England's Maro Itoje and Scotland trio Tommy Seymour, Blair Kinghorn and Stuart Hogg have been ruled out of their last Guinness Six Nations game on Saturday.
Itoje last week suffered a setback in his recovery from the knee injury sustained against Ireland on the opening weekend of the tournament and has now been omitted from a 31-man training squad for the final-round fixture at Twickenham.
"Maro Itoje has returned to his club Saracens to continue to rehabilitation," the Rugby Football Union has announced.
Eddie Jones was non committal when asked about Itoje's condition in the wake of Saturday's rout of Italy, declaring "we'll just wait and see", but the second row's Championship is now definitively over with only 53 minutes played.
It was hoped he would return to face the Azzurri only to injure the same right knee that was hurt in Dublin when taking part in a forwards drill at the squad's Surrey base.
Bizarrely, Jones raised the prospect of the British and Irish Lion being propelled back into the match-day 23 against Italy soon after he had been left out, stating that he could make a rapid recovery.
While the loss of Itoje is a blow, England can at least take heart from the performance of George Kruis at Twickenham on Saturday.
Jones singled out Kruis as his man of the match on the grounds of his work-rate and the 29-year-old is set to continue his second-row partnership with Joe Launchbury against the Scots.
Fly-half Marcus Smith has been called into title-chasing England's training squad for the first time in this Six Nations in a snub to Danny Cipriani, who does not appear to be in Jones' World Cup plans.
Meanwhile, Seymour, Kinghorn and Hogg have also been ruled out of Saturday's game.
Wing Seymour (rib) and utility back Kinghorn (ankle) both sustained injuries in Saturday's defeat to Wales and join full-back Hogg on the sidelines.
Hogg damaged his shoulder in last month's home defeat to Ireland and missed the 18-11 loss to Wales at Murrayfield.
"Consequently, head coach Gregor Townsend has added Glasgow Warriors pair Ruaridh Jackson and uncapped Kyle Steyn to the squad," Scottish Rugby said.
Scottish Rugby also confirmed Warriors duo Adam Ashe and Tim Swinson, plus Edinburgh pair James
Johnstone and Luke Crosbie, had been released back to their clubs.
Scotland will defend the Calcutta Cup against England at Twickenham in their final match of the tournament having won the corresponding fixture last year at Murrayfield 25-13.
Townsend's side beat Italy in their opening 2019 Six Nations match, but have since lost to Ireland, France and Wales and are fourth in the table.