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 half-back.
With first-choice fly-half 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 scrum-halves Mitchell and McParland out of contention.
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 and it is hoped he will recover in time to face Japan or New Zealand later in the month.
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To fill the vacancies created in his 36-man squad, head coach Steve Borthwick has called up Ben Spencer and Harry Randall.
Spencer is just three games into his comeback from shoulder surgery for Bath, but appeared as a replacement in the climax to England's July tour against Argentina, while Randall has been an ever-present for Bristol so far this season.
Both players are competing to provide support to Leicester's Jack van Poortvliet, who reclaimed the number nine 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 by starting for Exeter and Gloucester, they have been promoted into the squad.
As a result, Noah Caluori and Max Ojomoh drop out of the 36-man group that is to gather at England's Surrey training base on Monday evening.
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.
There is one change to the forwards named a week ago because of the withdrawal of rookie hooker Kepueli Tuipulotu due to a knee injury incurred during last week's three-day training camp.
Theo Dan comes in as his replacement and will add depth in the position behind Jamie George and Luke Cowan-Dickie.
Once England have negotiated inconsistent Australia, they will face Japan and New Zealand on successive Saturday before entering the Nations Championship's finals weekend from Nov. 27 until Nov. 29.
FULL ENGLAND SQUAD:
Forwards:
Fin Baxter (Harlequins)
Ollie Chessum (Leicester Tigers)
Arthur Clark (Gloucester Rugby)
Alex Coles (Northampton Saints)
Luke Cowan-Dickie (Sale Sharks)
Ben Curry (Sale Sharks)
Tom Curry (Sale Sharks)
Theo Dan (Saracens)
Ben Earl (Saracens)
Greg Fisilau (Exeter Chiefs)
Jamie George (Saracens)
Ellis Genge (Bristol Bears)
Joe Heyes (Leicester Tigers)
Maro Itoje (Saracens)
Emmanuel Iyogun (Northampton Saints)
George Martin (Saracens)
Asher Opoku-Fordjour (Sale Sharks)
Tom Pearson (Northampton Saints)
Henry Pollock (Northampton Saints)
Will Stuart (Bath Rugby)
Backs:
Seb Atkinson (Gloucester Rugby)
Fraser Dingwall (Northampton Saints)
Immanuel Feyi-Waboso (Exeter Chiefs)
George Ford (Sale Sharks)
Tommy Freeman (Northampton Saints)
George Furbank (Harlequins)
Will Joseph (Gloucester Rugby)
Joe Marchant (Sale Sharks)
Cadan Murley (Harlequins)
Harry Randall (Bristol Bears)
Henry Slade (Exeter Chiefs)
Fin Smith (Northampton Saints)
Marcus Smith (Harlequins)
Ben Spencer (Bath Rugby)
Freddie Steward (Leicester Tigers)
Jack van Poortvliet (Leicester Tigers)