Fire officials say the incident was reported around 9 a.m. Thursday.
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They say students were conducting an acid experiment when on of the beakers "popped," exposing four students to the unknown chemical.
The four students had to be treated for exposure to an unknown substance.
The students who were exposed to the spill only had minor injuries, but because officials were not able to immediately to identify the chemical, they took no chances and evacuated the entire building.
Students spent hours outside of the math and science building, waiting for the all-clear to get their belongings.
Nick Baines says his class was working to identify unknown acids at their workstations when he heard the sound of a beaker shattering at the table next to his.
"We looked at the ground and it was all over the ground," he says. "It was just a mess."
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Adds another student, Elinore Alms, "I thought it was glass that hit my hand, but then I looked down and it was a liquid of some sort."
Alms and Eunice Odozie are two of four students treated for exposure by first responders.
"I didn't know what splashed on my face. So the next thing I had to do was rush into water," says Odozie.
Per protocol, all four students were rushed to emergency showers - and required to wash for five minutes each.
The water from that shower damaged the lab and flooded the classroom below.
The Fresno fire department's hazmat teams spent several hours neutralizing and removing the chemicals in the classroom.
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However they say because the class was working with seven unidentified chemicals, it's unclear what the students were exposed to.
"We have a lot of concern here. This is a life safety hazard. We just want to make sure we're taking all the necessary precautions to make sure everybody is safe," says Fresno Fire Captain Robert Castillo.
Fresno County Environmental Health has been called into assist in the cleanup after the spill in the chemistry lab.
School officials canceled all classes in the building for the remainder of the day.
They will likely be resumed on Friday.