The OECD validation program of the H295R steroidogenesis assay: Phase 3. Final inter-laboratory validation study

Area of Science:

• Endocrinology
• Toxicology
• In vitro assay development

Background:

• Growing concerns over endocrine-disrupting chemicals (EDCs) in humans and wildlife.
• Initiation of national and international programs to develop new testing guidelines for EDCs.
• Need for validated in vitro assays to screen chemicals for endocrine activity.

Purpose of the Study:

• Validate the H295R steroidogenesis assay for detecting chemicals that inhibit or induce testosterone (T) and 17β-estradiol (E2) production.
• Prepare for the development of an Organization for Economic Cooperation and Development (OECD) test guideline.
• Assess the reliability and performance of the assay across multiple laboratories.

Main Methods:

• Utilized a pre-validated protocol to test 28 diverse chemicals using the H295R steroidogenesis assay.
• Involved seven international laboratories, assessing effects on T and E2 production.
• Evaluated confounding factors including cell viability and direct interference with antibody-based assays.

Main Results:

• The H295R assay successfully identified most known and unknown endocrine-active chemicals.
• High assay reliability with only 2% (T) and 7% (E2) of experiments excluded.
• Demonstrated low false positive (1/28 for T and E2) and no false negative rates.

Conclusions:

• The H295R steroidogenesis assay is a reliable in vitro screen for chemicals altering sex steroid hormone production.
• The assay shows potential for identifying chemicals affecting hormone homeostasis, comparable to in vivo studies.
• A revised protocol led to the development of an OECD draft test guideline.