Berahino was also left out of Pulis' squad for Tuesday's League Cup win over Port Vale.
"Our position remains unchanged. The sale of this player is not on our agenda," Baggies' chairman Jeremy Peace said in a brief statement on the club's website (www.wba.co.uk) that confirmed the rejection of the second bid from Spurs.
Tottenham's pursuit of the striker, who is contracted to West Brom until the summer of 2017 and scored 20 goals in all competitions last season, has led Pulis to criticise the transfer window being left open after the start of the season.
"I just think the transfer window should close.
"It just takes everything away from what we're here for and that's the football. So close the transfer window and let the season start and then everyone can concentrate on the season," the manager had said on Sunday.
Tottenham, who signed Cameroonian forward Clinton N'Jie from French side Lyon on Aug. 14, have been in the hunt for another striker to ease the burden on England international Harry Kane.
Spurs have sold Spaniard Roberto Soldado to Villarreal after the forward scored just seven goals in 52 league appearances since joining in 2013, and manager Mauricio Pochettino's only other striking option is the out-of-favour Emmanuel Adebayor, who has not featured for the club this season.
(Reporting by Simon Jennings in Bengaluru; Editing by John O'Brien)
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