Why Are a Minimum of 12 Subjects Required in a Bioequivalence (BE) Study?

When it comes to designing a pivotal BE study, one number stands out: 12 evaluable subjects. But why is this the golden minimum?

Regulatory Standard

 Agencies like the USFDA, EMA, and WHO require ≥12 subjects for crossover studies (or 12 per group for parallel design) to ensure the robustness of BE data.

Statistical Validity

This minimum helps ensure the study has ≥80% statistical power to detect differences in PK parameters like AUC and Cmax within the accepted 0.80–1.25 range.

Variability Matters

High intra-subject variability? You may need more than 12 subjects to maintain confidence in your results.

Pilot Studies Guide the Way

Data from pilot or relative bioavailability studies help fine-tune the sample size calculation for pivotal trials.

Avoiding Underpowered Studies

Fewer than 12 subjects? That's a red flag —you risk Type II errors, missing real differences between test and reference products.

80% Power Is the Benchmark

It’s the probability your study will correctly demonstrate equivalence—and it’s a must for regulatory acceptance.

Post-Hoc Power? Not Valid

A posteriori calculations won’t save you. Your sample size must be justified in advance—based on variance and expected ratios.

Confidence Interval Precision

More subjects = narrower 90% confidence intervals = better chance your BE data falls within the regulatory window.

Conclusion

While 12 subjects is the regulatory baseline, a solid BE study must go beyond the minimum, accounting for variability, design, and dosing strategy. It’s all about ensuring credible, reproducible, and approvable results.

Read also: Pilot BE Study of Pharmaceuticals

Resource Person: Vijay Agrawal