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.