Centre Ben Te’o and full-back Mike Brown will not have a final chance to impress head coach Eddie Jones after being left out of the squad to face Wales this weekend.
Scrum-half Ben Spencer and rookie forward Alex Dombrandt have also been excluded from the 33-man group.
Jones will name his World Cup squad of 31 the day after Sunday’s warm-up at Twickenham.
England then face Wales in Cardiff before games with Ireland and Italy.
After being left out of the recent training camp in Italy, Gloucester fly-half Danny Cipriani is again excluded, which is another blow to his World Cup chances.
However, the uncapped pair of Northampton flanker LeWIS Ludlam and Bath wing Ruaridh McConnochie are both named, and could make their Test debuts at Twickenham this weekend.
Scrum-half Willie Heinz is also in line for a surprise first cap after being included at the expense of Spencer.
While the RFU say those omitted could still force their way into Jones’ final party, the omissions of Brown, Te’o, Spencer and Dombrandt appear significant.
Te’o has long been a favourite of Jones’, while Spencer ended the 2019 Six Nations as England’s second choice scrum-half behind Ben Youngs.
Brown looks to be in a shoot-out with Anthony Watson for the final back-three spot, but has lost ground, while Dombrandt was always a long-shot to make the plane to Japan.
“Players excluded from being involved in this camp are naturally disappointed but may get another opportunity so they must be ready,” Jones said.
“By the time we fly out on 8 September we will be ready to win the Rugby World Cup. We are not there yet but we have four games to get ready.”
Star wing Jack Nowell will continue his rehabilitation from ankle surgery in camp and is not available for the match with Warren Gatland’s side.
Prop Mako Vunipola is also not in contention to face the Welsh, but is said to be progressing well from his hamstring problem.
England squad to face Wales
Forwards: Dan Cole (Leicester), Luke Cowan-Dickie (Exeter), Tom Curry (Sale), Charlie Ewels (Bath), Ellis Genge (Leicester), Jamie George (Saracens), Maro Itoje (Saracens), George Kruis (Saracens), Joe Launchbury (Wasps), Courtney Lawes (Northampton), LeWIS Ludlam (Northampton), Joe Marler (Harlequins), Kyle Sinckler (Harlequins), Jack Singleton (Saracens), Sam Underhill (Bath), Billy Vunipola (Saracens), Mako Vunipola (Saracens), Harry Williams (Exeter), Mark Wilson (Sale).
Backs: Joe Cokanasiga (Bath), Elliot Daly (Saracens), Owen Farrell (Saracens), George Ford (Leicester), Piers Francis (Northampton), Willi Heinz (Gloucester), Jonathan Joseph (Bath), Joe Marchant (Harlequins), Jonny May (Leicester), Ruaridh McConnochie (Bath), Henry Slade (Exeter), Manu Tuilagi (Leicester), Anthony Watson (Bath), Ben Youngs (Leicester).