Using LC-MS/MS to optimize the OECD 456 H295R steroidogenesis assay for sensitive and selective detection

7 June 2021

Assessing in vitro steroidogenesis endpoints is an important early step in establishing the endocrine disruptor (ED) potential of your crop protection product. Many regulators worldwide now require this information as part of the registration and approval process; for example, in the E.U., the updated pesticide and biocide directives now requests information on ED potential to be included. OECD test number 456 provides in vitro mechanistic information about the impact of a chemical on steroidogenesis and this can determine if subsequent in vivo studies are needed. A reliable, sensitive and selective analytic methodology for measuring steroid hormones is, therefore, a prerequisite for success with OECD 456 – read on to find out how such a method has been optimized by using high-end technology platforms.

Endocrine disruptor potential: focus on steroidogenesis

Endocrine disruption occurs when a chemical interferes with the body’s endocrine system to produce adverse effects such as impaired development or reproduction. The main systems at risk from endocrine disruption are those involving estrogen, androgen, thyroid hormones and steroidogenesis – collectively referred to as EATS modalities.

Steroidogenesis describes the production of steroid hormones, like estrogens and androgens, from cholesterol. Assaying a crop protection product’s impact on steroidogenesis can yield information about three EATS modalities.

The assessment of ED is generally based on a conceptual framework that organizes OECD test assays into five levels related to their biological complexity. Level 1 extrapolates information from existing data while Levels 2 and 3 involve tests to provide mechanistic data – Level 2 comprises in vitro assays, while Level 3 uses in vivo studies. Levels 4 and 5 yield system/population level data and the studies are often complex in nature. Identifying ED potential early in development – at Levels 1 or 2 of the OECD conceptual framework – is highly beneficial, as it allows you to direct subsequent higher-tier testing or stop further development of a substance with excess ED concerns.

The OECD 456 H295R steroidogenesis assay is the only Level 2 in vitro assay that examines steroidogenesis broadly – the aromatase assay (OPPTS 890.1200) only looks at one specific enzyme. OECD 456 detects substances that affect the production of 17β-estradiol (E2) and testosterone (T) to provide information for ED screening and prioritization.

Overview of the OECD 456 H295R steroidogenesis assay for ED screening and prioritization

The goal of the OECD TG 456 H295R steroidogenesis assay is to provide a ‘yes or no’ answer regarding the potential of a crop protection product or chemical to induce or inhibit T and E2 production. The results of the assay are expressed as relative changes in hormone production compared with the solvent control.

The in vitro H295R test system

A total of five different enzymes are responsible for the biosynthesis of sex steroid hormones. H295R is a human adreno-carcinoma cell line that expresses the genes encoding these key steroidogenesis enzymes. This means the H295R cell system produces all the steroid hormones found in the adult adrenal cortex and the gonads, making it a useful in vitro test system for steroidogenesis. Both increases and decreases in T and E2 production can be detected, and cytotoxicity occurring independent of any effects on steroidogenesis can be differentiated. In terms of limitations, H295R cells can produce gluco- and mineralo-corticoids, so changes in their production elicited by a test substance could influence levels of T and E2. In addition, ED potential resulting from effects on the hypothalamic-pituitary-gonadal axis cannot be ascertained from this assay.

OECD TG 456 design

Under standard cell culture conditions cells, 24 well plates are exposed for 48 hours to seven concentrations of the test chemical in triplicate. In parallel, two positive reference chemicals (prochloraz and forskolin) are run. These controls are known inducers and inhibitors of hormone production.

At the end of the exposure period, cell viability is assessed using MTT and the cell medium is analyzed for hormone concentrations.

The assay was validated using 5 proficiency chemicals:

  • Forskolin: strong inducer
  • Prochloraz: strong inhibitor
  • Atrazine: moderate inducer
  • Aminoglutethimide: moderate inhibitor
  • Bisphenol A
    • weak inducer (E2 production)
    • weak inhibitor (T production)
  • Benomyl: negative chemical (this was used instead of human chorionic gonadotrophin which is a controlled drug and potentially difficult to obtain).

Measurement of hormone concentration

The OECD 456 test guideline does not specify how hormone concentrations should be assessed; it simply highlights that a variety of methods are available. It does specify that results should be expressed as fold changes relative to the solvent control and should report the Lowest-Observed-Effect-Concentration (LOEC). If the assay is negative, No-Observed-Effect-Concentration (NOEC) – the highest concentration tested – should be reported.

OECD TG 456 also specifies that the test used must meet predefined performance criteria including having a limit of quantification (LOQ) of 100 pg/mL for T and 10 pg/mL for E2.

The methods commonly used for hormone analysis include ELISA and LC-MS. Whilst ELISA is rapid and sensitive for most routine applications, it does lack specificity and accuracy, showing cross-reactivity between structurally similar compounds. There have been numerous anecdotal reports of the lack of reliability in the performance of ELISAs for hormone assessment for OECD TG 456. In an effort to enhance analytical assessment of T and E2, the Bioanalysis team has developed and validated an LC-MS/MS methodology with higher sensitivity, selectivity and reliability than the existing ELISA methodology. Working collaboratively with the in vitro assessment team, this offers dedicated, flexible capacity for running robust OECD TG 456 assays. 

LC-MS/MS analytical approach for hormone assessment for the OECD TG 456 H295R steroidogenesis assay

There were multiple goals in developing LC-MS/MS methodology:

  • Develop a highly sensitive and selective assay for T and E2 that can measure to very low concentration levels – beyond those required by the OECD TG 456
  • Validate the methodology by demonstrating:
    • it is precise, accurate, selective and reliable
    • it is appropriate to support requirements for OECD TG 456

LC-MS/MS hormone bioanalytical test development

The assay was developed using API4500 mass spectrometer and the methodology was optimized to achieve the parameters as shown in Table 1.

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In an effort to further push the lower LLOQ for estradiol (5.00 pg/mL) to support the variability in estradiol levels in the event of downregulation, we utilized an API 6500+ mass spectrometer with SelexION, which has a differential mobility separation (DMS) device that provides an extra level of selectivity to minimize the matrix effect and achieve higher confidence in selectivity and sensitivity.

Validation of the LC-MS/MS bioanalytical methodology for T and E2 to support OECD TG 456

The first part of the validation of the LC-MS/MS methodology focused on the LC-MS/MS methodology and validated precision and accuracy, selectivity, linearity and sample storage stability.

In line with the OECD TG 456 requirements, the test was also validated using the six chemicals known to induce and inhibit hormone production listed previously. The samples generated in OECD TG 456 H295R steroidogenesis assay were analyzed using validated LC-MS/MS methodology and all the OECD TG 456 requirements were met.

Application and benefits of the LC-MS/MS hormone analysis methodology for OECD TG 456

This bioanalytical methodology is now standard practice for OECD TG 456 for all crop protection products and chemicals. The test is reliable, accurate, sensitive and selective, which are distinct analytical advantages over ELISA. It also does not suffer from the long lead times, batch-to-batch variability or reagent accessibility issues which are common with such immunoassays. This LC-MS/MS hormone analysis methodology for OECD TG 456 also benefits from being performed by a dedicated team of bioanalysts working in close collaboration with the in vitro safety assessment team. This joined up, collaborative approach optimizes outcomes for your studies – given that results from this in vitro study will direct subsequent expensive higher-level studies, you need the data to be robust, reliable and timely.


This LC-MS/MS validated method for the measurement of low concentrations of the steroid hormones T and E2 is accurate, sensitive and selective. It provides important information on the impact of a crop production product on steroidogenesis without the need to rely on special reagent and kit availability. In addition, the shorter total analysis times mean you receive this important mechanistic in vitro data faster, so you can determine the need for subsequent in vivo testing on ED potential more rapidly.