Question

_______the shirt waswashed twice, still he refused to wear it.

Answer 1

Although the shirt was washed twice, still he refused to wear it.

Answer 2

Although, the shirt was washed twice, still he refused to wear it.

However, it is much preferred if we use "though" in spoken English. "Although" is generally used in written English.

"Although" is a subordiating conjunction that means "in spite of something". It is actually a subordinate clause that needs a main clause to make a complete meaningful sentence.