2-Hydroxy-4-methoxybenzophenone (2H4MBP), also known as oxybenzone and benzophenone‑3, is approved by the U.S. Food and Drug Administration for use in sunscreens and other personal care products in concentrations of <6%, either alone or in combination formulations, and as an indirect food additive in acrylic and modified acrylic plastics that come into contact with food. Mechanistic screening studies have shown that 2H4MBP and its metabolites are capable of activating the estrogen receptor and antagonizing the androgen receptor to varying degrees. The objective of the present study was to characterize the potential for 2H4MBP to adversely affect any phase of development, maturation, and ability to reproduce in Sprague Dawley (Hsd:Sprague Dawley SD) rats administered 2H4MBP in 5K96 feed, a diet low in phytoestrogens, using the National Toxicology Program (NTP) modified one-generation (MOG) study design. 2H4MBP exposure via diet, rather than topical application, was selected for this study to sustain internal exposure; if applied topically, the internal dose would have been influenced by intra- and interanimal grooming behavior. Exposure concentrations were based on a dose range-finding study that demonstrated 25,000 ppm 2H4MBP did not induce excessive maternal toxicity or affect parturition, litter size, or pup viability. 2H4MBP intake by F0 females in the 3,000, 10,000, 25,000, and 50,000 ppm 2H4MBP groups, based on feed consumption and dietary concentrations from gestation day (GD) 6 through GD 21, was approximately 215, 695, 2,086, and 6,426 mg 2H4MBP/kg body weight/day (mg/kg/day), respectively; from lactation day (LD) 1 through LD 14, 2H4MBP intake was approximately 577, 1,858, 4,460, and 12,029 mg/kg/day, respectively. Exposure concentrations of 3,000, 10,000, and 30,000 ppm were selected for the subsequent MOG study; ethinyl estradiol (EE), a synthetic form of estrogen, was included at 0.05 ppm as a positive reference control.
Modified one-generation study
F0 exposure began on GD 6 and was continual. At weaning on postnatal day (PND) 28, F1 offspring were assigned to either reproductive performance (2/sex/litter), prenatal (1/sex/litter), or biological sampling (1/sex/litter) cohorts. Upon sexual maturity, F1 mating and pregnancy indices were evaluated. In the prenatal cohort, F2 prenatal development (litter size, fetal weight, and morphology) was assessed on GD 21. In the reproductive performance cohort, littering indices, F2 viability, and growth were assessed until PND 28. The likelihood of identifying potential 2H4MBP-induced adverse effects (similarity and magnitude thereof) at any phase of growth or development was increased by examining related endpoints in multiple pups within a litter throughout life, across cohorts, and across generations.
2H4MBP exposure at the tested concentrations did not induce any effects on mating or pregnancy indices. In the prenatal cohort, exposure to 30,000 ppm was associated with a slight but significant decrease in the mean numbers of corpora lutea and F2 implants and a slightly lower number of live fetuses on GD 21 than in the control group. In the reproductive performance cohort, total F2 mean litter size on PND 0 was also significantly decreased compared to the control group. 2H4MBP exposure might have affected litter size, although the effect was small in magnitude. Collectively, given the minimal apparent response that may or may not be a direct effect of 2H4MBP, this was considered equivocal evidence of an adverse effect on reproductive performance. EE exposure did not affect F1 live litter size on PND 0, but significantly decreased mean number of corpora lutea and total F2 implants were observed.
2H4MBP was associated with lower F1 and F2 preweaning and F1 postweaning mean body weights. At 30,000 ppm 2H4MBP, preweaning F1 mean body weights of both males and females were progressively lower over time, relative to their respective control groups. The response was lessened in F2 males and even more so in F2 females. The significantly decreased F1 postweaning mean body weights were not associated with concurrent lower feed consumption. The effects on body weights associated with exposure to 2H4MBP were considered some evidence of developmental toxicity. 2H4MBP intake by F0 females in the 3,000, 10,000, and 30,000 ppm 2H4MBP groups, based on feed consumption and dietary concentrations from GD 6 through GD 21 was approximately 205, 697, and 2,644 mg/kg/day, respectively; from LD 1 through LD 13, 2H4MBP intake was approximately 484, 1,591, and 5,120 mg/kg/day, respectively. 2H4MBP intake by the F1 generation postweaning (PND 28 through PND 91) in the 3,000, 10,000, and 30,000 ppm groups was approximately 267, 948, and 3,003 mg/kg/day (males) and 287, 983, and 3,493 mg/kg/day (females), respectively. 2H4MBP intake by the adult F1 females in the 3,000, 10,000, and 30,000 ppm groups was approximately 240, 825, and 2,760 mg/kg/day (GD 0 through GD 21) and 426, 1,621, and 5,944 mg/kg/day (LD 1 through LD 13), respectively.
Diaphragmatic hernias were observed at a low incidence in 2H4MBP-exposed animals in both the F1 and F2 generations but were not observed in any control animals. Most of the diaphragmatic hernias were associated histologically with hepatodiaphragmatic hernias. Low incidences of diaphragmatic and hepatodiaphragmatic hernias have been reported in control groups in other NTP MOG studies. Therefore, it is unclear whether the occurrences of diaphragmatic and hepatodiaphragmatic hernias in both the F1 and F2 generations were related to 2H4MBP exposure.
2H4MBP did not alter estrogen or androgen-mediated developmental markers, and no gross lesions were observed at adult necropsy consistent with perturbation of normal estrogen receptor- or androgen-receptor-mediated development. Expected estrogenic responses were observed in the EE group. In the 30,000 ppm group, adult weights of male androgen-dependent reproductive tissues were slightly lower than those of the control males, likely secondary to the apparent growth retardation, and occurred in the absence of histopathological findings. Sperm and spermatid counts were not affected by 2H4MBP exposure. The ability of F1 males in either cohort to successfully mate, resulting in pregnancy, also was not affected. Unlike findings reported for in vitro cell models, 2H4MBP had no apparent effect on estrogen receptor- or androgen-receptor-dependent processes, nor did it affect mating or pregnancy indices.
2H4MBP exposure in F1 rats was associated with significantly increased kidney weights, renal tubule epithelial regeneration, interstitial chronic active inflammation, renal tubule and pelvic concretions, renal tubule dilation, papillary necrosis, urothelial hyperplasia, and urothelial ulcers. F1 females also displayed renal tubule epithelial degeneration, pelvic dilation, chronic progressive nephropathy, and mineralization. 2H4MBP-exposed F1 males and females displayed significantly increased liver weights relative to their respective control groups. The absolute weight of the adrenal glands was significantly decreased in the 30,000 ppm female group relative to the control group in the reproductive performance cohort. Several other decreases in organ weights were not associated with histological correlates and were considered related to changes in body weights.
F2 fetal findings of hydronephrosis of the kidney and enlarged liver were observed in the 30,000 ppm group. F2 offspring in the 30,000 ppm group exhibited dilation of the renal pelvis. The observed fetal, PND 28, and adult necropsy findings were consistent with previously reported studies that identified the kidney and liver as target tissues of 2H4MBP-mediated toxicity.
Conclusions
Under the conditions of this modified one-generation (MOG) study, there was equivocal evidence of reproductive toxicity of 2-hydroxy-4-methoxybenzophenone (2H4MBP) in Hsd:Sprague Dawley SD rats based on a decrease in F2 litter size in both the prenatal and reproductive performance cohorts.
Under the conditions of this MOG study, there was some evidence of developmental toxicity of 2H4MBP in Hsd:Sprague Dawley SD rats based on the observed postnatal growth retardation. The relationship of the increased occurrence of diaphragmatic and hepatodiaphragmatic hernias in F1 adults and F2 pups to 2H4MBP exposure is unclear.
Exposure to 2H4MBP was not associated with signals consistent with alterations in estrogenic, androgenic, or antiandrogenic action. Exposure to 2H4MBP was associated with lower F1 and F2 mean body weights; this effect on body weight contributed to the apparent 2H4MBP-related decreases in male reproductive organ weights. Mating and littering were not significantly affected by 2H4MBP exposure. Exposure to 2H4MBP was associated with nonneoplastic kidney lesions in the F0, F1, and F2 generations. Expected estrogenic responses were observed in the EE group.
National Toxicology Program (NTP). 2022. NTP developmental and reproductive toxicity technical report on the modified one-generation study of 2-hydroxy-4-methoxybenzophenone (CASRN 131-57-7) administered in feed to Sprague Dawley (Hsd:Sprague Dawley SD) rats with prenatal and reproductive performance assessments in F1 offspring. Research Triangle Park, NC: National Toxicology Program. DART Report 05. https://doi.org/10.22427/NTP-DART-05