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As I wrote in the description, you can use any 2 colors. For intermediate colors, you can get a lower rating in the "Black and White" category.

I noticed that colors of the same color but with a different alpha won't be considered different colors, could you just overlay white on black with different opacities to get a greyscale effect?

Yes, this is possible, but the difference between a translucent color and gray will be noticeable to the user. I recommend using transparency for effects, such as particle system in unity.