Decrease, circulating testosterone levels

Testosterone is an endogenous steroid hormone that acts by binding the androgen receptor (AR) in androgen-responsive tissues (Murashima et al., 2015). As with all steroid hormones, testosterone is produced through steroidogenesis, an enzymatic pathway converting cholesterol into all the downstream steroid hormones. Briefly, androstenedione or androstenediol is converted to testosterone by the enzymes 17β-hydroxysteroid dehydrogenase (HSD) or 3β-HSD, respectively. Testosterone can then be converted to the more potent androgen, dihydrotestosterone (DHT) by 5α-reductase, or aromatized by CYP19A1 (Aromatase) into estrogens. Testosterone secreted in blood circulation can be found free or bound to SHBG or albumin (Trost & Mulhall, 2016).

Testosterone is produced mainly by the testes (in males), ovaries (in females) and to a lesser degree in the adrenal glands. The output of testosterone from different tissues varies with life stages. During fetal development testosterone is crucial for the differentiation of male reproductive tissues and the overall male phenotype. In adulthood, testosterone synthesis is controlled by the Hypothalamus-Pituitary-Gonadal (HPG) axis. GnRH is released from the hypothalamus inducing LH pulses secreted by the anterior pituitary. This LH surge leads to increased testosterone production, both in testes (males) and ovaries (females). If testosterone reaches low levels, this axis is once again stimulated to increase testosterone synthesis. This feedback loop is essential for maintenance of appropriate testosterone levels (Chandrashekar & Bartke, 1998; Ellis et al., 1983; Rey, 2021).

By disrupting e.g. steroidogenesis or the HPG-axis, testosterone synthesis or homeostasis may be disrupted and can lead to less testosterone being synthesized and released into circulation.

General role in biology

Androgens are essential hormones responsible for the development of the male phenotype during fetal life and for sexual maturation at puberty. In adulthood, androgens remain essential for the maintenance of male reproductive function and behavior but is also essential for female fertility. Apart from their effects on reproduction, androgens affect a wide variety of non-reproductive tissues such as skin, bone, muscle, and brain (Heemers et al 2006). Androgens, principally testosterone and DHT, exert most of their effects by interacting with the AR (Murashima et al 2015).

Testosterone levels can be quantified in serum (in vivo), cell culture medium (in vitro), or tissue (ex vivo, in vitro). Methods include traditional immunoassays such as ELISA and RIA, advanced techniques like LC-MS/MS, and liquid scintillation spectrometry following radiolabeling (Shiraishi et al., 2008).

The H295R Steroidogenesis Assay (OECD TG 456) is (currently; anno 2025) primarily used to measure estradiol and testosterone production. This validated OECD test guideline uses adrenal H295R cells, with hormone levels measured in the cell culture medium (OECD, 2011). H295R adrenocortical carcinoma cells express the key enzymes and hormones of the steroidogenic pathway, enabling broad analysis of steroidogenesis disruption by quantifying hormones in the medium using LC-MS/MS. Initially designed to assess testosterone and estradiol levels, the assay now extends to additional steroid hormones, such as progesterone and pregnenolone. The U.S. EPA’s ToxCast program further advanced this method, enabling high-throughput measurement of 11 steroidogenesis-related hormones (Haggard et al., 2018). While the H295R assay indirectly reflects disruptions in overall steroidogenesis (e.g., changes in testosterone levels), it does not provide mechanistic insights.

Testosterone can be measured by immunoassays and by isotope-dilution gas chromatography-mass spectrometry in serum (Taieb et al., 2003; Paduch et al., 2014). Testosterone levels may also be measured by: Fish Lifecycle Toxicity Test (FLCTT) (US EPA OPPTS 850.1500), Male pubertal assay (PP Male Assay) (US EPA OPPTS 890.1500), OECD TG 441: Hershberger Bioassay in Rats (H Assay).

This key event (KE) is applicable to all mammals, as the synthesis and role of testosterone are evolutionarily conserved (Vitousek et al., 2018). Both sexes produce and require testosterone, which plays critical roles throughout life, from development to adulthood; albeit there are differences in lief stages when testosterone exert specific effects and function (Luetjens & Weinbauer, 2012; Naamneh Elzenaty et al., 2022). Accordingly, this KE applies to both males and females across all life stages, but life stage should be considered when embedding in AOPs.

Notably, the key enzymes involved in testosterone production first appeared in the common ancestor of amphioxus and vertebrates (Baker, 2011). This suggests that the KE has a broader domain of applicability, encompassing non-mammalian vertebrates. AOP developers are encouraged to integrate additional knowledge to expand its relevance beyond mammals to other vertebrates.

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