research papers on food dyes

Potential impacts of synthetic food dyes on activity and attention in children: a review of the human and animal evidence

Affiliations.

1 Office of Environmental Health Hazard Assessment, California Environmental Protection Agency, 1515 Clay St, Oakland CA, and 1001 I St, Sacramento, California, USA. [email protected].
2 Office of Environmental Health Hazard Assessment, California Environmental Protection Agency, 1515 Clay St, Oakland CA, and 1001 I St, Sacramento, California, USA.
3 Center for Environmental Research and Community Health, School of Public Health, University of California, 2121 Berkeley Way, Berkeley, California, USA.
4 Department of Public Health, School of Social Sciences, Humanities and Arts, University of California, Merced, 5200 N Lake Road, Merced, CA, USA.
PMID: 35484553
PMCID: PMC9052604
DOI: 10.1186/s12940-022-00849-9

Concern that synthetic food dyes may impact behavior in children prompted a review by the California Office of Environmental Health Hazard Assessment (OEHHA). OEHHA conducted a systematic review of the epidemiologic research on synthetic food dyes and neurobehavioral outcomes in children with or without identified behavioral disorders (particularly attention and activity). We also conducted a search of the animal toxicology literature to identify studies of neurobehavioral effects in laboratory animals exposed to synthetic food dyes. Finally, we conducted a hazard characterization of the potential neurobehavioral impacts of food dye consumption. We identified 27 clinical trials of children exposed to synthetic food dyes in this review, of which 25 were challenge studies. All studies used a cross-over design and most were double blinded and the cross-over design was randomized. Sixteen (64%) out of 25 challenge studies identified some evidence of a positive association, and in 13 (52%) the association was statistically significant. These studies support a relationship between food dye exposure and adverse behavioral outcomes in children. Animal toxicology literature provides additional support for effects on behavior. Together, the human clinical trials and animal toxicology literature support an association between synthetic food dyes and behavioral impacts in children. The current Food and Drug Administration (FDA) acceptable daily intakes are based on older studies that were not designed to assess the types of behavioral effects observed in children. For four dyes where adequate dose-response data from animal and human studies were available, comparisons of the effective doses in studies that measured behavioral or brain effects following exposure to synthetic food dyes indicate that the basis of the ADIs may not be adequate to protect neurobehavior in susceptible children. There is a need to re-evaluate exposure in children and for additional research to provide a more complete database for establishing ADIs protective of neurobehavioral effects.

Keywords: Animal toxicology; Behavior; Children; Clinical trials; Synthetic food dyes.

Publication types

Systematic Review
Research Support, Non-U.S. Gov't
Attention Deficit Disorder with Hyperactivity*
Coloring Agents
Food Coloring Agents* / toxicity
Food Coloring Agents

Open access
Published: 29 April 2022

Potential impacts of synthetic food dyes on activity and attention in children: a review of the human and animal evidence

Mark D. Miller ORCID: orcid.org/0000-0002-9301-0093 1 ,
Craig Steinmaus 1 ,
Mari S. Golub 1 ,
Rosemary Castorina 2 ,
Ruwan Thilakartne 2 ,
Asa Bradman 2 , 3 &
Melanie A. Marty 1

Environmental Health volume 21 , Article number: 45 ( 2022 ) Cite this article

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Concerns about possible associations between exposure to synthetic food dyes and the exacerbation of symptoms of Attention Deficit/Hyperactivity Disorder (ADHD) in children have surfaced periodically since the 1970s. The concern prompted the California legislature to request a review by the California Environmental Protection Agency’s Office of Environmental Health Hazard Assessment (OEHHA) of available studies to evaluate whether the synthetic food dyes currently allowed in foods and medications in the United States impact neurobehavior in children [ 1 ]. This paper provides an overview of key portions of OEHHA’s peer-reviewed assessment, specifically the evaluation of the clinical trials of synthetic food dyes in children and available animal toxicology studies, as well as discussion of our hazard characterization and the possible public health implications of our findings.

Our evaluation focused on seven of the nine food dyes subject to FD&C batch certification by the US Food and Drug Administration (FDA) and approved for general use in food in the US (Table 1 ). These seven dyes contribute nearly all of the exposure to synthetic food dyes for the general US public [ 1 ]. The term “FD&C batch-certified” refers to the Food Drug and Cosmetic Act requirements for chemical analysis of each manufactured batch of food dye to ensure that specific contaminants are present below legal limits. OEHHA evaluated the literature to determine whether there is any evidence supporting the association of exposure to synthetic food dyes with adverse neurobehavioral impacts in children in the general population with or without a diagnosis of ADHD.

The literature review methods were designed to identify all the literature most relevant to the assessment of evidence on the neurological or neurobehavioral effects of the synthetic food dyes listed in Table 1 . The search was executed to identify peer-reviewed open-source and proprietary journal articles, print and digital books, reports, and gray literature that potentially reported relevant toxicological and epidemiological information. We also included Citrus Red No. 2 and Orange B/CI Acid Orange in the search terms since these food dyes are part of an overlapping literature that might contain information on the commonly used FD&C synthetic food dyes. PubMed MeSH browser (PubMed MeSH browser) and PubChem ( PubChem ) were used to identify subject headings, other index terms and synonyms for the food dyes of interest and their metabolites, as well as for the concepts related to exposure, food, mechanisms of action, and neurological outcomes. Preliminary searches were run and results reviewed to identify additional terms. The concepts were combined in the following manner:

((food/dietary terms) AND (specific food dye terms)) OR ((specific food dye terms) AND (neurological outcome terms) OR (general exposure terms) OR (mechanisms of action terms))

The detailed search strategy executed in PubMed on November 26, 2018 is summarized in the additional information (Table A.1). This search was run again to capture literature updates, on March 8, 2019 and April 22, 2019, and again in October 2020.

Additional databases (PubMed, Embase, Scopus) and other data sources (European Food Safety Authority (EFSA) Journal, EFSA Scientific Output, US FDA Safety Information Office, University of California, San Francisco Food Industry Documents Archive, and Dyes and Pigments Journal) were also searched; strategies were tailored according to the search features unique to each database and data source. Relevant literature was also identified from citations in individual articles. In addition, we searched NIH RePort to identify additional unpublished clinical trials or animal research. In our systematic review of the epidemiologic research on synthetic food dyes and neurobehavioral outcomes in children, we summarized the major strengths and weaknesses of each study, described any consistencies across study results, and if heterogeneity exists, identified its sources as far as possible [ 1 ].

Our epidemiologic review focused on clinical trials. A major advantage of this type of study is that investigators generally have control over the exposure which can help reduce bias and confounding compared to other study designs. Next, we conducted systematic evaluations of study methods and quality to ensure an emphasis on the high quality studies for our conclusions. In evaluating study quality, we utilized criteria based on the National Toxicology Program’s OHAT Risk of Bias Rating Tool [ 2 ]. We modified these to be specific to randomized clinical trials (RCT) on artificial food dyes and childhood neurobehavior. We examined several key characteristics of each study to assess study quality including design, participant selection, exposure levels, age groups, washout period, infractions, outcome metric, and funding (Table A.2). This table also includes key information on results including statistical significance, effect size, dose-response, and subgroups. The coding used in our statistical analyses and quality scoring is provided in Tables A.3 and A.4. These tables show the criteria used to evaluate study quality, which included randomization, placebo use, dropout rate, blinding, whether dose-response was assessed, outcome metric validation, replication, and adequate washout. All this information was considered in making our overall conclusions about the human study results.

In determining whether the study reported an association, we define association as either a statistically significant outcome ( p value <.05 or 95% confidence intervals that excluded 1.0 for relative risk estimates or 0 for mean differences) or an effect size ≥20% or standardized effect size ≥0.20. Most studies involved small sample sizes and thus may not have had sufficient statistical power to identify effects that are relatively small but still of public health importance. Because of this, in addition to statistical significance, bias and effect size were also considered in our evaluations of association and causal inference. There are several arguments against solely using statistical significance to identify associations [ 3 , 4 ].

We searched the animal toxicology literature and identified numerous studies of neurobehavioral effects in laboratory animals exposed to synthetic food dyes. These included studies of exposures during prenatal, infant, and juvenile development, examining neurobehavioral effects in the offspring manifest during development and/or later in adult animals. The availability of studies at different developmental stages allowed a comprehensive review of adverse developmental effects, although it limited the ability to compare results across study designs, as exposures during different developmental stages may manifest differently later in life. The OEHHA report reviewed all available studies and provided strengths and limitations for the individual studies [ 1 ].

Acceptable Daily Intakes (ADIs) for synthetic food dyes were established by the US FDA between the 1960s and the1980s based on general toxicology studies. OEHHA therefore also evaluated whether newer studies that included neurobehavioral assessment would be useful for developing updated acceptable exposure levels that explicitly account for and protect against neurobehavioral effects of individual food dyes. OEHHA compared the results of those specific studies to the existing US FDA ADIs, as well as ADIs developed by the Joint FAO/WHO Expert Committee on Food Additives (JECFA).

Review of clinical trial studies

In total, 27 clinical trials were identified that met each of the following criteria:

Human study

Clinical trial design

Participants were given a known quantity of synthetic food dyes or a diet low in or eliminating synthetic food dyes

A neurobehavioral outcome related to hyperactivity or inattention was assessed

The majority of participants were children ≤19 years of age

The effects of an active ingredient or elimination diet were compared to those of a placebo

Studies were excluded if they were:

Studies involving cohort, case-control, or cross-sectional designs

Studies that assessed the effects of a broad range of food groups, including elimination studies, and did not specifically evaluate synthetic food dyes. Any effect identified in such studies would be difficult to ascribe specifically to synthetic food dyes.

No exclusions were made based on the number of participants, participation rates, blinding, randomization, or source (e.g., government reports), although each of these factors was considered in our review of study quality and in our overall conclusions.

Figure 1 presents the results of our literature search as the number of clinical studies reporting adverse neurobehavioral outcomes by key study variables. Of the 27 studies meeting our criteria for inclusion, 25 involved challenge studies, which we consider most relevant as they directly challenge children with food dyes, and two involved diet elimination studies. Detailed descriptions of the 25 included challenge studies are provided in Table A.2. Table 2 below summarizes the characteristics and overall findings of the reviewed challenge studies. Several studies of exposure to dye mixtures also included other dyes not used in the US.

Number of clinical studies reporting positive associations by key study variables

The most frequent study locations were in the US (44%), followed by the UK (22%), and Australia and Canada (15% each). The mean number of participants was 44 (range 1–297). All studies used cross-over designs. In the cross-over design, each subject receives each treatment (including placebo) and, thus, the subjects serve as their own controls, which minimizes bias and confounding. Most challenge studies were double-blinded and the cross-over design was randomized, although in two studies the use of blinding was unclear. Randomization was either not done or was unclear in seven studies. Six studies assessed tartrazine only, whereas the rest studied mixtures of common dyes. The average dose assessed was 55.8 mg/day (range 1.2 to 250 mg/day, doses relevant to children’s exposure in the US). In all but one challenge study, participants were placed on an elimination diet during the study. Most studies (70%) used a validated or otherwise commonly accepted metric to assess neurobehavioral outcomes, with the most common being the Conners Parent scale.

Sixteen (64%) out of 25 challenge studies identified some evidence of an association and in 13 (52%), the association was statistically significant (Fig. 1 and Table 2 ; Table A.2). Associations (either large effect sizes or statistically significant results) were most commonly identified in studies that assessed neurobehavioral outcomes using information from the child’s parents. Out of eight challenge studies that provided results for both parents and teachers, four found associations only when examining parent reports [ 5 , 6 , 7 , 8 ], one found associations for both parent and teacher reports [ 9 ], two did not report an association for any outcome metric [ 10 , 11 ], and one found an association only for another metric [ 12 ].

Positive associations were also more frequently reported in studies published after the year 1990 (83.3 vs. 57.9%, p = 0.26), in studies that used validated metrics for assessing outcome (70.6 vs. 50.0%, p = 0.17) and in studies with larger numbers of participants (see Fig. 1 and Table 2 ). The reason why more recent studies tended to report associations compared to earlier studies is unclear.

While two positive studies tested mixes of dyes plus preservatives [ 13 , 14 ], the large majority did not include preservatives and many of these (59.1% overall), identified associations between these dyes and adverse effects on neurobehavior with 10 of them reporting associations that were statistically significant [ 5 , 7 , 15 , 16 , 17 ].

Rowe and Rowe [ 17 ] saw a dose-response pattern between increasing doses of 1, 2, 5, 10, 20, and 50 mg of Yellow No. 5 (tartrazine) per day and worsening behavioral scores. Only two other studies reported information on dose-response, one using multiple dyes and one with Yellow No. 5 alone, with neither finding a clear dose-response pattern [ 18 , 19 ] However, Rowe and Rowe used many more doses and had a larger sample size than the other two studies. These differences and other study design issues may have affected whether a dose-response could be seen.

We could not divide studies based solely on age as there was a wide range of ages studied with broad overlap across studies reviewed. However, based on sensitivity analyses examining age, in three studies, results varied minimally [ 11 , 17 , 20 ], while in three others, greater effects were seen in younger participants [ 5 , 14 , 21 ].

Nigg et al., 2012 meta-analysis

A high-quality meta-analysis [ 22 ] is supportive of the hypothesis that synthetic food dye exposures is associated with adverse behavioral effects in children. This study identified statistically significant summary associations for findings based on parent reports or on attention tests, with effect sizes about one-sixth to one-third of those seen for improvements from ADHD medications. Nigg et al. estimated that 8% of children with ADHD may have symptoms related to synthetic food dyes. Our report evaluated the same studies used in the Nigg et al. meta-analysis as well as two pilot or preliminary reports [ 7 , 19 ], two studies with only 1–2 participants [ 8 , 16 ], and a study published after the meta-analysis was published [ 10 ]. These five studies reported mixed results. It is unlikely their inclusion in a meta-analysis would dramatically affect its results because most of these studies had small sample sizes. Additionally, the Lok et al. study [ 23 ] did not present means and standard deviations for analyses comparing placebo to artificial food dyes, and as such would be difficult to include in meta-analysis with most other studies.

Bias and confounding

As documented in Tables S.2-S.4 we performed extensive evaluations of quality for each study. One strength of our findings is that they are based on clinical trials with cross-over designs and placebo control. Non-compliance can lead to exposure misclassification in clinical trials, but we found that infraction rates were generally low in the studies when they were reported. Potential confounding can be markedly reduced with the use of cross-over designs since subjects are being compared to themselves. Bias that may be introduced by the expectations of the researchers and participants is minimized by use of blinding and placebo control. We performed a sensitivity analysis in which we only included studies that were double-blinded and had the cross-over randomized, and found that our conclusions were similar to that of our analysis that included all studies (Table 2 , rows for RCDP).

Recruitment strategies and participation rates were not always clearly described in the studies, and most seemed to involve convenience samples. The use of convenience samples or low participation rates can introduce bias. However, in studies in which the participants, parents, and others were blinded, we found no clear evidence or obvious reason that convenience sampling or low participation might cause false positive results. While convenience sampling and low participation rates might affect the generalizability of some studies, we see no reason why they would affect the ability of a study to examine whether at least some children might be adversely affected by synthetic food dyes, especially given the cross-over design.

Adjustments for publication bias by Nigg et al. [ 22 ] attenuated summary effect sizes in the meta-analysis, although several remained statistically significant. However, these adjustment methods are imperfect. In addition, given the widespread interest in the potential health effects of synthetic food dyes, it seems unlikely that well-conducted clinical trials would remain unpublished resulting in publication bias.

Susceptibility

From the studies reviewed, it appears that not all children react to the dyes with adverse behavioral outcomes. Possible explanations for this sensitivity are not clear. Studies that included only children who were previously diagnosed with hyperactivity were not more likely to report positive associations between synthetic food dye exposure and poorer behavioral outcomes. Stevenson et al. [ 24 ] found that children (both 3 year-olds and 8/9 year-olds) with certain polymorphisms in histamine degradation genes had greater adverse responses to synthetic food dyes. In addition, gene polymorphisms in the dopamine transporter gene in 8/9 year-old children moderated the effects of the food dyes. Since histamine plays a role as a neurotransmitter in the brain and is involved in wakefulness, polymorphisms in the histamine degradation genes are a plausible basis for varied behavioral sensitivity to dyes associated with histamine release. Replication of this study and further research of the impacts of gene polymorphisms on response to food dyes are needed.

Review of animal toxicology studies

Animal toxicology studies were used by FDA as the basis for regulatory risk assessments of food dyes [ 25 ]. All current dye registrations were made between 1969 and 1986 based on studies performed 35 to 50 years ago. These studies were not designed to assess neurobehavioral endpoints. Dye registration was accompanied by derivation of an “acceptable daily intake” (ADI) based on these studies. FDA ADIs have not been updated since original dye registration, although there have been several reviews of specific effects since then, the latest in 2011 [ 25 ].

Our review of animal toxicology studies was intended to examine neurobehavioral toxicity of food dyes and included any study administering one or more of the FDA registered food dyes and measuring a behavioral endpoint. We obtained 25 reports from the peer-reviewed literature. Two reports could not be reviewed due to lack of study information. The 23 studies reviewed had the following characteristics:

Rodent models (rats or mice)

Oral administrations (diet or gavage)

Dosing with individual dyes (14 studies) or dye mixtures (9 studies) (Fig. 2 )

Dosing included at or below that in studies used to establish FDA ADIs

Durations ranging from a single dose to lifetime daily dosing (Fig. 3 )

Behavioral endpoints including preweaning motor development, spontaneous motor activity and/or learning and memory tests

Comparison of dosed and control groups

Number of animal developmental neurobehavioral toxicity studies by dye and year

Experimental designs of developmental neurotoxicity studies in animals with synthetic food dye exposures

The study designs varied (Fig. 3 ) and included exposures during prenatal, infant, juvenile and adult life stages, and examined neurobehavioral effects during development and/or adulthood. Due to the wide range of designs, an overall integration of findings was not possible but a broader picture of the potential for food dye neurobehavioral toxicity is seen. Details of the studies are presented in Table A.5 and A.6. Detailed evaluation and interpretation of each study is reported in the OEHHA document [ 1 ]. First author and dates of publication are shown.

Details from all the studies reviewed in this section are shown in Table A.5.

Findings from these studies have greatly advanced our knowledge of neurobehavioral effects of synthetic food dyes:

Long term consequences of exposure during pregnancy [ 26 , 27 , 28 , 29 ]. This is the first research using the classical developmental neurotoxicology (DNT) design where exposure begins during pregnancy to identify long-term effects of perinatal exposure. Prior regulatory developmental toxicology studies have been limited to effects on mortality, malformation, and growth. There are no studies in humans using exposure in pregnancy.

Effects of synthetic food dyes on behavior in adult rats after a single administration [ 30 , 31 ]. These are the only available animal studies measuring behavior shortly after a single dye administration.

Behavioral effects when synthetic food dyes are administered at juvenile/adolescent life stages [ 32 , 33 ]

Effects on behavior in adult rodents with chronic exposures [ 31 , 34 , 35 , 36 ]. Due to the emphasis on behavioral effects in children, more general studies of neurobehavioral toxicity in adults have been lacking but have recently been undertaken in animal models.

Prevention of effects of synthetic food dyes on behavior by antioxidants [ 35 , 36 ]. This line of investigation has also been pursued for other aspects of dye toxicity [ 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 ]

Brain changes associated with behavioral effects [ 26 , 29 , 30 , 31 , 34 , 35 , 36 ]. Emerging research in the last 10 years has begun to explore effects of synthetic food dye exposures on the brain at doses that affect behavior.

Individual dye studies

A series of neurobehavioral studies of individual dyes has been performed by one laboratory in Japan for 5 of the 7 food dyes approved for use in the US [ 46 , 47 , 48 , 49 , 50 , 51 ]. These studies used lifetime exposure beginning prior to parental mating. Details of the studies can be found in Table A.5. For three of the dyes, behavioral effects were identified at doses below those producing the toxicological effects used to establish the FDA ADIs. Several considerations limit the use of these studies in assessing food dye risk to children, including reproductive toxicity in the studies, multiple life stage exposure, dosing both before and during testing, and lack of litter-based statistics for preweaning endpoints.

Eight studies were from US laboratories, all published prior to 1987. We did not find any programmatic investigator-initiated research on neurobehavioral effects of food dyes currently being performed in the US.

The US FDA supported early studies of three synthetic food dyes (Yellow No. 5, Red No. 3, Red No. 40) that also used lifetime exposures beginning prior to or shortly after conception and continuing through adult animal testing [ 52 , 53 , 54 ]. Dosing was based on known non-behavioral toxicity of the dyes. Behavioral effects were reported for Red No. 3 [ 54 ] and Red No. 40 [ 53 ] using an extensive test battery.

The other 5 studies of individual dyes were conducted more recently and administered individual dyes to postpubertal (adolescent and adult) rodents [ 30 , 31 , 34 , 35 , 36 ]. These investigators were interested in specific hypotheses about food dye mechanism of action and included brain assays: brain microhistomorphometry [ 35 , 36 ]; measures of oxidative stress [ 34 ]; and measures of influence on the serotonin system [ 30 , 31 ].

Mixture studies

Early studies used dye mixtures designed to parallel US food dye exposure at that time [ 33 , 55 , 56 , 57 , 58 ]. More recent studies used dosing based on a multiple of regulatory ADIs [ 27 , 28 , 29 , 32 ]. Study details are presented in Table A.6. Animal mixture studies, like children’s mixture studies, are valuable for hazard identification, but not for ADI development, which is based on each dye individually.

The two studies using synthetic food dye mixtures that are most relevant to the studies in children reported behavioral effect during dye administration to immature rats [ 32 , 33 ]. Both studies reported effects on regulation of spontaneous motor activity. Shaywitz et al. [ 33 ] used a mixture based on human exposure and found greater activity in rats dosed at twice the estimated average exposure at that time in children. Erickson et al. [ 32 ] found increased movement time using a mixture of dyes in drinking water, each dye at a dose less than 2 times the FDA ADI.

One series of studies examined exposure to a mixture of synthetic food dyes during pregnancy [ 27 , 28 , 29 ]. The 9 dyes were administered at either the JECFA ADI [ 28 ] or 100 times the JECFA ADI [ 27 , 29 ]. Six of the seven FDA registered food dyes were included. Effects on activity and emotionality were reported with testing of one-month old (early adolescence) and three-month old (adulthood) offspring, but learning and memory tests were not affected.

Behavioral endpoints

Behavioral assessments were primarily conducted in a few domains: preweaning motor development (4 studies), spontaneous motor activity (21 studies), and trial-based learning and memory tests (18 studies). Some recent studies included emotionality tests [ 27 , 28 , 29 , 32 ].

Spontaneous motor activity, a sensitive and widely used test in developmental neurotoxicology, was the most frequently used test in animal dye studies because of the findings of hyperactivity in children’s studies. While the test apparatus and specific endpoints affected (vertical/horizontal activity, speed, distance, duration) varied, altered regulation of activity was seen in 17 of the 21 of the studies.

Sensitivity of learning and memory tests in developmental neurotoxicology is less consistent [ 59 , 60 ]. For the food dye studies we examined, tests included shock-motivated avoidance, food motivated mazes, and water mazes, with 12 of 18 studies reporting dye effects. Our review found that many of the test results could not be used for risk assessment due to design and statistical issues. For example, some studies did not use litter-based statistics.

Brain assays in behavioral studies

Many animal studies we reviewed conducted brain assays that evaluated a number of parameters with a focus on neurotransmitter systems. Early studies did not identify effects of synthetic food dye exposures on tissue catecholamine neurotransmitter concentrations [ 33 , 55 , 56 , 57 ]. More recent studies identified effects on gene receptor expression, enzyme activity in neurotransmitter systems, and localized changes in neurotransmitter levels [ 26 , 29 , 30 , 31 ]. Also, brain histomorphology assessed with contemporary methods has identified effects (decreased medial prefrontal cortex volume, decreased numbers of glia and neurons, changes in dendritic morphology) of the two most used food dyes, Red No. 40 and Yellow No. 5 [ 35 , 36 ]. Protective effects of antioxidants [ 35 , 36 ], as well as changes in brain anti-oxidant defense systems [ 34 ] provide evidence for oxidative stress as a mechanism of toxicity. Two other papers with no behavioral measures found markers of oxidative stress in the brain after in vivo treatment of rats with Yellow No. 5 [ 61 , 62 ].

Data from a human study provide evidence for a mechanism involving the neurotransmitter histamine. The investigators demonstrated that polymorphisms in the histamine degradation gene for histamine-N-methyltransferase influences response to a dye mixture [ 24 ]. In addition to its role in the inflammatory process, histamine is recognized for its role in regulating synaptic transmission alone and in concert with other neurotransmitters [ 2 ].

Considering both in vivo and in vitro research, other potential pathways for food dye neurotoxicity have been suggested [ 63 , 64 , 65 ].

Endocrine (thyroid, estrogen) mediated effects

Interference with neuronal proliferation and differentiation

Effects secondary to general physiological toxicity

Immune mediated effects

Interference with nutrient bioavailability

The relevance of the animal toxicology findings to humans ingesting synthetic food dyes in food and medications would be better understood with more information about food dye toxicokinetics. In particular, the breakdown of azo dyes in the gut prior to absorption requires toxicological examination of metabolites. Future studies should evaluate whether the parent compounds act on the gut to influence behavior via the gut-brain axis [ 66 ].

Hazard characterization

The studies that form the basis of the FDA (and JECFA) ADIs are many decades old and as such were not capable of detecting the types of neurobehavioral outcomes measured in later animal studies, or in clinical trials in children consuming synthetic food dyes.

Nonetheless, OEHHA first compared the US FDA ADIs and the No-Observed-Adverse-Effect Levels (NOAELs) from which they were derived to NOAELs from the animal toxicology studies that were reviewed [ 1 ]. Next, we compared the estimated food dye exposures (mg/kg/d) from food consumption to available regulatory benchmarks in a traditional Hazard Index approach for noncancer health effects. The Hazard Index approach divides estimated exposures by a toxicity benchmark. If that ratio is greater than 1, then it is indicative of a possible risk of adverse noncancer effects. Finally, we compared the ADIs to NOAELs and Lowest-Observed-Adverse-Effect Levels (LOAELs) observed in the few key animal and human studies of sufficient quality. This comparison should help inform future revisions of the ADIs aimed at protecting children from neurobehavioral effects.

Comparing neurobehavioral effect levels to FDA ADI NOAELs

To derive the ADI for each dye, US FDA divided NOAELs reported by investigators from animal studies by a factor of 100. While reviewing animal neurobehavioral toxicology studies, we compared the effective doses (LOAELs) to animal NOAELs used by US FDA to derive human ADIs (hereinafter referred to as ADI NOAELs). The purpose of this comparison was to see if neurobehavioral effects were found at doses that FDA determined were not causing effects in the older general toxicology studies . Tables 3 and 4 presents these comparisons for both developmental and adult neurotoxicology studies where a single dye was administered.

Comparing food dye exposures to available regulatory benchmarks

OEHHA [ 1 ] derived exposure estimates based on NHANES 2015–2016 Dietary Interview data, and information on food dye concentration data sourced from Doell et al. [ 67 ]. We calculated single-day and two-day average cumulative daily synthetic food dye intake estimates (mg/person/day) for the following demographic categories:

Pregnant women 18 years and older

Women of childbearing age (18–49 years)

Children: 0- < 2 years, 2- < 5 years, 5- < 9 years, 9- < 16 years, and 16–18 years

We estimated daily synthetic food dye intakes (mg/person/day) for

The typical-exposure scenario , which represents exposure to a given FD&C batch-certified synthetic food dye for a typical consumer, an individual who may not always eat products with the lowest or highest levels of that food dye but some combination of both.

The high-exposure scenario , which represents the highest exposure where the individual is only consuming products with the highest levels of that food dye.

We divided each individual’s FD&C batch-certified synthetic food dye intake estimate (mg/person/day) by their body weight (kg) reported in NHANES 2015–16 [ 68 ] to produce synthetic food dye dose estimates in units of mg/kg/day. The most commonly consumed dyes for the various age ranges of children expressed as the mean of typical-exposure scenario estimates were Red No. 40 (ranged from 0.11 to 0.3 mg/kg-day), Red No. 3 (ranged 0.02 to 0.54 mg/kg-d), Yellow No. 5 (ranged from 0.05 to 0.19 mg/kg-d) and Yellow No. 6. (ranged from 0.05–0.20 mg/kg-d) [ 1 ]. The 95th percentile of the high-exposure scenario estimates ranged from about 1 to 8 mg/kg-day for these four dyes. Children’s exposures tended to be higher than adult women.

We compared the synthetic food dye dose estimates to the US FDA and JECFA ADIs (Table 5 ) by calculating the ratio of the dose estimates to the established ADIs [ 25 , 69 , 70 , 71 , 72 ] as the Hazard Index. Hazard index > 1 signifies that the food dye exposure estimates (mg/kg/day) exceeded the established ADI.

With the exception of FD&C Red No. 3, all exposure estimates (mg/kg/day) from foods were below the US FDA or JECFA ADIs. The Hazard Indices (HI) that exceeded 1 for Red No. 3 are bolded in Table 6 . Children’s single day mean FD&C Red No. 3 exposure estimates for typical- and high-exposure scenarios ranged from 0.01 to 0.60, not exceeding the FDA ADI of 2.5 mg/kg-day. The 95th percentile exposure estimates ranged up to 3.16 (although it represents few children). For several age categories the mean single day typical- and high-exposure scenarios exceeded the JECFA ADI of 0.1 mg/kg-day, with HI ranging from 0.21 to 15; the 0 < 2 year age category had the highest HI.

Comparing US FDA ADIs to key neurobehavioral studies

There are several animal studies and one human study that could be used to evaluate whether existing ADIs are protective of neurobehavioral effects for Red No. 3, Red No. 40, Yellow No. 5 and Yellow No. 6. No suitable studies of green or blue dyes were found for this comparison.

Tanaka et al. [ 48 ] conducted a developmental toxicity study of Red No. 3 where various doses were administered via diet from preconception through PND 63 and reported increased activity measurements in female offspring. For adult female dams, more turning was reported in the high-dose group than in controls. Activity in male offspring was affected at 3 weeks of age ( p < 0.01 for linear dose trend), but not at 8 weeks of age. In the female offspring at 8 weeks, statistically significant dose-dependent dye-induced increases in activity were seen, but not at 3 weeks of age. These included number of activity bouts, distance traveled in each bout, greater speed, total time moving and total distance. This interesting finding of greater activity is particularly valuable because of the absence of more severe developmental toxicity.

The NOAEL was 24 mg/kg/day for the female offspring. This NOAEL is a factor of 10 higher than the FDA ADI of 2.5 mg/kg/day. If one were to apply the same methodology as US FDA (dividing the NOAEL by a factor of 100) to derive an ADI, the resulting ADI would be a factor of 10 lower.

The studies by Dalal and Poddar [ 30 , 31 ] (Table A.5) provide unique information on brain serotonin pathway changes, and on behavioral changes in young adult animals either following single gavage administration or following 15 or 30 day exposures to Red No. 3. In their first study, the investigators measured activity (vertical rearing frequency detected automatically) for 5 min at 30 to 60 min intervals up to 9 h post-dosing after single gavage doses of 0, 1, 10, 100 or 200 mg/kg. A dose-dependent pattern of diminished activity was observed that reached a low at 2 h after dye administration and then returned to baseline by 7 h (Fig. 1 in Dalal and Poddar (2009)). The effect of diminished activity was replicated in an experiment demonstrating reversal of this effect by inhibitors of monoamine oxidase (MAO), the enzyme that metabolizes serotonin. In the second report, the investigators administered the same doses daily for a period of 15 or 30 days and activity was measured following the last administration. Following the 15 or 30 day treatments, activity was increased rather than decreased in a dose-dependent fashion (Fig. 1 in Dalal and Poddar (2010)). One explanation for these contrasting results is the role of two neuronal corticotrophin releasing factor (CRF) receptors that determine an active versus passive response to stress [ 73 ]. The NOAEL from these studies is 1 mg/kg/day based on changes in vertical activity in male rats, on increased serotonin levels in specific brain regions, and increased plasma cortisone levels. The NOAEL of 1 mg/kg/day in these studies is lower than the FDA ADI of 2.5 mg/kg/day. If one were to use a 100-fold safety factor with this NOAEL, the ADI would be 0.01 mg/kg/day.

Red no. 40 and yellow no. 5

Noorafshan et al. [ 35 ] administered Red No. 40 to adult male rats ( N = 10 per dose group) at doses of 0, 7, or 70 mg/kg/day (Table A.6) with and without 200 mg/kg/day of the anti-inflammatory molecule taurine, by gavage for 6 weeks. Both Red No. 40 treated groups performed more reference memory errors and working memory errors in the radial arm maze than controls ( p < 0.01). Taurine administration mitigated this effect. Histomorphology and stereology found that, in the high dose Red No. 40 group, the medial prefrontal cortex volume was smaller, and there were fewer neurons and glial cells in this brain area. Interpretation of these results is somewhat complicated by the lack of information on body weight and brain weight. The LOAEL is 7 mg/kg/day for this study, which is the same as the US FDA and JECFA ADI of 7 mg/kg/day.

These investigators used the same protocol to evaluate the effect of another azo dye, Yellow No. 5 [ 36 ]. Adult male rats ( N = 10 per dose group) were gavaged with Yellow No. 5 at 0, 5, or 50 mg/kg/day for 7 weeks with and without vitamin E. Exploration time in the novel object test was decreased at the high dose (p < 0.01). More days were required for Yellow No. 5 treated rats (low- and high-dose groups were combined) to reach the learning criterion in the radial arm maze test, and more errors occurred during the learning and retention phases. The brain assays demonstrated a smaller volume of the medial prefrontal cortex in the high-dose group, and lower cell count and shorter dendrites with lower spine density at both doses; qualitative alterations in cell shape were described. These effects were ameliorated by concomitant administration of the antioxidant vitamin E. The LOAEL was 5 mg/kg/day, based on morphometry, the same as the US FDA ADI of 5 mg/kg/day and lower than the JECFA ADI of 10 mg/kg/day. If this study were to be used as the basis for setting an ADI, the resulting ADI would be considerably lower than the existing ADI. Changes in the medial prefrontal cortex can be directly related to the cognitive performance of the animals, as this part of the rodent brain is involved in spatial memory, decision-making and attention [ 35 , 74 ], and may predict similar effects in children.

One study in children used several doses and demonstrated a dose response effect on behavioral scores for Yellow No. 5 [ 17 ]. For this study, the investigators recruited 34 children whose parents had brought them to the Royal Children’s Hospital in Melbourne to be evaluated for hyperactivity and 20 children whose parents had no concern about behavior. The children, ranging in age from 2 to 14 years, were enrolled in a double blind, placebo-controlled repeated measures study of the effects of Yellow No. 5 on behavioral score. The investigators developed a Behavioral Rating Inventory for this study that included 11 items measuring irritability, 9 items that measured sleep disturbance, 4 items that measured restlessness, 3 items that measured aggression and 3 items that measured attention span. In addition, the investigators also used the Conners 10-item Abbreviated Parent-Teacher Questionnaire to assess behavior, which focuses on attention related problems. Children were placed on a dye-free diet for at least 6 weeks before the trial, and then given doses (randomly) of 0, 1, 2, 5, 10, or 20 mg Yellow No. 5 with 2 days in between each dosing. Parents rated the behavior daily using the two instruments.

The investigators found 24 children who had significant behavioral responses to dye challenge, based on ranking the behavioral scores for the six dye-challenge days paired with a set of placebo days; these children were labelled as reactors. The mean behavioral scores on dye-challenge days were significantly different than the scores for the placebo (day before) challenge for all dose/placebo pairs ( p < 0.05) in the reactors, while the nonreactors showed random fluctuations in behavioral scores. Using repeated measures ANOVA on the six dye-challenge scores with reactors and nonreactors as the between-groups factor, the authors report a significant between-groups effect ( p < 0.001). There was a dose-dependent effect and the mean score difference between the reactor and the nonreactor groups were significant at doses of 2 mg and higher (p < 0.05). There were no significant differences in mean behavioral rating between the groups on the placebo days. OEHHA identifies 1 mg tartrazine as a NOAEL. The children ranged from 2 to 14 years, with a mean of 7 years. To determine a NOAEL dosage, OEHHA divided the NOAEL of 1 mg by a reference body weight of 25.5 kg for the mean age of 7 years (US EPA, 2011, Table 8–10, based on NHANES 1988–1994); a NOAEL dosage of 0.04 mg/kg/day is obtained. This NOAEL is more than 100-fold lower than the US FDA ADI for Yellow No. 5 of 5 mg/kg/day.

While not all of the human trials demonstrated effects of mixtures of food dyes or of Yellow No. 5 on behavior, the findings of Rowe and Rowe [ 17 ] are supported by some of the other clinical trials in children (Table 7 ). Note that in all these studies, effects were observed at estimated doses lower than the US FDA ADI for Yellow No. 5 of 5 mg/kg/day. One study [ 9 ] reports that in a six-week open trial of the Feingold diet in 55 subjects, ages 3 to 15 years, who had been suspected of reacting to food dyes, 40 children demonstrated improvement when on the Feingold diet, based on assessment of attention span, activity level, distractability, frustration tolerance, and social and manipulative skills by therapists, and teacher and parent questionnaires. In the same study, 8 of the children were challenged with Yellow No. 5 using a double-blinded cross-over design, and two of these children were observed to exhibit strong behavioral responses to the dye. Based on reference body weights for children ages 3 to 15 years, the dosages employed in that study [ 9 ] would have been 0.9–2.7 mg/kg/day. In a double-blind crossover study of 22 children, 4 to 8 years of age, both objective tests for attention and parent and teacher ratings (Conners Parent Teacher Rating Scale) were administered before and after a 4 week dye-free diet, after a 2 week Yellow No. 5 (5 mg daily) challenge and after a 4 week washout dye-free diet [ 7 ]. The investigators report statistically significant effects of Yellow No. 5 based on parental ratings in a subgroup of children whose mothers had reported improved behavior while on the elimination diet. The dose for this range of ages and body weights to the children would be 0.2 to 0.3 mg/kg/day. Levy and Hobbs [ 75 ] reported that mothers’ ratings using the Conners scale were an average of 13% lower when children ( N = 8) ate placebo cookies compared to those containing Yellow No. 5, in a 2 week crossover trial with daily ratings by parents for a 3 h period after eating the cookies. While there were no statistically significant differences noted, the authors reported that this effect “just failed to reach the .05 level of significance”. The dose of Yellow No. 5 in this study was about 0.1 to 0.2 mg/kg/day.

Taken together, these studies provide support for an effect of Yellow No. 5 on behavior and for use of a neurobehavioral endpoint to determine a safe level of exposure for Yellow No. 5 to protect children who respond to this food dye.

Yellow no. 6

There is only one study of Yellow No. 6 with neurobehavioral endpoints [ 47 ]. Some neurobehavioral effects in offspring were reported for preweaning development and maze learning, but it was not possible to draw firm conclusions due to the statistical approach and varying group sizes in the study.

Goldenring et al. demonstrated that sulfanilic acid (1 mg/kg/day I.p.), a common metabolite of the azo food dyes Yellow No. 5 and Yellow No. 6, increased activity in pups following direct administration assessed three times during a treatment extending throughout juvenile development [ 55 ].

Honohan et al. reported gastrointestinal absorption of sulfanilic acid of 37.4% [ 76 , 77 ]. The 1 mg/kg intraperitoneal dose of sulfanilic acid used by Goldenring et al. would be equivalent to 2.7 mg/kg produced in the gastrointestinal tract, which in turn would result from metabolism of 7 mg/kg of orally administered Yellow No. 5. Thus, one could view 7 mg/kg−/day of Yellow No. 6 to be a free-standing LOAEL. This LOAEL is about twice the FDA (3.75 mg/kg/day) and JECFA (4 mg/kg/day) ADIs for Yellow No. 6. The study by Goldenring et al. [ 55 ] indicates the ADIs for Yellow No. 6 may not be adequately protective of neurobehavioral effects.

Current evidence from studies in humans, largely from controlled exposure studies in children, supports a relationship between food dye exposure and adverse behavioral outcomes in children, both with and without pre-existing behavioral disorders. There appears to be considerable interindividual variability in the sensitivity to synthetic food dyes. While there were a range of results in the studies we identified, the majority reported at least some evidence of an association, including higher quality studies. Importantly, none of the factors we examined (e.g., parent vs teacher report, publication year, validated outcome metric) explained the majority of the heterogeneity seen across the study results. For example, although a large fraction of the studies published since 1990 reported statistically significant results (5 of 6 challenge studies), many studies published before 1990 also reported statistically significant results (8 of 19). And, while studies using a validated outcome metric were more likely to report associations, several studies without validated outcome metrics reported similar associations. Despite the various study limitations, we were unable to identify strong evidence for any apparent biases or other factors that invalidated the positive results reported in the literature.

Studies of Yellow No. 5 alone provide evidence that this dye affects children’s behavior. Most of the challenge studies involved administering multiple dyes at the same time so no single offending agent could be identified from those studies. Regardless, studies involving mixtures more closely represent real-life scenarios, where most children are exposed to multiple dyes in a single day.

Importantly, impacts on behavior and/or neurotransmitter systems or cellular architecture in the brain have been observed in animal studies. Several studies examining exposures during development, during pregnancy only, or as adolescents or adults reported changes in activity using a variety of metrics either in the offspring or in the adolescent or adult animals. In utero exposure was observed to have behavioral effects in the adult offspring. Thus, the animal literature provides support for behavioral effects of synthetic food dyes, including those most often consumed.

Taken together, the scientific literature supports an effect of synthetic food dye exposures on neurobehavior in children at environmentally relevant exposure levels.

Comparing estimated exposures we derived from the 2015–16 NHANES dietary interview to the FDA and JECFA ADIs revealed that for most dyes we analyzed, exposures do not exceed the ADIs. The exception is Red No. 3, where the Hazard Index based on the mean ranged up to 15 for the youngest age groups (Table 6 ).

Comparisons of the effective doses in some of the animal studies that measured behavioral or brain effects following exposure to synthetic food dyes indicates that the basis of the FDA ADIs are not adequate to protect neurobehavior in susceptible children. Three of the studies using developmental exposures reported LOAELS that were below the NOAEL that was used for the FDA ADI. Almost all studies in mature animals that measured behavioral changes and/or changes in the brain found effects of the synthetic food dyes at doses lower than the NOAELs used by the US FDA for the derivation of the ADIs. Several studies observe effects on behavior in animals at doses close to or even lower than the existing FDA ADIs. As noted above, the animal studies that form the basis of the FDA ADIs were not capable of detecting the types of neurobehavioral outcomes observed in many human challenge studies.

For four of the dyes with adequate animal studies explicitly reporting neurobehavioral effects, applying results from these studies would result in lower ADIs and likely exceedances of those ADIs from typical food consumption by children. Consumption of over-the-counter medications and vitamins adds to the exposure from foods [ 78 , 79 ].

If the ADI for Yellow No. 5 were based on the one study that evaluated a dose-response in children for behavioral effects, the ADI would be considerably lower. The human challenge studies provide support for an effect of Yellow No. 5 on behavior and for use of a neurobehavioral endpoint to determine a safe level of exposure for Yellow No. 5 to protect children who respond to this food dye.

It is not possible to compare the results of the animal or human mixtures studies to an ADI for a single dye. However, Erikson et al. [ 32 ] reported increased activity in male rats administered synthetic food dye mixtures where each dye was given at less than twice the ADI NOAEL. Shaywitz et al. [ 33 ] and Goldenring et al. [ 56 ] found greater activity and decreased habituation in a rodent model following administration of mixtures at doses near the ADIs. These mixture doses are in the range of doses in human mixture studies. Doses used in the human mixture studies were designed to mimic actual exposures in children.

A broad range of potential mechanisms by which the synthetic food dyes may impact behavior in susceptible children have been proposed. Additional research is warranted including:

Animal testing in immature animals that includes a within-subjects design and measures of neurobehavior more similar to those in the human studies.

Studies of the toxicokinetics of food dyes in humans and animals using modern techniques and including exposures during different life stages.

Mechanistic studies and studies of underlying genetic susceptibility.

Additional adequately powered clinical trials in children of the FD&C batch-certified synthetic food dyes with a cross-over, placebo-controlled, double blinded design utilizing validated outcome measures, inclusion of behavioral assessments by parents, and objective tests of attention and other behavioral measures by trained psychometricians. Such studies should attempt to evaluate whether the response differs by age, gender, ethnicity, race, or socioeconomic status through a design that evaluates dosing on a mg/kg/day basis.

Studies that evaluate the potential long-term impacts of repeated exposures to food dyes in children.

Such research would provide additional data to inform appropriate acceptable daily intakes that explicitly protect children from neurobehavioral effects. In the short-term, the neurobehavioral effects of synthetic food dyes in children should be acknowledged and steps taken to reduce exposure to these dyes in potentially susceptible children.

Availability of data and materials

As this is a review, data sharing is not applicable to this article as no datasets were generated during the current study. Details of the studies we reviewed are contained in the supplementary tables. The study quality review and coding are available in the supplementary files. Exposure estimates were based on the National Health and Nutrition Examination Survey conducted in 2015 and 2016: CDC. 2017. NHANES 2015–2016 Demographics Data. Available: https://wwwn.cdc.gov/nchs/nhanes/search/datapage.aspx?Component=Demographics & CycleBeginYear = 2015: CDC. 2018. NHANES Dietary Data. Available: https://wwwn.cdc.gov/nchs/nhanes/Search/DataPage.aspx?Component=Dietary . CDC. 2019. National Health and Nutrition Examination Survey. Available: https://www.cdc.gov/nchs/nhanes/index.htm .

Abbreviations

Attention deficit hyperactivity disorder

Acceptable daily intake

Corticotrophin releasing factor

Developmental neurotoxicology

US Food and Drug Administration

The NOAEL used by FDA to derive the current FDA ADI

Food Drug and Cosmetic Act, referring to dyes that must be batch-certified per FDA regulations

Food and Agriculture Organization of the World Health Organization

Joint FAO/WHO Expert Committee on Food Additives

lowest-observed-adverse-effect level in a study

Mg of substance per kg body weight per day

Monoamine oxidase

National Institutes of Health

National Toxicology Program

National Health and Nutrition Examination Survey

No-observed-adverse-effect level in a study

Office of Environmental Health Hazard Assessment, California Environmental Protection Agency

Office of Health Assessment and Translation

Postnatal day

Randomized clinical trial

Clinical trials that are randomized cross-over design, double-blinded and placebo controlled

United Kingdom

United States Food and Drug Administration

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Heidbreder CA, Groenewegen HJ. The medial prefrontal cortex in the rat: evidence for a dorso-ventral distinction based upon functional and anatomical characteristics. Neurosci Biobehav Rev. 2003;27(6):555–79.

Levy F, Hobbes G. Hyperkinesis and diet: a replication study. Am J Psychiatry. 1978;135(12):1559–60.

Honohan T, Enderlin FE, Ryerson BA. Absorption, Metabolism and excretion of the azo food dyes amaranth, sunset yellow and tartrazine after oral administration to rats. Fed Proc 1976;35(3):No.682.

Honohan T, Enderlin FE, Ryerson BA, Parkinson TM. Intestinal absorption of polymeric derivatives of the food dyes sunset yellow and tartrazine in rats. Xenobiotica. 1977;7(12):765–74.

Lehmkuhler AL, Miller MD, Bradman A, Castorina R, Mitchell AE. Dataset of certified food dye levels in over the counter medicines and vitamins intended for consumption by children and pregnant women. Data Brief. 2020;32:106073.

Thilakaratne R, Castorina R, Gillan M, Han D, Pattabhiraman T, Nirula A, et al. Exposures to FD&C synthetic color additives from over-the-counter medications and vitamins in United States children and pregnant women. J Expo Sci Environ Epidemiol. 2022.

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Acknowledgements

The authors would like to acknowledge Marjannie Akintunde, Ph.D. for help organizing information from available animal toxicology studies for the OEHHA (2021) review, and Nancy Firchow for library services.

The California state legislature appropriated funding to conduct this review. The legislature had no input into or control over the design of the study, collection, analysis, or interpretation of the data, or writing, reviewing or editing the manuscript.

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MDM and MAM were involved in conception, interpretation of results, and substantially drafted, reviewed and edited the paper. CS designed and conducted the review of the clinical trials of food dyes in children. MSG designed and conducted the review of animal toxicology studies. RC, RT, and AB conducted the exposure assessment and subsequent calculations of hazard index. All authors reviewed the paper.

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Additional file 1: table a.1..

Search Strategy. This table illustrates the literature search strategy. Table A.2 . Clinical trials of synthetic food dyes and neurobehavioral outcomes in children: study details. This table provides study details for the 25 challenge studies in children reviewed by OEHHA. Table A.3 . Clinical trials of synthetic food dyes and neurobehavioral outcomes: coding. This table provides the variables and coding used in the study quality analysis. Table A.4 . Coding dictionary. This table defines the variables and numerical codes used in the study quality evaluation. Table A.5 . Individual dyes. Developmental and adolescent/adult studies. This table provides study details of the animal toxicology studies of individual dyes reviewed by OEHHA. Table A.6 . Dye mixtures. Developmental and adolescent/adult studies. This table provides study details of the animal toxicology studies of dye mixtures reviewed by OEHHA

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Miller, M.D., Steinmaus, C., Golub, M.S. et al. Potential impacts of synthetic food dyes on activity and attention in children: a review of the human and animal evidence. Environ Health 21 , 45 (2022). https://doi.org/10.1186/s12940-022-00849-9

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New report shows artificial food coloring causes hyperactivity in some kids

2 min. read ▪ Published May 24, 2021
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A report released in April 2021 by the state of California—with contributors from UC Berkeley and UC Davis—confirmed the long-suspected belief that the consumption of synthetic food dyes can cause hyperactivity and other neurobehavioral issues for some children.

The report also found that federal rules for safe amounts of consumption of synthetic food dyes do not reflect the most current research and may not be protecting children’s behavioral health.

Over the past 20 years, the percentage of American children and adolescents diagnosed with Attention Deficit/Hyperactivity Disorder (ADHD) has increased from an estimated 6.1% to 10.2%. Concerns over ADHD and other behavioral disorders led the California Legislature to ask the California Environmental Protection Agency’s Office of Environmental Health Hazard Assessment (OEHHA) to conduct the report, which is based on two years of extensive evaluation of existing studies on the seven synthetic food dyes currently approved by the FDA.

“Evidence shows that synthetic food dyes are associated with adverse neurobehavioral outcomes in some children,” said OEHHA Director Lauren Zeise. “With increasing numbers of U.S. children diagnosed with behavioral disorders, this assessment can inform efforts to protect children from exposures that may exacerbate behavioral problems.”

Researchers found that all of the FDA’s Acceptable Daily Intake levels (ADIs) for synthetic food dyes are based on 35- to 70-year-old studies that were not designed to detect the types of behavioral effects that have been observed in children. Comparisons with newer studies indicate that the current ADIs may not adequately protect children from behavioral effects.

“This is the most comprehensive study examining dietary exposure to artificial food coloring in vulnerable populations such as young children and pregnant women. We found that children tended to have higher exposures than adults, and some exposures might exceed regulatory guidelines,” said UC Berkeley Environmental Health Sciences Professor Asa Bradman, who contributed to the report. “We also observed higher exposures in lower-income populations, pointing to the need to improve consumption of, and access to, healthier food.”

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October 2015 Issue

Eating with Your Eyes: The Chemistry of Food Colorings

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By Brian Rohrig October 2015

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Would you drink black water? Clear Pepsi? How about using pink butter or green ketchup? Believe it or not, these products actually existed, and not that long ago either. But there is a reason these food fads did not last. Consumers prefer that the color of food matches its flavor.

The link between color and taste is logical. Since oranges are orange, we expect orange-colored drinks to be orange-flavored. Red drinks should taste like cherries, and purple drinks should taste like grapes. If a food is multicolored, it could be moldy and should not be eaten, unless you are eating blue cheese—which gets its distinct flavor from mold!

An astonishing amount of the foods we eat is processed. These foods are altered from their natural states to make them safe, say, to remove harmful bacteria, or to make them appealing and to prolong their shelf life. About 70% of the diet of the average U.S. resident is from processed foods. Much of what we eat would not look appealing if it was not colored. Think of food coloring as cosmetics for your food. Without coloring, hot dogs would be gray. Yum!

Natural Food Coloring

To avoid so much processed food, some have advocated using natural food coloring, whenever possible. Natural dyes have been used for centuries to color food. Some of the most common ones are carotenoids, chlorophyll, anthocyanin, and turmeric.

Carotenoids have a deep red, yellow, or orange color. Probably the most common carotenoid is beta-carotene (Fig. 1), which is responsible for the bright orange color of sweet potatoes and pumpkins. Since beta-carotene is soluble in fat, it is a great choice for coloring dairy products, which typically have a high fat content. So beta-carotene is often added to margarine and cheese. And, yes, if you eat too many foods that contain beta-carotene, your skin may turn orange. Fortunately, this condition is harmless.

Beta-carotene molecule

Click image to enlarge

Figure 1. Beta-carotene is composed of two small six-carbon rings connected by a chain of carbon atoms.

Chlorophyll is another natural pigment, found in all green plants. This molecule absorbs sunlight and uses its energy to synthesize carbohydrates from carbon dioxide and water. This process is known as photosynthesis and is the basis of life on Earth. Mint- or lime-flavored foods, such as candy and ice cream, are sometimes colored using chlorophyll.

The best natural source for deep purple and blue colors is anthocyanin. Grapes, blueberries, and cranberries owe their rich color to this organic compound. Unlike beta-carotene, anthocyanins—which form a class of similar compounds rather than a single chemical compound—are soluble in water, so they can be used to color water-based products. Blue corn chips, brightly colored soft drinks, and jelly are often dyed with anthocyanins.

More than 500 different anthocyanins have been isolated from plants. They are all based on a single basic core structure, the flavylium ion (Fig. 2). This ion contains three six-carbon rings, as well as many hydroxyl (–OH) groups that make the molecule polar (it has partially negative and partially positive charges) and water-soluble.

Anthocyanin

Figure 2. Chemical structure of an anthocyanin. R 1 and R 2 are functional groups, and R 3 is a sugar molecule.

Another natural food additive you have probably consumed is turmeric, which is added to mustard to impart a deep yellow color. Turmeric is obtained from the underground stem of a plant that grows in India, and it is commonly used as a spice in Indian food. Many U.S. food companies are using turmeric and other natural spices to color their products. Turmeric is also a great acid/base indicator. If you add a basic substance to mustard, it will turn red.

Bugs, anyone?

The next time you enjoy strawberry-flavored yogurt or cranberry juice, you may be eating bugs! But don’t worry. These insects did not contaminate your food by accident. An extract from a type of insect, known as the cochineal, was deliberately added by the food manufacturer.

For centuries, the Aztecs used these insects to dye fabrics a deep-red color . If you crush up 70,000 of these bugs, you can extract a pound of a deep-red dye, called carminic acid (C 22 H 20 O 13 ) (Fig. 3). This dye is safe to ingest, so it found its way into a variety of food and cosmetic products that required a red color. However, the thought of eating bugs is unappealing to some people. Starbucks formerly used cochineal dye in its strawberry-flavored products, but it has since removed this additive in response to customer complaints.

Carminic Acid

Figure 3. Chemical structure of carminic acid

To find out if your food contains bugs, look for carmine, carminic acid, cochineal, or Natural Red 4 on the ingredient label. While these substances are typically considered safe, in rare instances people can have a severe allergic reaction to them, leading to a life-threatening condition called anaphylactic shock.

Why go artificial?

Why bother with artificial, or synthetic, food colorings? Aren’t there enough natural colors to go around? A big reason to go artificial is cost. Synthetic dyes can be mass-produced at a fraction of the cost of gathering and processing the materials used to make natural colorings.

Another reason is shelf life. Artificial dyes might be longer-lasting than natural ones of the same color. Also, although nature produces an impressive hue of colors, those suitable for use as a food dye are limited. But there is no limit to the variety of colors that can be artificially produced in a lab. Considering the thousands of different substances that color our food, it may come as a surprise to discover that the U.S. Food and Drug Administration granted approval to just seven synthetic food colorings for widespread use in food. These food colorings are summarized in Table 1.

Food Colorings

Table 1. Food colorings approved by the U.S. Food and Drug Administration. FD&C stands for laws passed by the U.S. Congress in 1938, called the Federal Food, Drug, and Cosmetic Act.

Artificial food colorings were originally manufactured from coal tar, which comes from coal. Early critics of artificial food colorings were quick to point this out. Today, most synthetic food dyes are derived from petroleum, or crude oil. Some critics will argue that eating oil is no better than eating coal. But the final products are rigorously tested to make sure they contain no traces of the original petroleum. One dye that does not have a petroleum base is Blue No. 2, or indigotine, which is a synthetic version of the plant-based indigo dye, used to color blue jeans.

How to color food

What makes a good food coloring? First, when added to water, it must dissolve. If the dye is not soluble in water, it does not mix evenly. When a typical solute, such as salt or sugar, is added to water, it dissolves, meaning it is broken down into individual ions or molecules. For instance, individual molecules of sugar (C 12 H 22 O 11 ) are held together by relatively weak intermolecular forces. So when sugar dissolves in water, the attractive forces between the individual molecules are overcome, and these molecules are released into solution.

Food-coloring molecules are usually ionic solids, that is, they contain positive and negative ions, which are held together by ionic bonds. When one of these solids dissolves in water, the ions that form the solid are released into the solution, where they become associated with the polar water molecules, which have partially negative and partially positive charges.

Another important property of food coloring is that when it is dissolved in water, the color remains. The reason this happens is that food-coloring molecules absorb some wavelengths of light and let others pass through, resulting in the color we see (Fig. 4). But why wouldn’t sugar or salt absorb portions of the visible light and scatter the rest of it, like food-coloring molecules do? Absorption of light is caused by bringing an electron in a molecule, atom, or ion to a higher energy level. Sugar molecules or the ions in salt require a large amount of energy to do that, so they do not absorb visible light but only light of shorter wavelength—typically ultraviolet light.

Blue and Red Dye

Figure 4. A food dye will appear a particular color because it absorbs light whose color is complementary to the food dye's color, as illustrated here in the case of (a) a blue dye, and (b) a red dye.

Instead, food-coloring molecules typically contain long swaths of alternating single and double bonds (Figs. 1–3) that allow electrons in these molecules to be excited at relatively low energy. The energy required for an electron to jump from that excited state to the ground state corresponds to the energy of visible light, which is why food-coloring molecules can absorb light from the visible spectrum.

What does the future hold?

It is tempting to think that natural products are healthier than artificial ones. But that is not always the case. Cochineal extract is not the only natural dye that can pose a health risk. Serious allergic reactions have also been reported with annatto and saffron—yellow food colorings derived from natural products.

So what will the food of the future look like? Some advocacy groups, such as the Center for Science in the Public Interest, seek to ban all food coloring, because of limited evidence showing that food coloring encourages children to eat junk food. Others envision a different future. One company has already manufactured an edible spray paint called Food Finish, which can be applied to any food. It comes in red, blue, gold, and silver colors.

Eating involves more than just taste. It is a full sensory experience. Both food scientists and chefs will tell you that the smell, sound, feel, and, yes, the sight of your food are just as important as taste to fully appreciate what you eat. That Slurpee would not taste the same if it did not dye your tongue an electric blue. You really can’t help watching what you eat.

Selected references

McKone, H. T. The Unadulterated History of Food Dyes. ChemMatters, Dec 1999, pp 6–7.

U.S. Food and Drug Administration. Overview of Food Ingredients, Additives and Colors. Nov 2004; revised April 2010: http://www.fda.gov/Food/IngredientsPackagingLabeling/FoodAdditivesIngredients/ucm094211.htm#qa [accessed July 2015].

Fiegl, A. Scientists Make Red Food Dye from Potatoes, Not Bugs. National Geographic, Sept 19, 2013: http://news.nationalgeographic.com/news/2013/09/130919-cochineal-carmine-red-dye-purple-sweet-potato-food-science/ [accessed July 2015].

Borrell, B. Where Does Blue Food Dye Come From? Scientific American, Jan 30, 2009: http://www.scientificamerican.com/article/where-does-blue-food-dye/ [accessed July 2015].

Brian Rohrig is a science writer who lives in Columbus, Ohio. His most recent ChemMatters article, “Smartphones, Smart Chemistry,” appeared in the April/May 2015 issue.

Try this Activity!

Can the caramel color of soda be artificially produced.

The caramel coloring of most commercially manufactured colas is derived naturally from caramelized sugar. Suppose for a moment that you are the chemist who works for a bottling plant. You are in charge of formulating the color for the latest batch of carbonated beverages. Unfortunately, the shipment of natural caramel coloring that you were expecting did not arrive, so you have to make the caramel coloring artificially. Can it be done?

Red, blue, and yellow food coloring
Clear plastic cups
Eyedroppers
Sample of commercial cola
Prepare 3 cups of colored water using the food coloring.
Pour a sample of the cola in a separate cup. This sample will remain untouched, and will serve as the control you are trying to replicate.
Using eyedroppers, add colored water from the 3 cups to the single empty cup in an attempt to replicate the color of the cola.

Were you successful? What strategies did you use? Why do you think artificial coloring is typically not used in carbonated beverages?

—Brian Rohrig

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PMC10009361

Status of food colorants in India: conflicts and prospects

Ressin varghese.

School of Bio Sciences and Technology, VIT University, Vellore, Tamil Nadu 632014 India

Siva Ramamoorthy

Food colorants are imperative ingredients for attracting consumers and in deciding their preferences. Here we discuss the current status of natural colorants and synthetic food colorants on the Indian market by appraising the growth of the food colorant market both globally and nationally, based on published case studies on synthetic food colorants (SFCs), rules, and regulations implemented by Food Safety and Standards Authority of India on natural food colorants and SFCs. The substantial lacunae in the research on the impacts of SFCs in the Indian population identified through our literature survey signify the scope and need for appraisal of the issues prevailing in the Indian food colorant market as well as the necessity of renewing the food colorant policies. The illegal use of banned food colorants, the adulteration of natural food colorants, mislabelling of SFCs as natural colorants, and the permitted use of internationally banned food colorants, as well as the unawareness among consumers are serious issues recognized. Appropriate labelling to denote natural food colorants' presence, renewed standards of policy to determine the permitted use of food colorants, comprehensive regulations for the production and use of natural food colorants, stringent rules to constrain the production of toxic SFCs are obligatory to breakdown the dilemma on the Indian food market. Most importantly, awareness and responsiveness should be generated among consumers regarding the illegal use and adulteration of colorants and the need to use natural colorants. We also recommend a logo to designate the presence of natural colorants which will aid the consumers to make the right choice.

Introduction

Any food product is instinctively scrutinized from a visual sense before deciding on purchasing or consumption. Color remains one of the most prominent visual cues contributing to the sensory aspect of foodstuff. There is significant research underscoring that the color of the food psychologically manipulates impelling the expectation of flavor generated in our brain before tasting the food (Velasco et al. 2015 , 2016 ). Consumers’ inclination to a particular food item is primarily visual specific to the color of the food which aids the consumers to predict the flavor and taste of the food. Interestingly, the perception of colour is deep-rooted and comes intuitively to human beings. For ages, the colour of fruits, vegetables, and meat has remained a determinant factor to distinguish raw from ripe and fresh from old or spoilt.

The implication attached to colour to the food impacted the decision of worldwide food manufacturers to add in a variety of colour additives. The natural colorants derived from natural sources including plants and microbes were used to impart colour to food which was later replaced by synthetic colorants. Natural colorants are of two categories: organic (derived from living sources) and inorganic (gold, silver). Synthetic food colorants are chemicals processed from coal tar compounds and most of them contain dyes from the azo group (Dey and Nagababu 2022 ). Moreover, natural identical man-made colours like riboflavin are also available (Sezgin and Ayyildiz 2018 ).

A considerable hike in demand for packaged food fostered the massive use of food colorants. Henceforth, food colorants are added to every packaged and processed food product sold on the markets and almost every food item sold or manufactured by a food industry or a food selling outlet. Additionally, prepared food, e.g. in restaurants and other small food-selling outlets, contains food colouring agents to enhance their visual appeal to entice consumers. However, health hazards reported predominantly in children triggered by these fascinating synthetic colours need to be addressed (Arnold et al. 2012 ). The colossal use of permitted and non-permitted food colorants, their toxicities, and associated adulterations are global concerns with varying gravity in different countries.

India is considered a hub of food diversity in terms of the taste, smell, and colour of foods. The Indian food industry is gaining copious profit by marketing different geographical food styles. Bright attractive colours are the distinctiveness of Indian cuisines which aid the growth of the synthetic food colorant (SFC) industry (CATR 2019 ). Additionally, the market for natural food colorants is on the rise due to an increase in health awareness among consumers (The Hindu 2022 ). Besides the potential adverse health effects of SFCs, this hike led to another serious misbranding of natural colorants as synthetic. The misuse of the label ‘natural’ to attract consumers, the adulteration of natural colorants, and the lack of proper legal regulations in the processing of natural colours are serious issues demanding the imperative attention of authorities. There is a pressing need for proper labelling to distinguish the presence of natural colorants as well as to implement systematic standards on the processing and use of natural colorants. We have proposed a label for distinguishing the incorporation of natural colorants in food. Even though India is known for its multi-hued foods and natural resources of colours (Ramamoorthy 2010 ), hardly a few studies have been published regarding the status of SFCs and natural colorants.

Here we discuss the rapid growth of the Indian food colorant market, the reports on side effects, and the associated regulations in a comprehensive manner. Stringent regulations and government-funded research initiatives are required to tackle together this growing menace.

Rapid growth of the Indian food colorant market

Initially, food manufacturing industries utilized SFCs as additives, and nowadays most of them are not used in the U.S. and Europe, but available in India (The Indian Express 2022 ; Ramamoorthy 2014 ). The rationale for rather using synthetic colorants rather than natural colorants is more stability, less sensitivity to heat, light, and pH, lesser quantities producing higher intensities, and a cheaper mode of synthesis. Examples of prohibited colorants being sold and still in use in other countries include Patent Blue V, Quinoline Yellow, Ponceau 4R, Amaranth, Rhodamine B, and Azorubin (Sachan 2013 ). In 2023, the expected worldwide market size of the food colorant industry will be around $ 3.2 bn. and the compound annual growth rate (CAGR) is expected above 7% (Fig. 1 ). As per reports, developing countries like India, and China is extensively contributing to this surge (MRF 2018 ).

An external file that holds a picture, illustration, etc.
Object name is 3_2023_1427_Fig1_HTML.jpg

Expected CAGR food colourant market growth rate globally and in India (GMI 2022 ; MRF 2018 ). The coloured countries in world map denotes the major colour producers. (Synthetic colours-North America; Annatto-Brazil, Peru; Carotenoids and Anthocyanins-European countries; Safflower-Kazakhstan; Curcumin-India) (Color figure online)

Indian foods are well known for their versatile flavours and uniqueness depending on the country’s region. India’s rich culinary heritage could be seen in provincial cuisines which are a blend of herbs and spices. Unfortunately, the increased demand for packaged foods and competition on the food market has largely promoted the use of SFCs in Indian cuisines. The Indian food colorant market will achieve a CAGR of 5.3% by 2027, which is majorly impacted by synthetic colorants (CATR 2019 ). Further, India’s exports of food colours to the U.S., China, Indonesia, Brazil, Mexico, and Italy are increasing. These export market sizes have increased from $ 203 M to $ 263 M from 2014 to 2019 (CATR 2019 ). The market for natural food colorants is also growing steeply and is expected to reach $ 92.96 M by 2027, growing at a CAGR of 3.90% (EI 2018 ). Colorants like tartrazine, sunset yellow, quinoline yellow, indigo carmine, and amaranth are widely used to enhance the visual aesthetic of Indian food. There are hundreds of companies in India manufacturing these chemicals as food colours (CATR 2019 ). At the same time, the natural food colorant market is on the rise due to an increase in health awareness among Indian consumers and reports on the harmful side effects of synthetic colorants (Satyanarayana 2022 ).

Indian case studies on toxic effects of SFCs

Previous studies have shown the perilous impact of colorants of health. Feingold ( 1975 ) first proposed the parallel link of hyperactivity and increased intake of SFCs in children (Weiss 2012 ). The stemmed investigations, clinical studies, evidences are ongoing. It has been shown by Arnold et al. ( 2012 ) that young children respond to synthetic colorants exceedingly and symptoms included irritability, sleep issues, lack of attention, impulsivity, and hyperactivity [a clinical condition called “Attention-deficit/hyperactivity disorder” (ADHD)]. Using mixtures of Erythrosine, Ponceau 4R, Allura Red, Sunset Yellow, Tartrazine, Amaranth, Brilliant Blue, Azorubine, and Indigotine on female rats and their offspring showed a lessening spatial working memory and sex-specific anti-depressive, anxiolytic behaviors (Doguc et al. 2015 ). In addition, there are also reports on the synergism between colorants and increased incidence of asthma and allergies (Amchova et al. 2015 ). Incidences of cancer associated with SFCs Red 40, Yellow 5, and Yellow 6 were also published (Dey and Nagababu 2022 ). Concerns for genotoxicity after intake of colorants like titanium dioxide, erythrosine, and brilliant black (Silva et al. 2022 ) should be corroborated in in vivo studies. Numerous organic dyes are also regarded as micro pollutants in aquatic environments owing to their toxic effects on aquatic life forms and their subsequent consumers in the food chain (Tkaczyk et al. 2020 ).

Several synthetic food colorants banned in developed countries are regularly used as food ingredient in India, e.g. azo dyes (Pratt et al. 2013 ). Azo dyes possess one or more azo groups (–N=N–) in their chemical structures and are toxic. Tartrazine is one of the widely used azo dyes which is reported to have toxic impacts on the liver, renal function, lipid profiles, and behaviour (Amin and Al-Shehri 2018 ). Likewise, in vitro studies of sunset yellow in rodents resulted in decreased testicle size and deformed lipid profile (Mathur et al. 2005a ; b ). And indigo carmine has been shown to have atrioventricular blocking capacity (Takeyama et al. 2014 ). Regardless, many of these banned dyes are permitted to use in India within certain limits, irrespective of the discrepancies regarding the health impacts of long-term use, the perception of food vendors about the permitted level of use, and the flow of non-permitted food colours on black markets.

Extensive use of non-permitted carcinogenic, neurotoxic colorants is a common practice observed in different parts of India (Nandakumar 2015 ). Melanil yellow, a potent carcinogenic dye banned by the Government of India was found in turmeric, ladoo, and besan at high levels in unorganized food sectors of West Bengal corroborating the lack of quality control and ignorance of food regulations (Nath et al. 2015 ). An analytical study performed to investigate synthetic food colorant usage in different states of India revealed that candyfloss, sugar toys, beverages, mouth fresheners, ice candy, and bakery product samples contain exceeded the limit of colourants. Practices of blending colorants with non-permitted colours (e.g. azo dyes sunset yellow, tartrazine) in mass amounts are also highly prevalent (Dixit et al. 2011 ). Likewise, the exceeding limits of the above SFCs have been detected in samples of coloured crushed ice with 8–20% higher than permitted levels. Non-permitted colorants like rhodamine B, metanil yellow, orange II, malachite green, auramine, quinoline yellow, amaranth, and Sudan dyes was also detected in a variety of foods (Tripathi et al. 2007 ). An extensive survey conducted in bakeries, supermarkets, street food shops, and fast food joints in urban and rural areas of Hyderabad with different age groups of pre-school (1–5 years) and school kids (6–18 years), adult individuals (19–44 years and > 45 years of age) from high-, middle- and low-income groups showed an intake of tartrazine, erythrosine, and sunset yellow higher than the permitted limits of 100 ppm (Rao and Sudershan 2008 ).

The literature shows very few case studies and investigations on the SFCs used in India in the last decade. Rapidly changing lifestyles, high inclination toward packaged foods, and rampant modernization with hectic daily schedules are likely leading to an analogous growth of food colorant levels intake and subsequently, a surge of health disorders. All these factors underline the necessity of utilizing safe natural sources of food colorants.

Forging food and natural colorants with synthetic colorants

The growth of the natural colorant industry is due to an increased consciousness of serious health effects caused by the colossal use of synthetic dyes. However, the factor restraining the prompt growth of the industry is the high cost of natural colorants when compared to synthetic counterparts. Red is the most demanded food colorant followed by green on the Indian market (EI 2018 ). Additionally, the COVID-19 pandemic has influenced the Indian organic market reflecting in 40% increased growth (EI 2018 ). Ironically, consumers who are asserting natural colorants didn’t know what is natural or chemical.

Natural pigments are extracted from both edible as well as non-edible constituents of plants such as flowers, seeds, leaves, fruits, roots, etc. Other major sources apart from plant constituents include marine fungi, insects, and microalgae. Anthocyanins, betalains from grapes, blue berries (Albuquerque et al. 2021 ), and beta-carotenes from carrots include pigments extracted from edible matter. Pigments like crocin from Crocus sativus , bixin from Bixa orellana (Rodriguez-Amaya 2015 ), and lutein from the marigold flower (Adeel et al. 2017 ), carminic acid from cochineal insect (Cooksey 2019 ) are derived from non-edible matter. Natural pigments are often combined with carriers, emulsifiers, and antioxidants to maintain colour stability since they are highly sensitive to air, light, and temperature. Besides, the impeccable curative properties of natural colorants make them apt candidates for chemo preventive therapy through diets (Saini et al. 2020 ).

Natural pigments like carotenoids are often water-insoluble, and organic solvents are employed for the extraction of pigments, leading to the selective separation of the pigment alone, without the minerals or carbohydrates and proteins of the overall natural pigment source. Although the extraction process does not contribute to any structural or chemical change in the pigment, how suitable the highly purified pigment remains for direct intake is a subject of investigation. Antioxidants such as ascorbyl palmitate are added to prevent oxidative degradation of pigments. Water insoluble pigments are altered to water-soluble substances by means of polysorbates, fatty acid sucrose esters, and additional encapsulation through polysaccharides and plant extracts. In India, safety of these additives should be regulated more stringently.

These extraction procedures and the lacunae of abundant raw materials trigger forging. For instance, the anthocyanin level in grapes is 30–750 mg per 100 g. Henceforth, synthetic counterparts of pigments are now accessible. These colorants have a higher market value compared to natural colorants. For consumed carotenoids this means that 76% are synthetic (Leepica and Siva 2021 ). The high cost of naturally derived pigments, the seasonal production (anthocyanins can only be produced during the fruiting season), and the acceptable coloration provided in fewer amounts by SFCs (Beate et al. 2020 ) also subsidize adulteration.

One of the challenges in largely populated developing nations such as India is vigilant and comprehensive monitoring of every small food selling outlet like street food vendors, where detection of illegal and prohibited ingredients, colorants, and several other additives becomes arduous to keep track of and eliminate. Each Indian state having separate procedures for registration and licensing prevents the coordination and regulation of food laws between the states. This leads to a whole heap of ambiguities in the food safety system, and quality regulation fuelled by a disagreeable practice of adulterating natural colorants and marketing synthetic colorants labelled as “natural”.

All natural?

An adulteration is the addition of non-permitted food colour additives to a food product. The accumulation of permitted food colours in exceeding levels is also an adulteration that results in severe health hazards (Gizaw 2019 ). Adulterated milk with chalk or diluted water, coffee seeds spiked with tamarind or mustard seeds), ice cream with pepperoni or washing powder etc. are examples of food fraud in order to make more profit (India today 2018 ). Synthetic colorants emerged in order to deceive consumers regarding food freshness, and to increase the visual attractiveness of food, e.g. malachite is green in vegetables, Metanil yellow in dal, Sudan red in red chili powder (India today 2018 ). Nevertheless, recent strategies are trying to promote the ‘naturalistic fallacy’: Around 490 food samples and 62 samples of natural, herbal colours were collected by the Society of Pollution and Environmental Conservation Scientists (SPECS) from places like Dehradun, Vikas Nagar, Sahaspur, Doiwala, Rishikesh, Haridwar, Rajpur, Mussoorie were adulterated with toxic chemical synthetic dyes (The Tribune 2016 ). One of the world’s most demanded spice and food colorant saffron was found to be adulterated with Sudan dyes (Petrakis et al. 2017 ), and the food colours anthocyanin and betalain with the textile dye ‘Reactive 95’ (Müller-Maatsch et al. 2016 ). Turmeric labelled as natural and organic was highly adulterated with lead chromate (Erasmus et al. 2021 ). Many “organic” labelled foodstuffs like tea powder, jaggery, and edible oils were also reported to contain non-permitted colorants (Pradeshi 2019 ).

Some of the used analytical techniques in the quality assessments of food colorants include spectrometry, thin layer chromatography, ion chromatography, coupled plasma mass spectrometry, gravimetric analysis, and the more sensitive and robust reverse-phase high-performance liquid chromatography (HPLC), quadrupole time of flight mass spectrometry (Martins et al. 2016 ). Although a broad range of food colorants used in foods are keenly inspected and certified by food regulatory boards for health safety, still the presence of prohibited colorants and additives cannot be excluded. Therefore, stricter regulations are obligatory to endorse natural colorants and natural food products without their synthetic counterparts.

Safety of natural colorants

The use of natural colorants in India is good within the permitted limits and in permitted foods (Table 2 ). Unfortunately, cases of adding natural food colorants unrestrained manner have been reported in India, e.g. the addition of annatto to cow milk for a yellowish appearance to resemble buffalo milk (Singh and Gandhi 2015 ). Above and beyond, the label “natural” may not mean that the used pigment for the food colorant has undergone numerous clinical trials without any reports of allergies and carcinogenic effects. Reports of urticaria, angioedema, hypotension, anaphylaxis in hypersensitive individuals on the use of plant pigment pressures for broad systematic clinic-level research on natural pigments (Singh and Gandhi 2015 ). Extensive studies are still necessitated to regulate the admissible levels of pigments although it is natural.

FSSAI approved natural colours and their specifications

The need for more stringent regulations

Authorities.

The global organizations that govern international food standards include Codex Alimentarius Commission (CAC), an inter-governmental body established by FAO and WHO that devises the global food standards of which developing countries like India. Starting with the industrial revolution, the use of synthetic colorants became widespread. The United States first published the list of approved food colours in 1906 to curb the use of toxic chemical dyes. In 1957, the UK published legally approved colorants. In the early 1950s, a joint committee under the leadership of FAO and WHO was established to assess the safety of food additives including colorants (Lehto et al. 2017 ). In India, the Food Safety and Standards Act (2006) prescribes food regulations that are implemented by the Food Safety and Standards Authority of India (FSSAI), which is authorized and functions under the Ministry of Health and Family Welfare of India. As per FSSAI regulations, the final concentration of synthetic food colorants should not exceed 100 ppm in foods and beverages (FSSAI 2009 ).

FSSAI has published the list of natural and synthetic colorants with permitted limits in 2 regulations (2009, 2011). Table 1 provides details of FSSAI-approved synthetic colorants. The non-permitted colours are Fast red, Rhodamine B, Metanil yellow, Bromo-cresol purple, Green S, Sudan 1, Sudan 2, Sudan 3, and Sudan 4, as well as the overuse of permitted colours (Deva 2007). Table 2 shows FSSAI approved natural colorants in India. However, the regulations do not provide a proper definition to define artificial colouring substances. There is no distinction between natural colours and the synthetically produced natural colours. As per FSSAI, food products are sold under the label ‘contains permitted natural colours/permitted synthetic colours/contains permitted natural and synthetic colours’ without any depiction of the used colorant (FSSAI 2011 ). There are also no rules regarding the labelling of the colorant amount added to the food. Only the synthetic food colorant package should indicate the total dye content (FSSAI 2009 ).

FSSAI approved synthetic colorants, their chemical name, structure along with colour index (FSSAI 2009 ; 2011 )

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The international identification number of colours needs to be listed on the label. The presence of annatto colour in oils is labelled as ‘annatto colour in oil’. But there is no specification about other colorants. The readability of these terms for consumers is also questionable. The manufacturing and sale of synthetic colours should be under license and their packaging labelled as ‘food colours’. There are certain specifications regarding the diluents, filler materials for the preparation of synthetic colours. Around 29 diluents including sugar, salt, ethanol, lactose can be used for the preparation of colorants (FSSAI 2009 ). There are permitted levels of synthetic food colorants as well as natural food colorants. But, is there any limit for a maximum daily uptake of permitted food colours in India? Are there any reports on the cumulative effects of these pigments accumulating in our bodies?

Regulatory standards with a proper HACCP system implemented by the industry are the most appropriate means to ensure the industrial production of natural food colorants. In the context of natural colorants production, proper quality checking throughout the supply chain is mandatory, starting from the collection of raw materials to sales, to ensure adulterant-free natural colours. The minimum amounts of toxic metals, pesticides, contaminants, solvent residues, and adulterants should be the benchmark for the quality and approval of natural colorants. Chemicals will stabilize the natural colorants that underwent multiple processing to enhance their colouring. A crystal image of regulations limiting the additional chemicals added to natural colorants should also lessen the adulteration in colorants.

The unrestrained use of colorants in Indian street food markets urgently needs to be appraised. Analysis studies on street food vendors in Chennai underpin that 94% are unaware of even the hygiene practices, and 74% believe that applying for a license is an arduous task (Abraham and Krishnan 2017 ). The conditions are even worse in other metropolitan and rural areas of India, where tourists are largely attracted by street foods (Gupta et al. 2020 ). There should be initiatives from the regional level itself to curtail the sources of banned pigments and to conduct awareness programs for street vendors about the hazards. With the collective effort strengthened by officials at the regional and national level, street food vendors will undoubtedly alter the fortune of Indian cuisines and magnetize more foreigners.

Most of all, the food monitoring frequency should also be renewed on par with the bourgeoning number of small-scale as well as large-scale outlets. Regular updates of legal regulations, and redefining the rationale for permitted food colorants considering the increased dependency on fast food is also desirable. The negligible scientific reports on the use of natural, and synthetic food colorants purpose the requisite of large-scale funded research to understand the current scenario of Indian food colorants, as well to frame solutions considering primarily the health of consumers.

Consumers are impelling factors in the progress of SFC safety and the natural colorant market. A consumer perception investigation performed in Switzerland revealed that consumers prefer natural colour additives based on the risk and regulation factors (Bearth et al. 2014 ). In Germany, people prefer natural colorants derived from plants and not animal sources (Müller-Maatsch et al. 2018 ). A survey conducted in Ethiopia showed that 64.15% of people are not aware of the possible adverse consequences of food additives, and 70.96% would continue to consume them even after discerning the effects Moreover, a large part of consumers was not interested in checking the labels to determine food safety and quality (Getasew et al. 2016 ). This accentuates the need of presenting labelling with good readability and logos to specify the use of natural colorants for consumers to differentiate synthetic from natural colorants (Fig. 2 ). Moreover, awareness programs at regional and national levels will generate the active, knowledgeable, discriminative consumers.

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Proposed logo for denoting natural permitted food colorants in food packages. Pigments extracted from plants are used to colour candies. The candy package is labelled with a proposed logo

Future perspectives

Meeting the rising demands of natural colorants is challenging owing to the tedious extraction procedures, low raw material availability, and high cost. Fruitful strategies such as gene editing technologies like CRISPR/cas 9, optimization of tissue culture techniques, utilization of stress factors like sonication, ultrasound, and magnetic field, and recognition of molecular markers for breeding should be employed for augmenting colorant production. For instance, lipases and carotenogenic genes were expressed in Saccharomyces cerevisiae to induce production of β-carotene (Fathi et al. 2021 ). Production of another natural colourant annatto have been enhanced through application of abiotic elicitors like methyl jasmonate, salicylic acid, paclobutrazol (Parimalan et al. 2011 ). However, research is still going on for the replacement of natural with synthetic substitutes, with studies on natural pigments being at substandard levels and their original function uncertain. Instead, more focus should be given to elucidating promising natural sources for pigments. For instance, beta-carotenes from microalgae such as Chlorella vulgaris and Dunaliella salina (Damergi et al. 2017 ; Xu et al. 2018 ) Additionally, research in exploring the likelihood to produce pigments from endophytic microbial sources (Sujithra and Ramamoorthy 2022 ; Sujithra et al. 2021 ) and large-scale production in bioreactors are providing possibilities for safer food colorant production.

More profound knowledge of natural pigments could help the progress and expansion of healthier, safety-specific functional food products. Besides, the intake of natural colourants in food also provides several health benefits including cardioprotective activity (Varghese et al. 2022 ). There needs to be a focus on scientific innovations and upgradation in the processing and storing conditions of the food such that it would reduce or eliminate the inclusion of food additives and also assist in the contribution of risk-free, secure food products to consumers without compromising on their catch factors and demands. Due to this demand and change in consumer expectations, food industries have been collaborating with top food research institutions to apply scientific knowledge to produce high valued nutrient-enriched food products (Garnweidner-Holme et al. 2021 ). The collaboration also aids the industries to stay updated on the latest progress in food science research to make possible the commercialization of safe, natural, healthy, and functional ingredients and foods in industrial food products. Currently, microparticles are used to enhance the solubility of pigments by a 100-fold, encapsulation inside emulsion system and polymers is used for stability of pigments over longer storage periods. Gamma irradiations have also remained a reliable technique to extend shelf life and stability (Martins et al. 2016 ).

Considering the regulatory and societal aspects of food colorants in India, a regularly updated list of permitted and non-permitted food colorants is needed. There should be an unbiased, unambiguous standard founded on the toxicity levels for permitting the colorants and their use. A delegated apprehensible description of the norms in production and sources of natural colorants is obligatory. Most of all, proper labelling defining the colorant should be provided esteeming the rights of consumers to know the ingredients of the food they intake.

According to Hippocrates, ‘leave your drugs in the chemist’s pot if you can heal the patient with food’. Paradoxically, we are in the state of consuming medicines because of the food we intake. The extensive use of synthetic colorants without any constraints is the causative factor of many health disorders. Some consumers are making informed choices and opting for natural and organic food products and also have become very specific towards their inclination for natural food colorants and flavorings, mainly because of the reports on health and environmental threats that synthetic colorants potentially have. However, the misbranding of “natural” and adulteration of natural colorants are also issues to combat. Collective efforts of authorities, traders, and consumers are required to promote the use of natural colorants without any adulteration.

Acknowlegment

The authors are thankful for the VIT managment for their constant support in the work.

Author contributions

All authors contributed to the study’s conception and design. Conceptualisation, writing-original draft: RV; writing-review, and editing; supervision: SR.

Not applicable.

Declarations

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Food dyes and health: Literature quantitative research analysis
Food dyes: an overview. Food dyes are explored as follows: i) definition and role of food dyes; ii) categorization of food dyes; iii) quantitative research literature analysis. 2.1. Definition and role of food dyes. When shopping for food, the first sensory stimuli that consumers feel is color. A long time before they smell or taste the food ...
Potential impacts of synthetic food dyes on activity and attention in
OEHHA conducted a systematic review of the epidemiologic research on synthetic food dyes and neurobehavioral outcomes in children with or without identified behavioral disorders (particularly attention and activity). ... This paper provides an overview of key portions of OEHHA's peer-reviewed assessment, specifically the evaluation of the ...
Toxicology of food dyes
Substances. Food Coloring Agents. This review finds that all of the nine currently US-approved dyes raise health concerns of varying degrees. Red 3 causes cancer in animals, and there is evidence that several other dyes also are carcinogenic. Three dyes (Red 40, Yellow 5, and Yellow 6) have been found to be contaminated with benzidi ….
Natural Food Colorants and Preservatives: A Review, a Demand, and a
The five food dyes increased the formation of F2-isoprostanes from blood neutrophils at all tested concns. ... Molecular Nutrition & Food Research (2015), 59 (1), 36-47 ... This paper reviews the pharmacol. properties, such as antioxidant, anti-cancer, anti-lipidemic and antimicrobial activity of betalains derived from sources such as red ...
Applications of food color and bio-preservatives in the food and its
Quinoline Yellow: Quinoline yellow, a bright yellow dye with green shade.We provide excellent quality quinoline yellow food colors. These food odors are processed by making use of high grade ingredients and composition of NH-O-Na(SO 3)x, NaO 3 S and COONa-N-OH etc.. Carmoisine: Carmoisine, a red to maroon shade in applications, is admired for its usage in add beverages, ice cream, sweat meant ...
Insight into the Progress on Natural Dyes: Sources, Structural Features
Natural dyes are widely found on land and in the sea, and can be extracted from plants, animals, microorganisms, minerals, and some other materials. Most mineral dyes cannot be used in the food industry because they are harmful to humans. Most plant dyes, animal dyes, and microbial dyes are not only safe and reliable, but also have functions of ...
Natural bio-colorant and pigments: Sources and applications in food
These natural colourants are utilized in a variety of applications, including natural colors in processed foods, dietary supplements, food preservatives, and pharmaceuticals. Furthermore, the review emphasizes the necessity of quality-controlling colourants in the food industry. 2. Sources of natural colors.
Potential impacts of synthetic food dyes on activity and attention in
Finally, we conducted a hazard characterization of the potential neurobehavioral impacts of food dye consumption. We identified 27 clinical trials of children exposed to synthetic food dyes in this review, of which 25 were challenge studies. All studies used a cross-over design and most were double blinded and the cross-over design was randomized.
Potential impacts of synthetic food dyes on activity and attention in
Concern that synthetic food dyes may impact behavior in children prompted a review by the California Office of Environmental Health Hazard Assessment (OEHHA). OEHHA conducted a systematic review of the epidemiologic research on synthetic food dyes and neurobehavioral outcomes in children with or without identified behavioral disorders (particularly attention and activity).
DIET AND NUTRITION: The Artificial Food Dye Blues
Artificial dyes derived from petroleum are found in thousands of foods. 3 In particular breakfast cereals, candy, snacks, beverages, vitamins, and other products aimed at children are colored with dyes. Even some fresh oranges are dipped in dye to brighten them and provide uniform color, says Michael Jacobson, executive director at CSPI.
Food dyes and health: literature quantitative research analysis
Safer Solvent Blends for Food, Dye, and Environmental Analyses Using Reversed-Phase High Performance Liquid Chromatography. Liquid chromatography (LC) is a technique widely used to identify and quantify organic compounds in a complex mixture. Typical operations of high-performance liquid chromatography (HPLC) involve….
New report shows artificial food coloring causes hyperactivity in some
A report released in April 2021 by the state of California—with contributors from UC Berkeley and UC Davis—confirmed the long-suspected belief that the consumption of synthetic food dyes can cause hyperactivity and other neurobehavioral issues for some children. The report also found that federal rules for safe amounts of consumption of ...
Degradation of food dyes via biological methods: A state-of-the-art
The objective of this paper is to conduct a comprehensive assessment of the existing literature about the degradation of food colors through biological methods. 2. Food dyes in industry. Dyes are complex, un-saturated aromatic molecules that have good color, intensity, and solubility ( Muhd Julkapli et al., 2014 ).
(PDF) Natural Dyes Extracted from Flower Crops: A Review of Recent
Natural Dyes Extracted from Flower Crops: A Review of Recent Advances Section A-Research paper Eur. July 2023; ... natural dyes are also used in food and cosmetics. For exam ple, beetroot .
Eating with Your Eyes: The Chemistry of Food Colorings
Synthetic dyes can be mass-produced at a fraction of the cost of gathering and processing the materials used to make natural colorings. Another reason is shelf life. Artificial dyes might be longer-lasting than natural ones of the same color. Also, although nature produces an impressive hue of colors, those suitable for use as a food dye are ...
Synthetic Food Colors and Neurobehavioral Hazards: The View from
The FDA's response would have benefited from adopting the viewpoints and perspectives common to environmental health research. At the same time, the food color debate offers a lesson to environmental health researchers; namely, too narrow a focus on a single outcome or criterion can be misleading. ... Kinsbourne M. Food dyes impair ...
Natural dyes: their past, present, future and sustainability
Natural colorants and dyestuffs are a significant class of non-wood forest products used in the manufacture of confections, other food items, textiles, cosmetics, medicines, leather, paper, paint ...
Artificial Food Colors and Attention-Deficit/Hyperactivity Symptoms
This article summarizes relevant background information, further needs for research, and interim conclusions. 1. AFC Classification Systems. ... Petition to Ban the Use of Yellow 5 and Other Food Dyes, in the Interim to Require a Warning on Foods Containing These Dyes, to Correct the Information the Food and Drug Administration Gives to ...
Research on production of some natural food colorings
extraction parameters are as follows: 50 mL of ethanol, 1.0 g of dry materials, extraction. temperature of 70 ° C, extraction time of 60 minutes for the red and yellow colorings, and 90. minutes ...
A brief review on natural dyes, pigments: Recent advances and future
Natural dyes are being examined in almost all areas where synthetic dyes exhibited high efficiency. Dyes and pigments can make the world beautiful. They are being used since long time and find vide applications in various fields such as food, textile, artifacts and paper industries. This has resulted in an extensive research on development of ...
Status of food colorants in India: conflicts and prospects
Rapid growth of the Indian food colorant market. Initially, food manufacturing industries utilized SFCs as additives, and nowadays most of them are not used in the U.S. and Europe, but available in India (The Indian Express 2022; Ramamoorthy 2014).The rationale for rather using synthetic colorants rather than natural colorants is more stability, less sensitivity to heat, light, and pH, lesser ...
(PDF) A Review on Natural Dyes: Raw Materials ...
Most of the natural dyes showed a very good fastness property in researches. The dyes can be extracted from trees, bark, leaves, flowers, and many more sources. Most of the natural dyes exhibit ...
A review on classifications, recent synthesis and applications of
1. Introduction. The textile dyeing industry has been in existence for more than 4000 years. For all but the last 150 years, the dyes were obtained from natural sources, [1].The big change in dyes came following the discovery of Mauveine by William Henry Perkin, in 1856, while trying to find a route to synthesise Quinine, a drug used to cure Malaria, the scourge of many tropical countries, [2].