England forward Courtney Lawes' involvement in the Six Nations has been threatened by a calf injury he suffered playing for club side Northampton at the weekend.
The back-row has withdrawn from the squad preparing for England's Calcutta Cup tournament opener at home to Scotland on February 4.
It is yet another setback for the 33-year-old flanker, who missed England's November Tests because of concussion.
England also have issues at hooker, with George McGuigan the latest player in that position to pull out of new England coach Steve Borthwick's first squad.
Exeter's Luke Cowan-Dickie had already withdrawn because of an ankle injury, with Saracens' Jamie George doubtful after he was concussed during the team's defeat by Edinburgh on Sunday.
That left the uncapped McGuigan and Jack Walker as the only fit hookers left in the squad but the Rugby Football Union said on Monday that McGuigan had pulled out because of a knee problem.
Bath hooker Tom Dunn and Northampton back-row David Ribbans have been added to the squad.
Borthwick, speaking at the Six Nations launch in London, said both Lawes and McGuigan had yet to be ruled out of the tournament, with both players undergoing scans later Monday before seeing specialists "as soon as possible".
"Both are definitely out of training this week," said Borthwick. "Clearly neither injury looked good but we'll get the facts and then make a plan."
George will now begin the return to play protocols for concussion, threatening his involvement against Scotland.
"Jamie was good after the game and now follows the protocols. It can be 12 days. You have to assess what are symptoms and non-symptoms," Borthwick said.