"The British policymakers and British voters have to know that there will not be any kind of renegotiation," Juncker told reporters in Brussels.
Juncker said the EU would not go further than the renegotiation deal that Prime Minister David Cameron sealed with his counterparts at a summit in February.
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"We have concluded a deal with the prime minister, he got the maximum he could receive, we gave the maximum we could give," Juncker said after talks with new Austrian Chancellor Christian Kern.
"So there will be no kind of renegotiation, nor on the agreement we found in February, nor as far as any kind of treaty negotiations are concerned.
"Out is out."
Cameron had suggested on Wednesday that he might push for further reforms, especially on free movement, if Britain votes to stay in the EU in Thursday's referendum.
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