hydroxyphenone from rat urine. Life Sci 26:365-369 (1980).

(8.) Bolt HM, Janning P, Michna H, Degen GH. Comparative assessment of endocrine modulators with oestrogenic activity: I. Definition of a hygiene-based margin of safety (HBMOS) for xeno-oestrogens against the background of European developments. Arch Toxicol 74:649-662 (2001).

(9.) SCC. Opinion of The Scientific Committee on Cosmetics Concerning 2-Ethylhexyl-4-methoxycinnamate (S28) Brussels:Scientific Committee on Cosmetics, 1996.

(10.) SCCNFP. Opinion of the Scientific Committee on Cosmetic Products and Non-Food Products Intended for Consumers Concerning 3-(4'-Methylbenzylidene)-D,L-camphor Adopted by the Plenary Session of the SCCNFP of 21 January 1998. Brussels:Scientific Committee on Cosmetic Products and Non-Food Products, 1998.

(11.) Hoffmann B. Problems of residues and health risks of anabolic agents with sex hormone-like activities. In: Proceedings of the European Commission: Scientific Conference on Growth Promotion in Meat Production, Brussels, Belgium, 29 November-1 December 1995. Luxemburg:Directorate-General VI Agriculture, Office for Official Publications of the European Communities, 1996:271-296.

(12.) Lamming GE. Scientific working group reports on anabolic steroids in animal production. In: Proceedings of the European Commission: Scientific Conference on Growth Promotion in Meat Production, Brussels, Belgium, 29 November--1 December 1995. Luxemburg:Directorate-General VI Agriculture, Office for Official Publications of the European Communities, 1996:433-434.

(13.) Senate Commission of Food Safety of the Deutsche Forschungsgemeinschaft. Hormonally Active Agents in Food (Eisenbrand G, Daniel H, Dayan AD, Elias PS, Grunow W, Kemper FH, Loser E, Metzler M, Schlatter J, eds). Weinheim, Germany:Wiley-VCH, 1998.

(14.) Diel P, Schulz T, Smolnikar K, Strunck E, Vollmer G, Michna H. Ability of xeno- and phytoestrogens to modulate expression of estrogen-sensitive genes in rat uterus: estrogenicicty profiles and. uterotrophic activity. J Steroid Biochem Mol Biol 73:1-10 (2000).

(15.) Bolt HM, Mumtaz MM. Risk assessment of mixtures and standard setting: Working towards practical compromises. Food Chem Toxicol 34:1179-1181 (1996).

Hermann M. Bolt
Christine Guhe
Gisela H. Degen
Institute of Occupational Physiology at the
University of Dortmund (IfADo)
Dortmund, Germany
E-mail: [e-mail]