The match between England and the Barbarians at Twickenham on Sunday has been cancelled due to a breach of coronavirus protocols by Baa-Baas players.
The Rugby Football Union (RFU) investigated after members of the squad left the team hotel without permission or informing organisers about their whereabouts on Wednesday.
The players in question were separated from the rest of the group in the bubble upon their return.
However, as part of its investigation, the RFU confirmed on Friday that an earlier undisclosed breach of the COVID-19 regulations took place on Tuesday, with Barbarians players leaving the hotel without permission and not isolating from team-mates after they returned.
"The RFU has concluded that the players leaving their COVID-secure environment on October 20 without then isolating from the rest of the group on their return has resulted in the bubble environment being compromised, with the potential risk of COVID transmission from individuals outside the bubble to everyone in the Barbarians team and management," the RFU said in a statement.
"The RFU is therefore left with no alternative but to cancel the game."
This Sunday's Quilter Cup fixture has been cancelled following several breaches of the Covid code of conduct within the Barbarians squad.— England Rugby (@EnglandRugby) October 23, 2020
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CEO Bill Sweeney said: "We are incredibly disappointed to be calling a halt to this fixture, we know how much fans were looking forward to seeing the teams play. However, our priority is to protect the health and safety of the England squad and the other international teams they will go up against this autumn.
"There has been a great deal of effort put into COVID codes of conduct and planning for games, including cooperation with Premiership clubs to release additional players to fulfil the fixture safely, and we are all incredibly frustrated and disappointed that the actions of a number of Barbarians players mean we no longer feel it is safe for the game to go ahead."
The RFU says it will not release information on the players involved until their investigation is completed.
England's 2003 World Cup-winning coach Clive Woodward was left in "disbelief" at the behaviour of those concerned.
"I'm sure they're feeling incredibly stupid and incredibly embarrassed. It's just stupidity, they've been ridiculously stupid," Woodward told talkSPORT.
"My reaction was just kind of gobsmacked really. These are professional players and they know what's at stake. It's live on TV, it's a game that everyone was looking forward to.
"I can't believe they've been so naive and stupid to think they can get away with it. People could see them, they're well-known people. I just shake my head in disbelief."