Former EU Commission President Jose Manuel Barroso says a second referendum is "unthinkable"
David Cameron has reportedly been told that he will face a leadership challenge unless he tones down his attacks on Boris Johnson and other "Out" campaigners.
Tory ministers and senior backbenchers have even threatened a "no confidence" vote if the Prime Minister fails to halt "blue on blue" attacks on fellow Conservatives, according to the Sunday Times.
A senior backbencher told the newspaper they would have "no problem" getting the 50 signatures needed to force a vote, adding: "Cameron's position will be untenable even if he wins the referendum if he carries on like this."
It comes after confirmation the Foreign Secretary Philip Hammond swore at one leading Tory Eurosceptic in a row over a confidential EU document.
Conservative divisions over the EU have deepened after Mr Hammond called a leading Eurosceptic a "total s***".
Sky News understands the Foreign Office has referred Sir Bill Cash to the parliamentary authorities following an alleged "abuse" of House of Commons rules over his handling of a confidential EU document.
Sir Bill is a key figure in the Grassroots Out organisation, which is campaigning for the UK to leave the European Union.
The row between Philip Hammond - who is campaigning for the UK to remain in the EU - and Sir Bill Cash, a member of the Grassroots Out organisation, bubbled over after the Foreign Secretary appeared in front of the European Scrutiny Committee, which is chaired by Sir Bill.
Labour MP Kate Hoey confirmed to Sky News that she overheard Mr Hammond swearing at Bill Cash and saying "you deliberately ignored me".
Mr Hammond was apparently angry that Sir Bill published a Brussels document confirming the Prime Minister's deal was legally binding, after the Foreign Secretary had asked him to keep it confidential.
A Foreign Office source confirmed they will be referring the incident to parliamentary authorities, adding: "The legal advice in question confirms that the deal the Prime Minister secured is legally binding and irreversible.
"The wider issue is whether Parliamentary authorities may not look into the issue of a Committee breaching longstanding rules in place that allow for the Government to provide sought-after advice, in confidence, to better inform committees of Parliament.
"This is a process matter, not an issue of how the Government’s EU deal is legally binding, that has already been recognised."
Second referendum 'unthinkable'
Britain will have only one chance to vote in a referendum on its membership of the European Union, the former President of the European Commission has told Sky News.
Some high profile campaigners have suggested that voting "Out" could strengthen the UK's negotiating hand ahead of a potential second referendum.
But Jose Manuel Barroso, a Portuguese politician who served as EC president from 2004 until 2014, told Sky's Dermot Murnaghan: "I don't believe that (a second referendum) could happen.
"That would be really ... unthinkable. I would say it's practically impossible.
"British people have the final word. If they want to remain they are most welcome, but if the British people want to leave the European Union, we have to respect that."
Former Conservative Party leader Michael Howard has previously suggested that an Out vote would not be binding.
He told Sky News: "The EU does have form on this. They went back to the people of Ireland and Denmark when they voted in a way that wasn’t to the liking of Europe's leaders."
London Mayor Boris Johnson also implied a vote to leave the EU could strengthen Britain's negotiating hand. He later rowed back on that position.
Unusually, the comments of Europhile Mr Barroso were welcomed by the UK Independence Party.
Suzanne Evans told Sky News the idea of a second referendum was "nonsense".
She added: "I'm quite pleased to hear Barroso says 'once is it, and whichever it goes, that's the way it is'.
"I think if we get a leave vote in this referendum, and I'm absolutely convinced we can, I'm convinced all the cards are coming our way, then out must mean out."
In a rare moment of agreement between the two rival camps, Alan Johnson of Labour's pro-EU campaign also said there would be "no second chances, it will be decisive".
Prime Minister David Cameron said last week that the referendum on the UK's future in the EU, to be held on 23 June, would be "final".
Speaking in the Commons, he said: "Sadly, Mr Speaker, I have known a number of couples who have begun divorce proceedings, but I do not know of any who have begun divorce proceedings in order to renew their marriage vows."