The amputee star, who touched first for what would have been first place at the SA championships, appealed against the decision.
But one official said the appeal had been unanimously rejected by a six-man committee. Du Toit effectively swam her nearest rival, Michelle Webber, out of space in the final sprint for the touch pad.
The swimmer, 28, explained that she had intentionally swum in her opponent's slip, and each time Webber had moved to her right, she had followed her to stay in the slip.
"It's allowed," said Du Toit, who yesterday started competing in the pool events to seal her selection for the Paralympics.
"The men's race at the Beijing Olympics (in 2008) was won like that. I didn't hit her, I didn't scratch her. It's been done to me all the time in international races.
"It's about finding your line, it's finding your position. I walked away from that race thinking I can actually swim internationally now," added Du Toit, who competed at the 2008 Olympics.
She will attempt to qualify for the women's 10km open-water race at the London Olympics in Portugal in June.
"I don't understand it. At the end of the day I don't know what I did."
Du Toit, who plans to retire after the Paralympics, attended her first senior SA championships in 1997 and missed only one national gala, after her accident in 2001.
Meanwhile, rising swim star Chad le Clos is too competitive to play down his chances of an Olympic medal.
The 20-year-old is being touted as SA swimming's great medal hope for the 2016 Olympics in Rio de Janeiro. London is about gaining experience more than anything.
And Le Clos likes to point out that he still has to qualify for the Olympics.
"The most important thing for me is to get out there this week and get times," he said after his 400m individual medley heat at the SA championships yesterday.