THI and immune system effects
When sugars are heated with ammonia compounds a trace by-product called 2-acetyl-4(5)-(1,2,3,4-tetrahydroxybutyl)imidazole, or THI, forms. In animal studies THI causes a marked fall in circulating lymphocytes, the white blood cells central to immune defence, by sequestering them in non-lymphoid tissues. EFSA concluded that this effect was the main uncertainty driving a lower acceptable daily intake specifically for E150c compared with other caramel colour classes. EU specifications cap THI in E150c at 10 mg/kg of colour.
THI caused profound lymphopenia and suppression of cell-mediated immunity in rats, with reduced B and T lymphocytes in blood, spleen and lymph nodes.
1988AnimalBär et al., Food and Chemical Toxicology, and multiple PubMed studies (PMIDs 3147240, 1553755, 1864015, 9429899)
EFSA set a lower individual ADI of 100 mg/kg bodyweight/day for E150c (versus 300 mg/kg bw/day for the group) specifically because of uncertainties about the immune effects of THI observed in animal studies.
2011Regulatory reviewEFSA ANS Panel, Scientific Opinion on re-evaluation of caramel colours (E150a,b,c,d), EFSA Journal 2011;9(3):2004
4-methylimidazole (4-MEI) and cancer classification
The ammonia reaction during manufacture also produces traces of 4-methylimidazole (4-MEI). Two-year animal feeding studies by the US National Toxicology Program found clear evidence of lung tumour formation in male and female mice at high doses. Based on those animal studies, the International Agency for Research on Cancer classified 4-MEI as Group 2B, meaning possibly carcinogenic to humans, in 2013. No human epidemiological studies have confirmed a cancer link from food-level exposure, and the IARC classification relies solely on animal evidence.
Two-year NTP feed studies found clear evidence of lung carcinogenicity (alveolar/bronchiolar neoplasms) in male and female B6C3F1 mice exposed to 4-MEI; evidence in rats was equivocal.
2007AnimalUS National Toxicology Program, Technical Report TR-535 (toxicology and carcinogenesis studies of 4-methylimidazole)
IARC classified 4-methylimidazole as Group 2B, possibly carcinogenic to humans, based on sufficient animal evidence and limited human data.
2013RegulatoryIARC Monographs on the Evaluation of Carcinogenic Risks to Humans, Volume 101
A quantitative risk assessment found that routine consumption of certain caramel-coloured beverages in the US resulted in 4-MEI exposures exceeding California's Proposition 65 threshold of 29 micrograms/day, with estimated lifetime excess cancer risks ranging from approximately 1 in 500,000 (Coca-Cola) to 1 in 5,000 (Malta Goya).
2015ObservationalSmith et al., PLoS ONE, 2015 (PMC4333292)
Analytical studies found 4-MEI concentrations in Class III (ammonia) caramel of approximately 163-180 mg/kg of colour material; the EU cap for 4-MEI in E150c is 200 mg/kg.
2023LabAkbari et al., systematic review, PMC10314196; Petruci et al. 2013; Wu et al. 2016
Dietary exposure and ADI exceedance
EFSA's 2011 opinion found that anticipated dietary exposure for children and adults could exceed the ADI for E150c. A 2012 refined assessment using industry-provided use levels found lower estimates overall, but still concluded that toddlers and adults with high E150c food consumption could exceed the 100 mg/kg bodyweight/day ADI for this class. The combined exposure to all four caramel colour classes stayed within the 300 mg/kg bw/day group ADI.
Using refined industry-supplied use-level data, EFSA found that toddlers and adults remained capable of exceeding the individual ADI of 100 mg/kg bw/day for E150c, even after exposure estimates were substantially lowered compared to the 2011 assessment.
2012Regulatory reviewEFSA ANS Panel, Refined exposure assessment for caramel colours (E150a, c, d), EFSA Journal 2012;10(12):3030