The on-again, off-again Subway tuna lawsuit is on again, and this time the plaintiffs are revealing their test results. They claim that 19 of 20 tuna samples from Subway locations throughout Southern California had no detectable tuna DNA, and all of the samples contained at least one other animal protein, whether chicken, pork or cattle.

Subway’s marketing, labeling and advertising, the lawsuit contends, duped the plaintiffs “into buying premium priced food dishes based on the representation that the tuna products contained only tuna and no other fish species, animal products, or miscellaneous ingredients.”

If true, the claims would raise more than legal issues for one of the largest fast-food chains in the world. It would raise ethical, religious and dietary issues for consumers who, for example, avoid red meat or don’t eat pork because of their beliefs.

But representatives for Subway say the amended lawsuit — the third one filed this year alleging fraud, unjust enrichment and other claims under federal and California state laws — is the only thing that’s misleading the public.

“The plaintiffs’ latest attempt to state a claim against Subway is just as meritless as their prior attempts,” Mark C. Goodman, an attorney representing Subway, said in an email statement to The Washington Post. “These claims are false and will be proven to be completely meritless if the case gets past the pleading stage.”

Goodman, a partner with Baker & McKenzie in San Francisco, said the sandwich chain will file a motion to dismiss the claims, which “are fatally vague, are based on flawed testing and misstate the contents of Subway tuna sandwiches, salads and wraps.”

In a statement forwarded to The Post, a spokeswoman for Subway said the disputed product is, in fact, “high-quality, wild-caught, 100% tuna. . . . The fact remains that Subway tuna is real and strictly regulated by the FDA in the U.S., and other government entities around the world.”

Both Subway and its attorney note that this is the plaintiffs’ third attempt to prove their case. In their original complaint, Karen Dhanowa and Nilima Amin, residents of Alameda County in the Bay Area, claimed that samples from Subway locations in California contained no tuna at all, but a “mixture of various concoctions that do not constitute tuna, yet have been blended together by defendants to imitate the appearance of tuna," based on independent lab tests.

The attorneys who filed the first lawsuit in January declined at the time to reveal their lab results. There were also no lab tests attached to the revised complaint in June, which narrowed its claims to allege that Subway’s tuna was not 100 percent sustainably caught skipjack and yellowfin tuna. A federal judge dismissed the revised complaint in October, saying the plaintiffs did not prove they had purchased the tuna products based on misrepresentations from Subway. U.S. District Court Judge Jon S. Tigar’s ruling made no official comment about Subway’s tuna or its ingredients.

The latest complaint, filed on Monday, explicitly lays out the results of DNA barcoding tests conducted by the Barber Lab at UCLA’s Department of Ecology and Evolutionary Biology. Fifty grams of tuna were collected from 20 Subway locations in Southern California and analyzed by the lab. Nineteen of the samples, the lab determined, had “no detectable tuna DNA sequences whatsoever,” according to the lawsuit.

What’s more, the complaint alleges, the lab found that all 20 samples contained “detectable sequences of chicken DNA," while 11 samples contained pork DNA and seven included cattle DNA. The presence of other animal DNA, the complaint argues, undermines Subways’ marketing and labeling, which implies customers will get only “tuna” when they order such a sandwich, salad or wrap. Customers, the lawsuit alleges, just have to look at the illuminated menu above the counter to see that Subway promises tuna to all those who want it.

Between 2013 and 2019, the complaint notes, Amin ordered and ate more than 100 tuna products from a Subway location in Palo Alto, Calif., for “health and weight loss purposes.”

“Specifically, each time plaintiff Amin visited a Subway restaurant to order a sandwich, she looked at the menu, acknowledged the food option identified as being ‘tuna,’ ordered a sandwich or wrap because it was identified as being ‘tuna,’ and consumed the tuna products, all with the understanding and belief that what she was eating was, in fact, only tuna,” the lawsuit states.

The plaintiffs allege that Subway stores across California get their tuna from “the same supply chain," where, at some point, adulteration can occur.

“Defendants do not take sufficient measures to control or prevent the known risks of adulteration to its tuna products. On the contrary, they actively perpetuate actions and steps that encourage mixing or allowing non-tuna ingredients to make their way into the tuna products,” the lawsuit contents.

Goodman, Subway’s attorney, cast doubt on the plaintiffs’ DNA barcoding tests, which are often used to determine the species in fish fraud cases. DNA barcoding is apparently effective for determining a species from “fresh or living tissue,” whether cow, pig or fish such as tuna. But experts say the test may not be as reliable on “finished” or cooked products, which can degrade the DNA.

Paul H. Barber, an evolutionary and conservation geneticist with the Barber Lab, did not respond to an email from The Post asking about the reliability of his tests.

At least two media outlets have also analyzed the tuna from Subway. Inside Edition tested samples from three Subway locations and determined each contained tuna. But the New York Times also analyzed a sample and found that “no amplifiable tuna DNA was present," although that could be because the sample was so processed that testing equipment couldn’t detect the species.

Since the lawsuit first hit the media in January, Subway has gone on the offensive. The company launched a webpage designed to refute allegations about its tuna. Subway even has its own experts to defend the fish.

“Recently, in 2021, AFT tested over 150 pounds of Subway tuna salad (more than 50 individual tests/samples) and detected and identified tuna in every sample. My professional opinion, which is confirmed by our testing, is that Subway tuna products are indeed tuna,” LeeAnn Applewhite, chief executive of Applied Food Technologies, said on SubwayTunaFacts.com.

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