Jonny Wilkinson, who has been battling to recover from an ankle ligament sprain suffered in training last week, was again omitted - although it is not known at this stage whether he could be drafted in after Barkley was sent for a scan.
A decision on Barkley's fitness will be made on Wednesday.
Other options at fly-half would be Andy Farrell or a move for Mike Catt from inside centre.
Mathew Tait and Dan Hipkiss, meanwhile, are both suffering from illness, hence Ashton's decision not to name a seventh replacement at this stage.
England captain Phil Vickery has been named to start the game, despite facing a disciplinary hearing in Paris later on Tuesday.
Vickery was cited for kicking American centre Paul Emerick, and could be handed a ban that would rule him out of the Springboks showdown.
If Vickery is ruled out, then Ashton confirmed Matt Stevens would be promoted off the bench with Perry Freshwaster joining the replacements.
Wasps back-row forwards Lawrence Dallaglio and Joe Worsley have failed to gain places in the matchday 22. Martin Corry has replaced Worsley at blindside flanker and Harlequins' Nick Easter takes over from number eight Dallaglio.
Dallaglio was ineffective against the USA, and a miserable afternoon for him concluded when he was sin-binned by referee Jonathan Kaplan.
Easter had an impressive tour for England in South Africa earlier this summer, and he now has a chance to show his quality in England's biggest game since they won the 2003 World Cup.
Changes in the back division see Jason Robinson moved from wing to full-back instead of Mark Cueto, with Wasps' Paul Sackey taking over from Robinson on the left wing.
Bath lock Steve Borthwick and Harlequins' scrum-half Andy Gomarsall are on the bench.