The European Commission told the two firms that the cooperation they launched in 2010 to make the air-conditioning coolant may "have limited its availability and technical development in breach of EU anti-trust rules."
The Commission, the 28-member EU's executive branch, said it sent a "statement of objections" to the companies, which amounts to a formal warning over suspected violations of the bloc's competition rules.
Since last year, EU norms demand that car makers use the refrigerant called R1234yf, which is made by Honeywell and DuPont, on the grounds that it produces far less greenhouse gases than older coolants.
"These effects include a limitation of the available quantities of the new refrigerant that would have otherwise been brought to the market, as well as a limitation of related technical development," it said.
R1234yf is already at the centre of a row that has seen luxury German automaker Daimler claim the new refrigerant is too flammable, and has caused tensions with France which briefly banned some Mercedes cars that used the substance.
In the latest salvo in September, the Commission warned Germany it risked being taken to the European Court of Justice for breaching environmental rules unless it forced Daimler to use the coolant.
It said it had sent Germany a "formal request" -- the second official stage in possible infringement procedures after an earlier written warning in January -- and gave it two months to comply.
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