Danny Care, Alex Lozowski and Zach Mercer have all been omitted from England's 28-man training squad ahead of Saturday's clash with Australia.
Nathan Hughes returns after serving a six-week suspension for punching and conduct prejudicial to the interest of the game. The Wasps No 8 replaces Mercer in the squad.
Piers Francis is also included in the squad in place of Lozowski. The Northampton fly-half won the most recent of his four caps during the summer tour to South Africa, making a replacement appearance in the first Test at Ellis Park.
Asked if Hughes would be fit enough for the clash with the Wallabies, live on Sky Sports Action on Saturday, England head coach Eddie Jones said: "He's not in bad nick. We'll train him Tuesday and Wednesday and see how he is but he is definitely in contention.
"He's a reasonably experienced player in our squad. He's played 20-odd Tests so we'll just take him and see how he is."
Lozowski has paid the price for failing to stop centre Ryoto Nakamura scoring Japan's opening try at the weekend, a mistake which saw him replaced by Owen Farrell at half-time as England overturned a 15-10 deficit to win 35-15.
Care scored England's first try against Japan but has lost his place in the squad, with Richard Wigglesworth, a second-half replacement against the Brave Blossoms, poised to supply scrum-half cover for Ben Youngs against Australia.
Asked about the omission of Care, Lozowski and Mercer, Jones said: "They're not part of the best 25 that we have selected for this week. They've all got areas of their game that they need to work on, specifically for each individual. They go away knowing what they need to work on."
England's 28-man squad:
Forwards (15): Charlie Ewels (Bath Rugby), Jamie George (Saracens), Dylan Hartley (Northampton Saints), Alec Hepburn (Exeter Chiefs), Ted Hill (Worcester Warriors), Nathan Hughes (Wasps), Maro Itoje (Saracens), Courtney Lawes (Northampton Saints), Ben Moon (Exeter Chiefs), Brad Shields (Wasps), Kyle Sinckler (Harlequins), Elliott Stooke (Bath Rugby), Sam Underhill (Bath Rugby), Harry Williams (Exeter Chiefs), Mark Wilson (Newcastle Falcons).
Backs (13): Chris Ashton (Sale Sharks), Joe Cokanasiga (Bath Rugby), Elliot Daly (Wasps), Owen Farrell (Saracens), George Ford (Leicester Tigers), Piers Francis (Northampton Saints), Jonny May (Leicester Tigers), Jack Nowell (Exeter Chiefs), Henry Slade (Exeter Chiefs), Ben Te'o (Worcester Warriors), Manu Tuilagi (Leicester Tigers), Richard Wigglesworth (Saracens), Ben Youngs (Leicester Tigers).
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