A month after the the probe into the poisoning of pet food began, government officials announced this week that a second contaminant had been found in protein additives that have sickened or killed hundreds of dogs and cats. The announcement came on the heels of another devastating discovery: that batches of rice protein concentrate used in pet food were also laced with the first known culprit, melamine, a nitrogen-based compound used in commercial and industrial plastics. The Food and Drug Administration (FDA) says the chemicals may have been deliberately added to the gluten in an attempt to artificially inflate the protein levels in the products.

"We have found cyanuric acid, which is somewhat related to melamine," said Stephen Sundlof, director of the FDA's Center for Veterinary Medicine. Both compounds have high levels of nitrogen, which are a measure of protein in a food product. Wheat, rice and corn gluten are forms of vegetable protein that are used as binders in soft (or wet) pet food. They can also be added to dry food to enhance the protein content, says Dave Griffin, owner of the independent pet store Westwood Pet Center, in Bethesda, Md. Griffin, who has worked in the pet industry for 35 years, adds that because of lax labeling requirements, pet food manufacturers are not required to specify the source of protein - whether it's from meat or meal.

Brent Hoff, a clinical toxicologist and pathologist at the University of Guelph in Ontario, Canada, confirmed the presence of cyanuric acid in both the rice protein concentrate as well as in crystals found in the urine and kidneys of sick animals. Late last month, those crystals, which are brown and round in shape, were found to be made up of 30 percent melamine; the composition of the other 70 percent has yet to be determined, though it is known to contain cyanuric acid as well as amilorine and amiloride, which are by-products of melamine.

Cyanuric acid may have been added separately to the feed, however it's also likely it was present because it can result from the bacterial degradation of melamine, says Richard Goldstein, a kidney specialist at the Cornell University School of Veterinary Medicine. Much like melamine, cyanuric acid, which is typically used in chlorination during pool cleaning, is not known to have a high toxicity. "People swallow it all the time" in pool water, Goldstein says. However, he adds, "It does have a toxic effect on the kidneys in very high doses.... Combining it with melamine may cause it to crystallize and hang out in the kidneys a lot longer than normal."

Hoff and his colleagues at Guelph are continuing to analyze the crystals found in sick pets to determine "how close the crystals are to the precipitate [the solid that results when two chemicals react] of melamine and cynauric acid." For now, though, Hoff cautions, "We haven't got it down pat."

The FDA also announced that it is taking preemptive steps to try to prevent further damage by testing protein ingredients for melamine in a variety of pet and human food, which contains protein additives - like wheat gluten and rice protein concentrate - that are imported from outside the U.S. David Acheson, chief medical officer for the agency's Center for Food Safety and Applied Nutrition, says the new measures are to determine "where else may this be" in order to keep the contaminant from sickening any more pets, or perhaps, people as well.

The number of pet food manufacturers that have yanked their products from shelves in the wake of the scandal continues to grow. Numbers of pet deaths are estimated to be in the hundreds, with one estimate counting as many as 39,000 cats and dogs as being affected by the poisoned food. The FDA for its part, however, still publicly links only 16 deaths to the contaminated products.

In addition to the pet food scare, the FDA revealed that the urine of hogs in North Carolina, South Carolina and California has tested positive for melamine. The implication is that salvaged melamine-containing pet food was sold to hog producers in those states, as well as farms in New York, Utah and possibly Ohio. The FDA has quarantined all of the farms that received the tainted feed. Stephen Sundlof, director of the FDA's Center for Veterinary Medicine, said that a poultry farm in Missouri may have received contaminated food, as well. But he stressed that officials are "working to assure that there is no further distribution of the meat from these farms."

During a teleconference with reporters, the FDA's Acheson said several times that the agency would be monitoring both human and pet food for melamine, even though so far the FDA has announced finding the toxin only in pet food. Products will be inspected if they contain wheat, rice and corn gluten, corn meal, rice bran and soy protein. These ingredients are widely added to a variety of human foods, such as bread, cereals, pizza dough, pasta, protein bars, baby formulas and vegetarian products.

"As part of this approach," Acheson said, "FDA and the state authorities are going to raise awareness with manufacturers and processors about the importance of knowing all there is to know about their suppliers." Thus far, contaminated wheat gluten and rice protein concentrate has been discovered in North America, and melamine-containing corn gluten was also used in pet food in South Africa. All of the tainted protein additives have been sourced to Chinese manufacturers.

The FDA said that it was possible that the protein additives were deliberately spiked with melamine to make them appear to have higher protein levels. "The motivation would be economic in that you can take a product that is low in protein...and add a substance that from a chemistry standpoint makes the product appear to have a higher protein content than it does so it can be marketed at the price," Sundlof confirmed.

The FDA will finally get a shot at getting to the root of the matter now that Chinese officials relented to requests to allow inspectors into the country to probe gluten suppliers implicated in the potential scandal. The FDA reported that it had finally received letters of invitation from the Chinese government, which are necessary to obtain visas. Once in the country, the agency plans to investigate the manufacturing practices of the two suppliers of the melamine-containing rice and wheat gluten, which have been imported by the U.S., to determine if and how cross-contamination may have occurred.

According to Julia Ho of the FDA's Office of International Programs, the Chinese government has been conducting its own investigations and has "also embargoed all the wheat gluten as well as the rice protein concentrate from those two companies for export."

The Center for Science in the Public Interest, a Washington, D.C.-based group that advocates health and food safety, recommended that the FDA bar the import of grains from China. If U.S. pets must serve as the puppies in the coal mine, we urge FDA to heed the warning and take action now to ban grains and other grain products until the Chinese government and producers can guarantee that these imports are free of illegal and dangerous substances, the group said in a written statement.

David Elder, director of FDA's Office of Enforcement, says that shipments from the two companies are already being stopped at the border and that similar products from China are being carefully screened. "We believe," he said, "that the safety net is in place to make sure that no additional products in this general category are going to get into the commerce of the United States."

The pet food recall has swelled dramatically since the FDA first announced the discovery in late March of melamine in wheat gluten added to wet foods manufactured by Menu Foods, a Canadian-based company that makes "cuts-and-gravy"-style chow for many product lines from premium brands like Iams to generics, such as Food Lion. This past week, Wilbur-Ellis, a San Francisco-based company that imports ingredients for feed, told the FDA, that it had received a pink bag with the word "melamine" stenciled on it in a shipment of rice protein concentrate; the gluten shipment, which subsequently tested positive for the toxin, came from its Chinese supplier Binzhou Futian Biology Technology Co. Ltd. (The tainted wheat gluten came from another Chinese supplier, Anying Biological Technology Development Company. (For full information on all North American brands recalling pet food, please click here.) The manufacturer Royal Canin South Africa, a subsidiary of the French pet nutrition company Royal Canin, also announced last week that it had discovered melamine in corn gluten that it received from a Chinese supplier. The food manufactured with that ingredient is now implicated in the deaths of at least 30 dogs in two weeks, according to the South African Veterinary Association.

Sundlof says that the fact that melamine has turned up in all three of these protein lends "credibility'' to theories that it was intentionally planted. "That will be one of the theories we will pursue when we get into the plants in China,'' he said. The FDA's Elder added that, "the shipment of an adulterated product'' is illegal under the Food, Drug and Cosmetics Act of 1938, and anyone found to be in violation of the law could face criminal prosecution and penalties.

According to Lora Sporny, a professor of nutrition education at Columbia University, protein is the only source of nitrogen in food "Whenever someone looks at nitrogen in food, almost always they are looking at the amount of protein in food," she explains. Sundlof concurred, telling Scientific American yesterday that, "Traditionally the way that food firms have analyzed for protein is by looking for total nitrogen. Maybe that's something that needs to be changed in light of this outbreak."

According to Ron Salter, a vice president at Wilbur-Ellis, his company did an early spot check on a shipment of rice gluten from its new Chinese supplier shortly after hooking up with the company in the summer of 2006. He declined to comment on the depth of the analysis, other than to say that it checked for levels of protein ash, moisture and fiber. He said the rice gluten was purchased based on an agreed-upon minimum amount of protein content, though he added, that he does not buy products based solely on protein concentration. "Definitely from here on, going forward,'' he says, "if we continue to go with anything from China, we will definitely be doing as many checks as we can."