SN/T 5156-2019 in English
VALIDChemicals- H295R steroidogenesistest method
- Issued on:2019-10-25
- Implemented on:2020-05-01
- File Format:PDF
- Delivery:Via email within 1~3 business days
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Introduction
Standard Overview and Technical Background
SN/T 5156-2019 standard is based on OECD Test Guide No.456 (2011) and uses H295R cell line to evaluate the interference of chemicals on steroid hormone synthesis. This cell model can simultaneously detect changes in the synthesis of 17β-estradiol (E2) and testosterone (T), and is suitable for screening the endocrine disrupting activity of environmental chemicals.
Core Test Principles
| Detection Indicators | Key Enzyme Systems | Interference Mechanisms |
|---|---|---|
| 17β-estradiol | CYP19 aromatase | Affects the conversion of androgens to estrogens |
| Testosterone | CYP17 lyase | Interferes with the metabolic pathway of pregnenolone |
Typical Application Cases
In the evaluation of bisphenol A, H295R cells showed that 1 μM exposure increased E2 production by 30% and inhibited testosterone synthesis, indicating that it has estrogen-like effects and anti-androgenic activity.
Key points of the experimental operation
- Cell culture: Use DMEM/F12 medium (containing 1.2g/L NaHCO3, 15mM HEPES), maintain at 37℃, 5% CO₂ conditions
- Exposure treatment: The test compound must be dissolved in DMSO (final concentration ≤0.1%), and at least 7 concentration gradients should be set
- Quality control: Each plate must contain solvent control, positive control (thiram/pregnenolone) and QC samples
Data analysis requirements
| Parameters | Acceptance criteria | Statistical methods |
|---|---|---|
| LOEC | ≤30% coefficient of variation | Dunnett's test |
| Cell viability | ≥80% survival rate | MTT assay |
Implementation recommendations
Notes:
- The activities of key enzymes such as CYP11A and CYP17 need to be detected simultaneously to confirm the target
- LC-MS/MS method is recommended to improve the sensitivity of hormone detection
- For poorly soluble substances, β-cyclodextrin can be considered as a cosolvent
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