ANN had a 2-part Lupin III history feature in which i also participated through the forum and i then remembered that Lupin Part I form 1971 is considered to be the first Seinen or let´s at least say "non-child-targeted" (TV) anime. Wiki sources the same so it must be true... NOT. (The changes 2nd half was also greatly toned down.)

What about the nearly equally dark and serious Ninpu Kamui Gaiden from 1969?
It may be a Shounen adaptation and based on the way more "upbeat" first manga series but it isn´t what i would call kidZ entertainment and the entire cast is also made up of self serious adults/murderers. It also featureless less humor and a bloody battle to the death within the OP! Can an anime historian point me to even older anime that counts?
I am also fine with toons but Disney´s Fantasia from 1940, the original Luney Toons or The Flintstones can easily be named so let´s cool the jets here.

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