Flanker Tom Curry will not make his England return against Wales in Cardiff on Saturday having once again withdrawn from the squad with injury.
Curry - who missed England's first two Six Nations games - has a leg issue and is replaced in a trimmed-down 26-man group by twin brother Ben.
Fly-half George Ford is left out of the squad having only been called back into the England fold on Sunday.
Wing Cadan Murley remains and could be in line for his international debut.
Starting wing Ollie Hassell-Collins had already been ruled out of Saturday's match with a knee injury, leaving room for Murley in the squad.
Former captain Courtney Lawes could make his first England appearance since July after he made his return to full training following a string of injuries.
Lawes has been out with concussion as well as neck, glute and calf issues but England defence coach Kevin Sinfield told BBC Sport "he is ready to go" against Wales.
Lawes comes into a competitive pack, where there is no room for Ben Earl, who was a replacement against Scotland and Italy.
Experienced scrum-half Ben Youngs is once again left out as Jack van Poortvliet and Alex Mitchell are preferred.
Sinfield added that Tom Curry's unspecified leg issue is not "major", explaining that the flanker would undergo further tests to understand the problem.
'We think the game will go ahead'
Under new head coach Steve Borthwick, England lost their Six Nations opener against Scotland before beating Italy.
Wales lost both their opening games and have cast some doubt on Saturday's fixture because players are threatening to strike as they are in a dispute with Welsh rugby bosses over contracts.
England captain Owen Farrell said his side have been preparing as normal, adding: "We do think the game will go ahead.
"Obviously it's not nice for the players what's going on in their camp and that's why they're taking the action that they are.
"Hopefully that all works itself out for everyone and it will be a good game on Saturday."
England squad
Forwards: Chessum, Cole, B Curry, Dombrandt, Genge, George, Isiekwe, Itoje, Lawes, Ludlam, Sinckler, M Vunipola, Walker, Willis.
Backs: Arundell, Farrell, Lawrence, Malins, Marchant, Mitchell, Murley, Slade, Smith, Steward, Van Poortvliet, Watson.