Juventus chairman Andrea Agnelli says the European Super League (ESL) project cannot proceed as AC Milan, Inter Milan and Atletico Madrid joined the six Premier League clubs in withdrawing.
Agnelli was one of the chief architects of the breakaway plans, which involved 12 clubs from England, Spain and Italy.
Barcelona and Real Madrid are yet to comment.
"To be frank and honest no, evidently that is not the case," said Agnelli, on whether the ESL could still happen.
"I remain convinced of the beauty of that project, of the value that it would have developed to the pyramid, of the creation of the best competition in the world, but evidently no. I don't think that project is now still up and running."
Agnelli has been replaced as chairman of the European Club Association by Paris St-Germain president Nasser Al-Khelaifi, the Qatari who resisted repeated attempts to persuade the French club to become founder members of the breakaway league.
Atletico Madrid and Inter Milan announced their withdrawal on Wednesday morning, followed by statements from AC Milan and Juventus.
Agnelli was described as a "snake and a liar" by Uefa president Aleksander Ceferin on Monday after the announcement of the breakaway plans on Sunday evening.
The Juventus chairman resigned his position as chairman of the European Clubs' Association on Sunday and refused to take calls from Ceferin.
In a statement, Juventus said there were "limited chances that the project be completed in the form originally conceived" but that the club "remains convinced of the soundness of the project's sport, commercial and legal premises".
The six Premier League clubs involved all withdrew within hours of each other on Tuesday following a furious backlash against the plans.
Manchester City were the first club to pull out after Chelsea had signalled their intent to do so by preparing documentation to withdraw.
The other four English sides - Arsenal, Liverpool, Manchester United and Tottenham - then followed suit late on Tuesday evening.
In announcing their withdrawal on Wednesday, Atletico Madrid said "harmony is essential" between the club and the fans, and added that the first-team squad and coach Diego Simeone had backed their decision because "sporting merits must prevail over any other criteria".
In their statement, Serie A side Inter Milan said they were "committed to giving fans the best football experience", adding: "Our engagement with all stakeholders to improve the football industry will never change."
AC Milan said "the voices and the concerns of fans around the world have clearly been expressed" about the plans and the club "must be sensitive to the voice of those who love this wonderful sport".
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The 12-team Super League was announced on Sunday to widespread condemnation.
"Despite the announced departure of the English clubs, forced to take such decisions due to the pressure put on them, we are convinced our proposal is fully aligned with European law and regulations," the ESL said earlier on Wednesday, adding it was "convinced that the current status quo of European football needs to change".
"Real Madrid president Florentino Perez is insisting on the idea of keeping the group together to push for change," says Spanish football expert Guillem Balague.
"Barcelona say they agreed to the ESL, but only if the season ticket holders' assembly approve it, which could be their way out."
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Liverpool, Manchester United and Manchester City chiefs apologised to their supporters on Wednesday.
Ceferin welcomed the reversal by the clubs, saying: "I said yesterday that it is admirable to admit a mistake and these clubs made a big mistake.
"But they are back in the fold now and I know they have a lot to offer not just to our competitions but to the whole of the European game.
"The important thing now is that we move on, rebuild the unity that the game enjoyed before this and move forward together."
Speaking at prime minister's questions, UK prime minister Boris Johnson said the withdrawals were "the right result for football fans, for clubs and for communities across the country".
He also promised a "root-and-branch investigation" into football governance and what the government could do to "promote the role of fans in that governance".
Labour leader Sir Keir Starmer added the proposals "would have destroyed football" and that "we now need to get on with the other changes that are necessary".
The League Managers' Association (LMA) welcomed the decision of the clubs to withdraw, saying: "There is no place in our game for clandestine collusion, driven by opportunism, with such a blatant disregard for the history and integrity of our game."
The LMA also called for a club licensing system that sets out a "clear and unified governance structure, with consistent game-wide checks and balances".
"The future of football should be one that upholds the values of integrity and transparency. Owners, and those responsible for the direction of the game, must be more connected to, and significantly more accountable to the game itself," it added.
The Football Supporters' Association said "the cabal of billionaire owners overplayed their hand" and all efforts needed to be poured into the review of football governance in order to "rebalance the power structure of the domestic game".
In a statement, the Professional Footballers' Association praised players "who stood up for the game they love" and former players who "used their platforms effectively and often devastatingly" to "passionately articulate why the preservation of the domestic game is so important".
The English Football League said it hoped events over the past few days would lead to "much needed change to the distribution of wealth" in domestic leagues to ensure all clubs can have a "sustainable future".
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