In this article, you’ll learn how DOT Bottle “B” testing works, from the role of the Medical Review Officer (MRO), to how results are communicated to the employer, and even who is responsible for covering the cost of the additional testing. Understanding this process helps ensure both fairness for the donor and compliance for the employer.

DOT drug tests use a split specimen system: every urine collection is divided into two bottles (the “A” primary and the “B” backup) during the collection. The B bottle serves as a reserved backup sample. Its purpose is to protect both employer and employee by enabling an independent re-test if needed (for example, if the donor challenges a positive result).

In practice, the B bottle stays sealed and stored at the lab unless the donor or MRO specifically requests it. This split-sample safeguard helps ensure accuracy and fairness in DOT testing.

Non-Negative Result & MRO Review: If the A specimen yields a non-negative result (e.g. a screening positive requiring confirmation, or an invalid/adulterated finding), the lab performs confirmatory testing. If the confirmatory test is positive (or indicates adulteration/substitution), the Medical Review Officer (MRO) conducts an interview with the employee per 49 CFR §40.135–§40.145. The MRO then makes a verification decision. If the MRO verifies a positive result or a refusal due to adulteration/substitution, the DOT rules mandate informing the employee of their right to split-specimen testing. 

MRO Notifies Employee of Rights: Upon verification of a positive/adulterated/substituted result, the MRO must notify the donor (and explain the findings) and explicitly offer the split-specimen test. The MRO must tell the employee how to request it (provide phone/answering service contact) and that they have 72 hours from notification to make the request. The MRO also informs the donor that the employer will ensure the test occurs and that the employee does not have to pay up front

MRO Issues Final Result: The MRO reviews the split-specimen result and takes action by regulatory category. In most straightforward cases, if the B-sample reconfirms the original finding (Category 1), the MRO reports the reconfirmed positive or refusal to the employer and employee as the final result. If the B-sample fails to reconfirm the A-sample result, the MRO must cancel the test and notify the employer and donor that the test is cancelled. (Cancellation might trigger an immediate “direct-observation” recollection if tampering is suspected.) In rare cases where the B bottle is adulterated or invalid, the MRO follows special procedures (Categories 3–5) in 49 CFR §40.187, which generally result in cancellation and a new observed collection

Donors information:

72-Hour Request Window: Regulations require the MRO to give the donor 72 hours from notification to request split testing. During this period, the MRO must remain accessible. If the donor calls or writes in time, the split test proceeds as above. If the donor does not request within 72 hours, they may still present evidence of serious barrier (illness, etc.) to excuse the delay. If the MRO accepts this, the split test is still done. If not, no further action is taken by donor; the original verified result stands.

10-Day Finalization Rule: Under DOT rules (49 CFR §40.133(a)(3)), if the MRO cannot reach the employee despite reasonable efforts, the MRO may verify a positive or refusal result without an interview once 10 days have passed from the date the MRO received the laboratory report. This means the “10-day clock” applies to the MRO’s contact attempts—not as extra time for the employee to respond. In practice, if the MRO is unable to contact the employee within that 10-day period, they are permitted to finalize and report the result at that point.

Reporting Positive Results to the Clearinghouse:

Once a test is verified positive, DOT law requires that it be entered into the FMCSA Clearinghouse. Specifically, 49 CFR §382.705(a)(1) mandates that the MRO (not the employer) report verified positive drug test results within two business days of verification. In other words, after the MRO has completed verification (including any split-specimen analysis), the MRO must submit the driver’s name, SSN or CDL #, date of test, and positive result to FMCSA’s Clearinghouse. If the donor requested the B-bottle test, the MRO will typically delay the final verification until the B results arrive. In that case, the “verification date” is when the MRO closes the case after split analysis. Once reported, the employer need not separately report that same positive drug test to Clearinghouse – the MRO’s report covers it

When the MRO reports a positive drug test to the Clearinghouse, that’s all the employer needs to do – no extra reporting required from them.

Who Pays for B-Bottle Testing

Under DOT rules (49 CFR §40.173(a)), the employer bears financial responsibility. They are accountable for ensuring that the MRO, first laboratory, and second laboratory promptly perform split-specimen functions once an employee requests a split test. Employers cannot make testing conditional on employee payment; rules explicitly prohibit requiring employees to pay the lab or MRO, or even reimburse the employer, before the test. If an employee is unwilling or unable to pay, the employer must still proceed and cover the cost. While company policy or labor agreements might allow for partial recovery from the employee afterward, testing must not be delayed due to lack of funds.