A mixture of 15 phthalates and pesticides below individual chemical No Observed Adverse Effect Levels (NOAELs) produces reproductive tract malformations in the male rat

Humans carry residues of multiple synthetic chemicals at any given point in time. Research has demonstrated that compounds with varying molecular initiating events (MIE) that disrupt common key events can act in concert to produce cumulative adverse effects. Congenital defects of the male reproductive tract are some of the most frequently diagnosed malformations in humans and chemical exposures in utero can produce these effects in laboratory animals and humans. Here, we hypothesized that in utero exposure to a mixture of pesticides and phthalates, each of which produce male reproductive tract defects individually, would produce cumulative effects even when each chemical is present at a no observed adverse effect level (NOAEL) specific for male reproductive effects. Pregnant Sprague-Dawley rats were exposed via oral gavage to a fixed-ratio dilution mixture of 5 pesticides (vinclozolin, linuron, procymidone, prochloraz, pyrifluquinazon), 1 pesticide metabolite (dichlorodiphenyldichloroethylene (DDE)), and 9 phthalates (dipentyl, dicyclohexyl, di-2-ethylhexyl, dibutyl, benzyl butyl, diisobutyl, diisoheptyl, dihexyl, and diheptyl) during the critical window of rat fetal masculinization (gestation day 14–18). The top dose (100% dose) contained each compound at a concentration 2-fold greater than the individual chemical NOAEL followed by a dilution series that represented each chemical at NOAEL, NOAEL/2, NOAEL/4, NOAEL/8, NOAEL/15, NOAEL/100, NOAEL/1000. Reduced fetal testis gene expression occurred at NOAEL/15, reduced fetal testis testosterone production occurred at NOAEL/8, reduced anogenital distance, increased nipple retention, and delayed puberty occurred at NOAEL/4, and severe effects including genital malformations and weight reductions in numerous reproductive tissues occurred at NOAEL/2. This study demonstrates that these phthalates and pesticides acted cumulatively to produce adverse effects at doses below which any individual chemical had been shown to produce an effect alone and even though they have different MIEs.

Impact/Purpose

Our research group has published several research studies on the combined effects of in utero exposure to chemicals that have converging Key Events within an androgen receptor signaling Adverse Outcome Pathway network. This manuscript describes recent experiments within this line of inquiry with a study design based on individual chemical No Observed Adverse Effect Levels (NOAELs). We hypothesized that a broad mixture (15 compounds) of phthalates and pesticides would produce permanent, adverse male reproductive effects when each chemical was administered at or below a dose at which the individual chemicals produce no effects in isolation (i.e., below NOAELs). We conducted experiments with pregnant female rats to assess effects on androgen signaling and reproductive tract development in offspring at fetal, newborn, juvenile, and adult time points. We found that a complex mixture of 15 anti-androgenic phthalates and pesticides with at least 3 different MIEs produced permanent malformations in F1 male reproductive tissues when each chemical was present at a dose which had been individually shown to produce no adverse male reproductive effects. This paper demonstrates the importance of assessing the toxicity for multiple chemicals with overlapping Key Events in an AOP network. We found that when individual chemicals that disrupt androgen receptor signaling via multiple MIEs are administered as a mixture to pregnant female rats during the critical window of fetal development of the reproductive tract, permanent adverse effects occur in the offspring. Critically, these effects occur at doses lower than those at which the individual chemicals have been shown to produce effects in individual exposures.

Citation

Conley, J., C. Lambright, N. Evans, M. Cardon, E. MedlockKakaley, V. Wilson, AND E. Gray. A mixture of 15 phthalates and pesticides below individual chemical No Observed Adverse Effect Levels (NOAELs) produces reproductive tract malformations in the male rat. Elsevier B.V., Amsterdam, NETHERLANDS, 156(106615):1, (2021). [DOI: 10.1016/J.ENVINT.2021.106615]

DOI: A mixture of 15 phthalates and pesticides below individual chemical No Observed Adverse Effect Levels (NOAELs) produces reproductive tract malformations in the male rat