The 30-year-old ex-Olympic sprinter was told of his fate after Tigers coaches and staff decided he would not be ready for first-team action this season.
Chambers received praise from officials at the Jungle for his attitude in training and his cameo appearance in a friendly match against York, but the club now want to focus on this year's Super League competition.
Tigers CEO Richard Wright said: "Many people watched Dwain in the trial game and he has certainly won over some of the doubters, and proved that the trial was more than simply a 'publicity stunt.'
"During his trial period Dwain has been a joy to have around. His work ethic and professionalism have been exemplary. He has got on well with all members of the club's staff that he has come into contact with and he has been in great demand, proving a popular visitor to local schools. He has also given us a huge media profile. I believe that he feels that he has gained from his time with us, so there are no losers.
"However our coaching team feels that he would need a significant amount of additional time to prove whether or not he would be capable of making the grade in Super League, and time is something that we don't have. Furthermore we have an outstanding group of young players coming through whom we need to focus upon and give our attention. As a result we have reluctantly come to the decision not to continue with the trial.
"All the club wish Dwain the very best for the future in whatever path he chooses to follow."
Speaking on behalf of Dwain, his manager Nick Collins said: "Whilst disappointed, Dwain is comfortable with and understands the decision. He has had a great time and made some good friends during his brief period with the club. He leaves the club in a positive mood and with a smile on his face."