Alex Mitchell and Archie McParland have been ruled out of Sunday's autumn opener against Australia in an escalation of the disruption faced by England at scrumhalf.
With first-choice five-eighth Fin Smith engaged in a race against time to recover from a calf strain in time for the Wallabies' visit to Allianz Stadium, England have been forced to rule halfbacks Mitchell and McParland out of contention.
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Mitchell damaged his hamstring when on Northampton duty against Gloucester on Saturday and is to undergo a scan, placing his involvement in the second phase of the Nations Championship in doubt.
McParland missed the 28-14 victory at Franklin's Gardens because of a calf issue sustained during England's training camp last week.
To fill the vacancies created in his 36-man squad, head coach Steve Borthwick has called up Ben Spencer and Harry Randall.
Both players are competing to provide support to Leicester's Jack van Poortvliet, who reclaimed the No.9 jersey during the summer and would have been competing with Mitchell for the starting role against Australia.
With Immanuel Feyi-Waboso and Seb Atkinson having proved their fitness from respective hamstring and knee problems they have been promoted into the squad.
Smith is being given until Wednesday to demonstrate he can pull the strings in the Ella Mobbs Trophy showdown with George Ford, Marcus Smith and Charlie Atkinson the alternatives if he is ruled out.
Once England have negotiated Australia, they will face Japan and New Zealand on successive Saturday before entering the Nations Championship's finals weekend on 27-29.
"These fixtures take on added significance this autumn as they represent our final opportunity to face southern hemisphere opposition before next year's World Cup," Borthwick said.
"Facing Australia next Sunday is an exciting challenge. They have played a significant amount of Test rugby in recent months and we know the quality and experience they will bring."
Backs: Seb Atkinson, Fraser Dingwall, Immanuel Feyi-Waboso, George Ford, Tommy Freeman, George Furbank, Will Joseph, Joe Marchant, Cadan Murley, Harry Randall, Henry Slade, Fin Smith, Marcus Smith, Ben Spencer, Freddie Steward, Jack van Poortvliet
Forwards: Fin Baxter, Ollie Chessum, Arthur Clark, Alex Coles, Luke Cowan-Dickie, Ben Curry, Tom Curry, Theo Dan, Ben Earl, Greg Fisilau, Jamie George, Ellis Genge, Joe Heyes, Maro Itoje, Emmanuel Iyogun, George Martin, Asher Opoku-Fordjour, Tom Pearson, Henry Pollock, Will Stuart
