Evaluation of the anti-androgenic and cytotoxic effects of benzophenone-3 in male Sprague-Dawley rats.

Benzophenone-3 (BP-3, 2-hydroxy-4-methoxybenzophenone, oxybenzone) is one of the most widely used types of benzophenone organic sunscreen. However, this compound is a potentially harmful toxicant. The aim of this study was 2-fold to: (1) utilize a Hershberger bioassay in vivo in castrated male Sprague-Dawley rats to investigate the anti-androgenic activities of BP-3, and (2) use in vitro a methyl tetrazolium assay to compare the toxicity between Leydig cells (TM3 cells) and mouse fibroblast (NIH-3T3) cell lines. In the Hershberger assay, rats were divided into 6 groups (each of n = 7): a vehicle control, negative control, positive control, PB-3 low (40 mg/kg), BP-3 intermediate (200 mg/kg), and BP-3 high (1000 mg/kg)-dose. The weight of the ventral prostate was significantly decreased at BP-3 doses of 200 or 1,000 mg/kg/day. In addition, the levator anibulbocavernosus muscle weights were also significantly reduced at BP-3 doses of 40, 200, or 1,000 mg/kg/day. In the MTT assay, the viability of NIH-3T3 mouse fibroblast cells was within the normal range. However, the TM3 mouse testis Leydig cell viability was significantly lowered in a concentration-dependent manner. Therefore, data indicate that BP-3 might exert in vivo anti-androgenic and in vitro cytotoxic effects in cells associated with the male reproductive system compared to normal non-reproductive cells.Abbreviation: BP-3: benzophenone-3; CG: Cowper's gland; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; GP: glans penis; LABC: levator anibulbocavernosus muscle; MTT: methyl tetrazolium; NC: negative control; PC: positive control; SV: seminal vesicle; TP: testosterone propionate; VC: vehicle control; VP: ventral prostate.

Citrate ester substitutes for di-2-ethylhexyl phthalate: In vivo reproductive and in vitro cytotoxicity assessments.

Di-2-ethylhexyl phthalate (DEHP) is frequently used as a plasticizer for wrapping films, in toys, and in medical devices. Previous studies demonstrated that DEHP in mouse reduced testicular and epididymis weights, suppressed levels of serum testosterone, luteinizing hormone, and follicle-stimulating hormone, and decreased synthesis of testosterone by Leydig cells. Due to these anti-androgenic effects of DEHP on the reproductive system, the aim of this study was to examine whether substitutes such as acetyl triethyl citrate (ATEC) and acetyl tributyl citrate (ATBC) also damaged the reproductive system. In particular, this study investigated the anti-androgenic effects and cytotoxicity of DEHP substitutes using castrated male Sprague--Dawley rats employing the in vivo Hershberger assay and in vitro mouse Leydig (TM3) cells and mouse fibroblast (NIH-3T3) cell lines. In the Hershberger assay, rats were administered testosterone propionate and ATEC or ATBC at 20, 100, or 500 mg/kg b.w./day or DEHP (500 mg/kg b.w./day). Controls received testosterone antagonist flutamide (positive control), testosterone only (negative control), or corn oil only (vehicle control). ATEC/ATBC treatment produced no significant differences compared with testosterone in 5-androgen-dependent tissues weights including ventral prostate, seminal vesicles, levator ani-bulbocavernosus muscle, Cowper's glands, and glans penis. In the 500 mg/kg ATBC group, there was a significant reduction in liver weight. The MTT assay revealed that cell viability of both TM3 and NIH-3T3 cells treated with ATEC was not markedly altered. However, ATBC significantly reduced TM3 and NIH-3T3 cell viability in a concentration-dependent manner. Further, ATBC reduced cell viability to greater extent in TM3 versus NIH-3T3 cells. Based upon the observed effects of citrate ester substitutes on reproductive tissue responses and cytotoxicity, ATEC compared to ATBC may be a better alternative to DEHP for potential commercial uses. ABBREVIATIONS: ATEC: acetyl triethyl citrate; ATBC: acetyl tributyl citrate; CG: Cowper's glands; DEHP: di-2-ethylhexyl phthalate; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; GP: glans penis; LABC: levator ani-bulbocavernosus muscle; MTT: methyl tetrazolium; NC: negative control; NT: untreated control; PC: positive control; SV: seminal vesicle; TP: testosterone propionate; VC: vehicle control; VP: ventral prostate.

Risk Assessment of Ethylhexyl Dimethyl PABA in Cosmetics.

Ethylhexyl dimethyl para-aminobenzoic acid (PABA) is an oily yellow liquid derivative of water-soluble PABA commonly used in sunscreen. Ethylhexyl dimethyl PABA is widely used as an ingredient in many cosmetics at an average concentration of 1.25% (0.5-2.0%) in Korea. Previous studies, including those involving animals, have demonstrated that ethylhexyl dimethyl PABA is toxic to the following four organs: testis, epididymis, spleen, and liver. In addition, experiments using human keratinocytes found that ethylhexyl dimethyl PABA inhibits cell growth and DNA synthesis at low concentrations, and halted the cell cycle of MM96L cells (human melanoma cell line) at the G1 phase. Despite limited clinical data in humans, many studies have confirmed increased mutagenicity of ethylhexyl dimethyl PABA following exposure to sunlight, which suggests that this molecule is likely to contribute to onset of sun-induced cancer despite protecting the skin through absorption of UVB. For risk assessment, the no observed adverse effect level (NOAEL) chosen was 100 mg/kg bw/day in a 4 weeks oral toxicity study. Systemic exposure dosage (SED) was 0.588 mg/kg bw/day for maximum use of ethylhexyl dimethyl PABA in cosmetics. Based on the risk assessment and exposure scenarios conducted in this study, the margin of safety (MOS) was calculated to be 180.18 for a sunscreen containing 8% ethylhexyl dimethyl PABA, which is the maximum level allowed by the relevant domestic authorities.