Halep was first suspended in October 2022 for testing positive for the banned substance roxadustat, which stimulates the production of red blood cells, at the US Open in August last year.
The ITIA, on Friday said the two-time Grand Slam winner has been further charged “relating to irregularities in her athlete biological passport”.
The athlete biological passport programme monitors biological data over time to identify potential doping violations.
Nicole Sapstead, the ITIA’s senior director for anti-doping, said: “We understand that today’s announcement adds complexity to an already high-profile situation.
“From the outset of this process – and indeed any other at the ITIA – we have remained committed to engaging with Ms Halep in an empathetic, efficient, and timely manner.”
Halep, 31, said after her positive test last October that she would “fight until the end” to clear her name.
“Knowingly, I have not taken any banned substance,” the Romanian star told tennismajors.com in an interview published last month.
“I am a big supporter of clean sports and have always been against doping. I had no clue at the beginning about where this substance came from.”
On Friday, Halep said on Instagram that she felt “helpless facing such harassment” and denied the charges, suggesting she has been “a victim of a contamination”.
“I have lived the worst nightmare I have ever gone through in my life. Not only has my name been soiled in the worst possible way, but I am facing a constant determination from the ITIA, for a reason that I cannot understand, to prove my guilt while I haven’t ever thought of taking an illicit substance.”
Halep said she hoped to have the chance to prove her innocence at a hearing scheduled for the end of May.
Halep won the French Open in 2018 and Wimbledon the following year.
She had climbed back into the world’s top 10 before she was suspended, having dropped out of the top 20 in 2020 following injury problems.
The ITIA said it was continuing discussions with an independent tribunal and Halep’s representatives.