First occurrence of subliminal messages in film?

Subliminal messages first came screaming into American consciousness
in the mid-1950's when American market/motivational researcher James
Vicary demonstrated the tachistoscope, a device used to flash
consciously invisible messages on a screen which could be seen only by
the subconscious mind. During one six-week test in a Fort Lee, New
Jersey movie theater, involving 45,699 patrons, messages were flashed
on alternate days over Kim Novak's calming face: "Hungry? Eat
Popcorn," and "Drink CocaCola" During the six weeks, popcorn sales
rose 57.7 percent and Coca-Cola sales rose 18.1 percent

Experimental research in the late 1950's yielded data strongly
emphasizing the enormous potential for the mass communications
industry or subliminally induced print and electronic media content.

By the late 1960's, subliminal messages had been exhaustively tested
in eight areas of human behavior: dreams, memory, value norm anchor
points, conscious perception verbal behavior, emotions, drives, and
perceptual defenses. There is at present, no serious question that
human behavior can be influenced by stimulation or information at
which individuals, groups or even societies are completely unaware.

Then in the late 1970's and 1980, stories leaked out to the public
about subliminal messages reducing shoplifting losses in retail stores
across the country, an article appeared about the use of subliminal
messages in advertising.

Numerous follow-up experiments in other media formats have confirmed,
time and time again the effectiveness of this powerful communications
tool.(source)

Some Instances of subliminal messages are listed here - Wikipedia

From the list the oldest one is

In Warner Brothers' 1943 animated film Wise Quacking Duck, Daffy
Duck spins a statue which is holding a shield. For one frame the words
"BUY BONDS" are visible on the shield.