Food Chemistry: X 31 (2025) 103039

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Food Chemistry: X
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Two decades of monitoring acrylamide in breakfast cereals: Impact of

market reformulation and compliance with EU regulation☆
Marta Mesías * , Alicia García , Francisco J. Morales
Institute of Food Science, Technology and Nutrition, ICTAN-CSIC, José Antonio Novais 6, 28040 Madrid, Spain

A R T I C L E I N F O A B S T R A C T

Keywords: This study assesses acrylamide levels in 60 ready-to-eat breakfast cereals marketed in Spain in 2025 and com­
Breakfast cereals pares them with data from 2006 and 2018 to evaluate trends related to reformulation, processing type, and grain
Acrylamide composition. Acrylamide concentrations ranged from <15 to 569 μg/kg, with the highest levels in puffed cereals
Exposure
and products made with wheat and spelt. Median concentrations have decreased notably since 2006, with 95 %
Cereal processing
Consumers
of samples now complying with EU benchmark levels. Paired comparisons of cereals from the same brands (2018
vs . 2025) suggest a general reduction in acrylamide content, especially in store-brand products. Estimated di­
etary exposure was 0.007 μg/kg bw/day, posing no neurotoxic or carcinogenic risk for average consumers,
although high consumers may exceed the safety threshold. The results highlight the impact of formulation and
processing on acrylamide formation and underscore the importance of continued monitoring and targeted
mitigation strategies in cereal manufacturing.

1. Introduction Breakfast cereals can be categorised based on the processing tech­

nology used, which influences their physical structure, sensory charac­
Breakfast cereals are widely consumed across all age groups due to teristics, and nutritional value. The main types include flaked, extruded,
their convenience, long shelf-life, and appealing sensory and nutritional puffed, granola, muesli and coated cereals (Lewis et al., 2021). These
attributes. They are typically produced from cereal grains such as maize, manufacturing processes allow for a wide variety of shapes, textures,
wheat, rice, or oats, and often contain added sugars, dried fruits, cocoa, and densities, but also influence the formation of process contaminants
and other ingredients that enhance flavour and consumer acceptance such as acrylamide, due to the different thermal treatments applied
(Shrestha et al., 2024). While especially popular among children and during production (Stadler & Gökmen, 2024).
adolescents, often representing a regular part of their daily breakfast Acrylamide is primarily formed via the Maillard reaction between
routine, they are also commonly consumed by adults. Households with reducing sugars and the free amino acid asparagine, especially at tem­
children are the most frequent purchasers, reflecting the product’s peratures above 120 ◦ C under low-moisture conditions (EFSA (European
strong popularity among school-aged children in Europe (Bellisle et al., Food Safety Agency), 2015). Its presence in heat-treated foods has raised
2018; Cuadrado-Soto et al., 2020; Inchley et al., 2020). Adult con­ public health concerns, as acrylamide has been classified as “probably
sumption, while somewhat lower, remains significant, particularly carcinogenic to humans” (Group 2 A) by the International Agency for
among those seeking quick and convenient breakfast options (Bellisle Research on Cancer (IARC (International Agency for Research on Can­
et al., 2018). In recent years, the adult consumer segment has driven cer), 1994).
market demand for cereals with improved nutritional profiles, such as In response to growing concern, the European Commission adopted
those formulated with alternative grains or pseudocereals, enriched Regulation (EU) 2017/2158, which established benchmark levels for
with fibre, protein, or whole grains, and characterized by reduced sugar acrylamide in various food categories, including breakfast cereals, and
content or clean-label ingredients. Consequently, the compositional and requires food business operators to adopt mitigation strategies (EC
technological characteristics of breakfast cereals have evolved consid­ (European Commission), 2017). In parallel, FoodDrinkEurope (FDE)
erably (Croisier et al., 2021). developed the Acrylamide Toolbox, offering practical guidance to food

☆
This article is part of a Special issue entitled: ‘Acrylamide Research’ published in Food Chemistry: X.
* Corresponding author.
E-mail address: mmesias@[Link] (M. Mesías).

[Link]
Received 14 July 2025; Received in revised form 12 August 2025; Accepted 15 September 2025
Available online 17 September 2025
2590-1575/© 2025 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license ([Link]
nc-nd/4.0/).
M. Mesías et al. Food Chemistry: X 31 (2025) 103039

producers on reducing acrylamide levels through ingredient optimisa­ were anonymised to prevent attribution of specific acrylamide levels to
tion and processing adjustments (FDE (FoodDrinkEurope), 2019). These manufacturers, thereby ensuring neutrality in the interpretation of
regulatory efforts have coincided with reformulation trends in the cereal results.
industry and public health initiatives aimed at improving the nutritional Samples were coded and characterized based on the following at­
profile of processed foods. As a result, manufacturers have increasingly tributes: processing type (rolled, extruded, granola, flakes, biscuits,
adopted strategies to reduce sugar, salt, and acrylamide content, sticks and puffed), major cereal (oat, maize, rice, rye, spelt, wheat or
particularly in breakfast cereals targeting children and health-conscious mixed), type of grain (refined or wholegrain), gluten-free status (yes/
adults (Croisier et al., 2021; MAPA (Ministerio de Agricultura, Pesca y no), without added sugar (yes/no), presence of honey (yes/no), presence
Alimentación), 2025). This aligns with broader public health policies of sweeteners (yes/no), and intended consumer population (children /
encouraging food reformulation to reduce intake of nutrients of concern, general). Nutritional data, as declared on packaging labels, were
such as sugars, sodium, and saturated fats, particularly in products recorded and included energy (kcal/100 g), protein (g/100 g), carbo­
consumed by children (Santos et al., 2021). hydrates (g/100 g), sugars (g/100 g), fats (g/100 g), saturated fats (g/
In this context, continuous monitoring of acrylamide levels in 100 g), fibre (g/100 g), and salt (g/100 g). Furthermore, samples were
breakfast cereals is crucial not only to verify compliance with regulatory classified using European Commission criteria for nutritional claims (EC
thresholds but also to assess the long-term effectiveness of mitigation (European Commission), 2006): source of protein (≥ 12 % energy from
strategies implemented by the industry (EFSA (European Food Safety protein), high protein (≥ 20 % energy from protein), low sugar (< 5 g/
Agency), 2015). Previous studies conducted by our research group re­ 100 g), sugar-free (< 0.5 g/100 g), source of fibre (> 3 g/100 g), and
ported a 60 % reduction in acrylamide concentrations in Spanish high fibre (≥ 6 g/100 g). All samples were homogenized by fine
breakfast cereals between 2006 and 2018, leading to a measurable grinding, vacuum-sealed in polyethylene containers, and stored at 4 ◦ C
decrease in estimated dietary exposure. However, despite this progress, until analysis.
15 % of the products analyzed in 2018 still exceeded the benchmark
levels established by Regulation (EU) 2017/2158 (Mesías et al., 2019), 2.3. LC-ESI-MS-MS determination of acrylamide
highlighting the need for ongoing assessment of current market
conditions. Sample extraction and clean-up were performed according to Mesías
This study evaluates the acrylamide content in a representative se­ and Morales (2015), with minor modifications. Ground sample (0.500 g)
lection of commercially available ready-to-eat breakfast cereals was mixed with 9.4 mL of water in polypropylene centrifuge tubes. The
currently marketed in Spain. The results are compared with historical mixture was spiked with 100 μL of a 5 μg/mL [13C₃]-acrylamide meth­
data to assess changes in acrylamide levels over the past two decades anolic solution (internal standard) and homogenized (Ultra Turrax, IKA
(2006–2025), and to identify trends related to evolving product for­ T10 basic, Germany) for 10 min. Subsequently, 250 μL each of Carrez I
mulations, such as grain type, fibre content, and sugar levels. Compli­ (15 g potassium ferrocyanide/100 mL water) and Carrez II (30 g zinc
ance with current EU benchmark levels is also assessed. In addition, the acetate/100 mL water) solutions were added. Samples were centrifuged
study provides updated estimates of acrylamide dietary exposure from at 9000 ×g for 10 min at 4 ◦ C. The aqueous phase was purified using
breakfast cereals in the Spanish population and applies the Margin of Oasis-HLB cartridges (preconditioned with 1 mL methanol and 1 mL
Exposure (MOE) approach to characterize the associated health risk, in water). A 1 mL aliquot of the supernatant was loaded at 2 mL/min; the
line with EFSA recommendations. initial drops were discarded and the remainder collected. The eluate was
filtered through a 0. 22 μm membrane into amber LC–MS vials.
2. Material and methods Analyses were performed on an Agilent 1200 LC system coupled to
an Agilent triple quadrupole mass spectrometer (Agilent Technologies,
2.1. Reagents and chemicals Palo Alto, CA, USA). Chromatographic separation was achieved using an
Inertsil ODS-3 column (250 × 4.6 mm, 5 μm; GL Sciences Inc., Tokyo,
Potassium hexacyanoferrate (II) trihydrate (98 %, Carrez I) and zinc Japan) maintained at 30 ◦ C. Isocratic elution was carried out with 0.2 %
acetate dihydrate (>99 %, Carrez II) were purchased from Sigma (St. formic acid in water at a flow rate of 0.4 mL/min, with an injection
Louis, MO, USA). [13C₃]-labelled acrylamide (99 % isotopic purity) was volume of 5 μL. Detection was conducted in positive electrospray ioni­
obtained from Cambridge Isotope Laboratories (Andover, MA, USA). zation mode, with a spray needle voltage of 1.0 kV, a nebulizing nitro­
Formic acid (98 %) was sourced from Panreac (Barcelona, Spain), and gen flow of 12.0 L/min, and a source temperature of 350 ◦ C.
deionized water was produced using a Milli-Q Integral 5 purification Quantification was performed in Multiple Reaction Monitoring
system (Millipore, Billerica, MA, USA). Reversed-phase Oasis HLB car­ (MRM) mode, monitoring transitions of m/z 72.1 → 55.1 for acrylamide
tridges (30 mg, 1 mL) were acquired from Waters (Milford, MA, USA), and m/z 75.1 → 58.1 for the [13C₃]-labelled internal standard. Frag­
and 0.45 μm cellulose syringe filters were obtained from Análisis Vínicos mentor voltages were set at 50 V for acrylamide and 76 V for the internal
(Tomelloso, Ciudad Real, Spain). All other chemicals, solvents, and re­ standard, with corresponding collision energies of 11 V and 8 V,
agents used were of analytical grade or HPLC grade. respectively. Method accuracy was verified through participation in the
Food Analysis Performance Assessment Scheme (FAPAS). The most
2.2. Samples recent proficiency tests included coffee (test ID 30117), crispbread
(30118), and potato crisps (30133), with z-scores of − 0.1, 0.1, and −
A total of 60 commercially packaged breakfast cereal samples, 0.2, respectively, confirming the high reliability of the analytical
marketed across Spain and produced by more than 20 manufacturers, method. The limit of quantification (LOQ) was 15 μg/kg. All samples
were purchased from various supermarkets in February 2025. The se­ were analyzed in duplicate, and results were expressed as μg acrylamide
lection included products from both prominent commercial brands and per kg of sample.
widely distributed store-brand lines, reflecting those with substantial
market presence in the Spanish breakfast cereal sector. To minimize 2.4. Evolution of acrylamide content in Spanish breakfast cereals
potential confounding effects, cereals containing chocolate coatings, (2006–2025)
dried fruits, or added nut were deliberately excluded, and only un­
coated, ready-to-eat cereal formats were selected for analysis. Whenever The acrylamide content in breakfast cereals marketed in Spain and
possible, brands assessed in previous longitudinal studies were priori­ analyzed in the present study was compared with data previously re­
tised to ensure comparability across sampling years. Although all ported by our research group in 2018 (n = 60) (Mesías et al., 2019) and
products corresponded to distinct commercial items, brand identities 2006 (n = 60) (Rufián-Henares et al., 2006), in order to assess the

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M. Mesías et al. Food Chemistry: X 31 (2025) 103039

temporal evolution of acrylamide levels in this food category. Overall,

the mean and 95th percentile values obtained for the set of samples
analyzed in different years were compared. More specifically, to assess
the evolution of acrylamide levels in cereals from the same commercial
brand between 2018 and 2025, a Wilcoxon signed-rank test was con­
ducted. This analysis included 34 paired samples, selected from a total of
over 60 collected observations.

2.5. Evaluation of acrylamide exposure from breakfast cereals

Estimated dietary exposure to acrylamide from breakfast cereals was

calculated by combining the average acrylamide concentrations ob­
tained in the current study with per capita consumption data (1.51 kg/
person/year) reported by the Spanish Ministry of Agriculture, Food and
Environment (MAPA (Ministerio de Agricultura, Pesca y Alimentación),
2025). An average body weight (bw) of 70 kg was assumed for intake
estimation, with results expressed as μg/kg body weight/day (EFSA
(European Food Safety Authority), 2012).
Health risk associated with acrylamide intake was assessed using the
Margin of Exposure (MOE) approach, which is the preferred method for
substances that are both genotoxic and carcinogenic (EFSA, 2015). The
MOE is calculated by dividing the benchmark dose lower confidence
limit (BMDL10) by the estimated dietary exposure. In this study, the
BMDL10 values recommended by EFSA were applied: 170 μg/kg bw/day
for neoplastic effects (carcinogenicity) and 430 μg/kg bw/day for non-
neoplastic effects (specifically neurotoxicity).

2.6. Statistical analysis

Given the non-normal distribution of acrylamide concentration data

and heterogeneity in group sizes and variances, all statistical analyses
were performed using non-parametric methods. Group-wise compari­
sons of acrylamide levels by attributes with two variables were con­
ducted using the Kruskal–Wallis H test, followed by post hoc pairwise
analyses using Dunn’s test with Bonferroni-adjusted significance
thresholds (α = 0.05) to control for Type I error inflation. Group-wise
comparisons of acrylamide levels by attributes with more than two
variables were conducted using the Mann–Whitney U test. Median and
interquartile range values were reported as measures of central ten­
dency, and effect sizes were calculated using rank epsilon squared (ε2),
classified according to standard thresholds for small, moderate, and
large effects. To evaluate compliance with benchmark levels within
regulatory classification groups, one-sample Wilcoxon signed-rank tests
were applied to assess whether acrylamide concentrations fell below
regulatory benchmark levels and the longitudinal changes in acrylamide
content between matched breakfast cereal products from 2018 and
2025. All statistical procedures were conducted using SPSS version 29
(SPSS, Chicago, IL) and the R software (version 4.3.2), with imple­ Fig. 1. Acrylamide content in breakfast cereals grouped by (a) the type of
mentation via the following packages: rstatix (for rank-based tests and breakfast cereal and (b) the major cereal.
effect size estimation), exactRankTests (for exact non-parametric infer­
ence), stats (base functions), and ggstatsplot, ggplot2, and ggpubr (for which documented levels between < 23 μg/kg and 556 μg/kg in
graphical representation of results). commercially available breakfast cereals. Additional studies from other
countries have shown comparable variability in acrylamide levels:
3. Results and discussion 41–362 μg/kg (Basaran & Sadighara, 2024), < 27–643 μg/kg (Anwarul
Hasan et al., 2022), 33–744 μg/kg (Nica-Badea, 2022), and 82–486 μg/
3.1. Acrylamide in breakfast cereals: Current levels and influencing kg (Merhi et al., 2020).
factors Acrylamide formation in cereal products is primarily driven by fac­
tors related to formulation and processing. Key determinants include the
Acrylamide concentrations in the analyzed breakfast cereal samples type of cereal grain used, which affects the natural content of asparagine
ranged from below the limit of quantification (LOQ, 15 μg/kg) to 569 and reducing sugars, as well as added ingredients such as sugars or
μg/kg, with a mean value of 114 μg/kg (CI95 %: 87–140 μg/kg) and a honey. Thermal processing parameters, especially baking, extrusion,
median of 83 μg/kg (Fig. 1). Two samples presented acrylamide levels toasting, and puffing, play a major role due to their direct impact on
below the LOQ. One sample (337 μg/kg) was classified as an outlier, and Maillard reaction pathways (FDE (FoodDrinkEurope), 2019; Stadler &
another (569 μg/kg) as an extreme value. Overall, the acrylamide con­ Gökmen, 2024).
centrations observed were within the range reported in recent European In a first approximation, samples were grouped into seven format
studies, such as those conducted in Germany (Lipinski et al., 2024), categories based on their physical structure and processing method:

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M. Mesías et al. Food Chemistry: X 31 (2025) 103039

rolled cereals (n = 8), extruded cereals (n = 13), granola (n = 3), flakes extruded oat products. The single rye-based sample showed a relatively
(n = 19), biscuits (n = 4), sticks (n = 4), and puffed cereals (n = 9) low acrylamide level (41 μg/kg); however, as this product was a rolled
(Fig. 1a). Acrylamide levels varied markedly across these groups. Rolled cereal, likely subjected to mild thermal treatment, it cannot be consid­
cereals exhibited the lowest concentrations (LOQ–58 μg/kg; mean: 31 ered representative of rye-based products in general. Among the cereal
μg/kg), followed by extruded cereals (24–186 μg/kg; mean: 60 μg/kg) types, spelt and wheat exhibited the highest acrylamide concentrations.
and granola (32–118 μg/kg; mean: 78 μg/kg). Intermediate to high Spelt-based cereals averaged 140 μg/kg, with values ranging from 31
values were found in flakes (33–255 μg/kg; mean: 104 μg/kg), biscuits μg/kg in rolled products to 337 μg/kg in puffed formats. Wheat-based
(57–182 μg/kg; mean: 105 μg/kg), and sticks (23–226 μg/kg; mean: 151 cereals had the highest overall mean (202 μg/kg), with a range
μg/kg). Puffed cereals showed the highest acrylamide levels of all cat­ extending from 33 μg/kg in biscuits to 569 μg/kg in puffed products.
egories (122–569 μg/kg; mean: 284 μg/kg). The diversity in acrylamide These differences were statistically significant and reflect both the
levels across cereal types can be explained by their underlying pro­ higher asparagine content of wheat and the processing techniques used.
cessing technologies and thermal intensity (Croisier et al., 2021; Lipinski Finally, cereals formulated with mixed grains showed intermediate
et al., 2024). The type and purpose of processing technologies for ready- values, ranging from non-detectable acrylamide levels in rolled products
to-eat breakfast cereals are extensively described by Ng (2023). Rolled to 182 μg/kg in biscuits.
cereals are produced by steaming whole grains followed by mechanical The dataset evidenced distinct patterns in the use of cereal grains
flattening. The process involves moderate temperatures and minimal across breakfast cereal formats. Maize appears to be predominantly
Maillard reaction, resulting in low acrylamide levels. Extruded cereals utilized in the production of flakes (n = 9) and extruded cereals (n = 6),
are formed by forcing grain-based dough through high-pressure, high- indicating its suitability for both flaking and extrusion technologies.
temperature conditions (typically 120–180 ◦ C), leading to partial gela­ This observation is supported by global industry data, which identifies
tinization and significant browning reactions. Granola is baked at maize as the most widely employed grain in breakfast cereal
moderate temperatures (130–160 ◦ C), usually with added oils and manufacturing, accounting for approximately 36.7 % of ingredient
sweeteners, which can promote acrylamide formation. Flakes undergo usage in 2024 (Mordor Intelligence, 2025). The technological adapt­
several stages, including cooking, drying, rolling, and toasting. The final ability of maize is further supported by its low fibre content and
toasting step can reach high surface temperatures and contributes to favorable starch gelatinization properties, which make it especially
acrylamide variability within this group. Biscuits are typically rich in compatible with extrusion-based processes. Wheat, by contrast, is rep­
sugar and fat and are baked at 180–200 ◦ C. The combination of resented more broadly across formats, with presence in flakes (n = 5),
composition and temperature explains their moderately high acrylamide biscuits (n = 2), sticks (n = 4), and puffed cereals (n = 7), reflecting its
levels. Sticks are often extruded and toasted or baked, and their elon­ versatility in high-temperature processing. Puffed cereals are commonly
gated shape with increased surface area can enhance thermal reactivity. produced from wheat, rice, or spelt due to their ability to expand under
Puffed cereals are produced via oven puffing or gun puffing. Oven extreme thermal conditions. These formats are typically manufactured
puffing involves rapid heating at atmospheric pressure to vaporize in­ through extrusion and rapid expansion techniques. Oats are primarily
ternal moisture. Gun puffing cereal grains to a sudden pressure drop associated with rolled cereals (n = 3), granola (n = 2), and to a lesser
after being superheated, causing explosive expansion. Both methods extent with extruded and flaked formats, consistent with their use in
apply short (30–90 s) but intense heat (200–300 ◦ C), which strongly minimally processed or baked products. Rolled cereal formats are typi­
favors acrylamide formation (Konkubaeva et al., 2025; Ng, 2023). cally produced from oats, rye, or wheat, given these grains’ structural
A Kruskal-Wallis test for independent samples revealed a significant tolerance to steaming and flattening without fragmentation. Spelt and
(p < 0.0001) and large effect size (ε2 = 0.385) of cereal shape on the rice, though less frequent, are each linked to select formats, spelt with
acrylamide levels (Fig. 1a). Subsequent post-hoc pairwise comparisons puffed and flaked cereals, and rice with extruded and flaked types. Rye
using Bonferroni correction due to the heterogeneity of the sample was observed exclusively in the rolled cereal category (n = 1), sug­
groups identified statistically significant differences between puffed gesting limited integration into industrial processing variants.
cereals and both rolled and extruded cereals (p-adjusted < 0.05). No The use of whole grains versus refined grains can significantly in­
other pairwise comparisons were significant after correction for multiple fluence acrylamide formation in cereal products. Whole grains generally
testing, indicating that puffed cereals stand out with distinct acrylamide contain higher levels of free asparagine and reducing sugars, particu­
profiles. These results are consistent with previous studies (e.g., Lipinski larly in the bran and germ fractions, which are largely removed during
et al., 2024), reinforcing the link between processing conditions and refining. Consequently, products made with whole grains are typically
acrylamide formation in breakfast cereal matrices. expected to exhibit higher acrylamide levels after thermal processing
In addition to processing conditions, the type of cereal used in the compared to those made with refined flours (Žilić et al., 2020). How­
formulation also plays a critical role in acrylamide formation, due to the ever, this trend was not observed in the present study, as whole grain
natural presence of its main precursors: asparagine and reducing sugars. samples did not show significantly higher acrylamide concentrations
Asparagine content has been identified as the limiting precursor in (Table 1). Similarly, no significant differences were found when samples
cereal-based matrices. Cereals such as rice tend to have low levels of were grouped by fibre or protein content according to the nutritional
asparagine and reducing sugars, and are therefore expected to yield profile declared by the manufacturers of breakfast cereals (Table 2).
lower acrylamide formation. Rye, wheat, and spelt typically contain Statistically significant differences were only observed in the “source of
higher concentrations, leading to greater acrylamide potential under fiber” and “gluten-free” categories, although the limited number of
similar processing conditions (Žilić et al., 2020; Stockmann et al., 2018, samples in these groups may have affected the robustness of the results
Mesias & Morales, 2025). In this study, acrylamide content varied (Table 1). Neither the presence of honey nor the intended target con­
significantly by grain type (Fig. 1b). Contrary to expectations, the mean sumer group had a significant effect on acrylamide levels. However, a
acrylamide concentration in rice-based samples was relatively high clear association was observed with sugar content: samples with higher
(120 μg/kg). This group included puffed, extruded, and flaked products, sugar levels tended to exhibit higher acrylamide concentrations. In fact,
underscoring the substantial influence of processing intensity and a positive and significant correlation was found between acrylamide and
formulation beyond the raw material itself. Maize-based cereals sugar content in the breakfast cereals (p = 0.014). This finding aligns
exhibited the lowest average acrylamide levels (54 μg/kg), with with the established role of reducing sugars as key precursors in acryl­
extruded corn cereals showing values around 39 μg/kg, and corn flakes amide formation during thermal processing (Stadler & Gökmen, 2024).
reaching 69 μg/kg. Oat-based cereals presented intermediate values, The classification of high protein content, sugar-free, and the presence of
averaging 71 μg/kg, although concentrations varied widely depending sweeteners was not considered due to the low number of samples in
on processing, from non-detectable levels in rolled oats to 187 μg/kg in these groups.

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M. Mesías et al. Food Chemistry: X 31 (2025) 103039

Table 1 leading market segment, accounting for nearly 60 % of total cereal sales.
Average acrylamide content (μg/kg), minimum, maximum and median in ce­ This shift reflects evolving consumer preferences, with increasing de­
reals samples grouped according to different factors. mand for gluten-free, whole grain, sugar-free, and high-fibre products.
Factor n Mean ± SD Minimum Maximum Median This trend is also evident in the findings of the present study. Compared
Type of grain ​ ​ ​ ​ ​
to previous assessments conducted by our research group, breakfast
Refined 30 125 ± 127a 24 569 55 cereals marketed on the Spanish market exhibit lower levels of sugar
Wholegrain 30 102 ± 70a < LOQ 255 89 (13.0 g/100 g in 2025 vs. 15.8 g/100 g in 2018) and saturated fat (0.6 g/
Source of protein ​ ​ ​ ​ ​ 100 g in 2025 vs. 0.8 g/100 g in 2018) (Table 2). In addition, a higher
< 12 % total energy 46 112 ± 106a < LOQ 569 84
prevalence of products with elevated fibre content has been observed. In
> 12 % total energy 14 118 ± 94a 23 337 97
Low sugar content ​ ​ ​ ​ ​ 2025, 67 % of the analyzed cereals contained more than 5 % fibre,
<5% 20 73 ± 74a < LOQ 337 47 compared to 57 % in 2018 (Mesías et al., 2019) and only 22 % in 2006
>5% 40 134 ± 109b 23 569 114 (Rufián-Henares et al., 2006). Furthermore, 50 % of the breakfast ce­
Without added sugar ​ ​ ​ reals sampled were formulated with whole grains. These results are
Yes 18 76 ± 83a < LOQ 337 40
No 42 129 ± 106b 23 569 101
consistent with those reported by Croisier et al. (2021), who observed an
Source of fibre ​ ​ ​ increase in the proportion of whole grain breakfast cereals containing
<3% 5 49 ± 34a 24 107 32 ≥8 g of whole grain per serving, from 67 % to 74 %, between 2013 and
>3% 55 119 ± 105b < LOQ 569 88 2020 in products marketed in Sydney.
High fibre content ​ ​ ​ ​ ​
Along with changes in nutritional composition, the most significant
<6% 26 109 ± 24a 24 569 51
>6% 34 117 ± 15a < LOQ 337 104 aspect of the current market is the profile of the main grains included in
Gluten free ​ ​ ​ ​ ​ breakfast cereals. While in 2006 breakfast cereals were formulated only
Yes 55 44 ± 33a < LOQ 95 33 with rice, corn, and wheat (Rufián-Henares et al., 2006), the current
No 5 120 ± 104b < LOQ 569 88 samples include other grains such as spelt, oats, and rye, a trend that was
Presence of honey
already widespread by 2018 (Mesías et al., 2019). This diverse and
​ ​ ​
Yes 13 151 ± 159a 31 569 88
No 47 103 ± 79a < LOQ 337 82 expanding consumption pattern underscores the importance of contin­
Target consumer ​ ​ ​ uous monitoring of breakfast cereals, evaluating them not only from a
Children 15 152 ± 156a 24 569 56 nutritional standpoint but also regarding the presence of processing
General population 45 101 ± 75a < LOQ 337 85
contaminants such as acrylamide.
Analyses were performed in duplicate. Data are means ± S.D. Different letters
within a factor indicate statistically significant differences (p < 0.05). LOQ: 3.3. Benchmark compliance of breakfast cereals according to EU
Limit of quantitation (15 μg/kg). regulation

According to Commission Regulation (EU) 2017/2158, breakfast

Table 2
cereals are classified into three groups based on the major cereal used in
Nutritional profile of breakfast cereals (per 100 g), as declared by the
their formulation. A benchmark level of 300 μg/kg has been established
manufacturer.
for breakfast cereals containing bran, whole grains, and gun-puffed
Mean ± SD Minimum Maximum
grains (Group 1), as well as those formulated with wheat and rye
Energy (kcal) 375 ± 24 296 429 (Group 2). Breakfast cereals primarily formulated with maize, oats,
Proteins (g) 9.0 ± 2.8 4.5 16.0 spelt, barley, or rice fall under Group 3, with a benchmark level of 150
Carbohydrates (g) 73 ± 12 35 88.8
Sugars (g) 13 ± 12 0.3 40.9
μg/kg (EC, 2017). In the present study, samples were classified
Fats (g) 3.3 ± 2.9 0.6 13.3 accordingly (Fig. 2): 39 products were assigned to Group 1 and 21 to
Saturated fats (g) 0.6 ± 0.4 0.1 2.2 Group 3, with no samples meeting the criteria for Group 2. Within Group
Fibre (g) 8.5 ± 7.4 0.1 40.0 1, samples were further subdivided into whole grain cereals (G1-
Salt (g) 0.6 ± 0.5 0.0 1.8
wholegrain, n = 30) and puffed cereals (G1-puffed, n = 9). When
assessed on a sample-by-sample basis, all products met the benchmark
3.2. Evolution of breakfast cereal formulation and acrylamide content thresholds, with the exception of three puffed cereal samples from
Group 1. The observed compliance with EU benchmark levels for
Breakfast cereal consumption contributes significantly to the intake acrylamide in breakfast cereals can be attributed to several key factors
of essential nutrients such as vitamins and minerals in both children and related to both raw material composition and processing conditions
adults, largely due to the widespread practice of fortification. However, (FDE, 2019). Cereals classified in Group 1, which contain bran, whole
despite their nutritional benefits, breakfast cereals can also contain grains, and rye or wheat, tend to have higher levels of free asparagine
considerable amounts of added sugars and salt, components associated and dietary fibre, both of which can promote acrylamide formation. This
with adverse health effects, including dental caries, obesity, type 2 explains the generally higher benchmark threshold established for this
diabetes, and cardiovascular diseases (Croisier et al., 2021). In response group (300 μg/kg) compared to Group 3 cereals (150 μg/kg) (EC, 2017).
to increasing public health concerns, global initiatives aligned with To assess compliance more rigorously, one-sample Wilcoxon signed-
World Health Organization (WHO) recommendations have been rank tests were conducted to compare acrylamide concentrations in each
launched to improve food environments, particularly those shaping regulatory group against their respective benchmark levels. In Group 1
children’s dietary habits. Within this framework, the European Com­ (benchmark threshold = 300 μg/kg), acrylamide levels were signifi­
mission has promoted innovation, product reformulation, and healthier cantly lower than the reference value (p < 0.001; n = 39), with a large
marketing practices to support the development of improved food op­ effect size (r = 0.784; Supplementary Fig. 1a). Similarly, Group 3
tions with lower sodium, sugar, and saturated fatty acid content, while products (benchmark threshold = 150 μg/kg) exhibited acrylamide
also highlighting dietary fibre and whole grains in cereal-based products concentrations significantly below the regulatory threshold (p < 0.001;
(Santos et al., 2021). n = 21), also with a large effect size (r = 0.877; Supplementary
This initiative is reflected in the current trend of the Spanish market. Fig. 1b). These results indicate strong compliance with EU safety stan­
According to the latest Spanish Food Consumption Report (2024) dards and underscore the effectiveness of current mitigation strategies in
(Mercasa, 2025), breakfast cereals targeted at adults have become the reducing acrylamide content across both benchmark groups.
Compared to previous years in the Spanish market, a progressive

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M. Mesías et al. Food Chemistry: X 31 (2025) 103039

the use of asparaginase enzymes to reduce asparagine content in raw

materials, optimization of baking temperatures and times, and selection
of cereal varieties with lower precursor levels, have been effective in
decreasing acrylamide concentrations. Moreover, differences in cereal
matrix composition affect heat and mass transfer during processing; for
example, Group 3 cereals often have higher moisture content and
different starch structures, which can limit acrylamide formation by
altering reaction kinetics. The progressive decline in acrylamide levels
observed from 2006 to 2025 reflects the combined effect of these miti­
gation measures alongside regulatory pressures encouraging continuous
improvement. Notably, the narrower variability range in recent data
suggests greater control and consistency in processing conditions. These
mechanistic insights not only reinforce the regulatory compliance
demonstrated by our results but also highlight the importance of tailored
strategies depending on cereal type and formulation to effectively
manage acrylamide risk.

3.4. Paired sample-based comparison of acrylamide content in recurrent

commercial cereal products

To assess changes in acrylamide content between 2018 and 2025 in

breakfast cereals from the same commercial brand, a Wilcoxon Signed-
Rank Test was performed. The test evaluates whether the median dif­
ference between paired observations significantly differs from zero,
Fig. 2. Acrylamide content in breakfast cereals (BC-all) and grouped according making it particularly suitable for matched samples. It is important to
to the classification indicated in the Regulation (EU) 2017/2158 for Group 1
note that samples were not strictly matched by lot, formulation, or
(BC-G1), Group 3 (BC-G3), and Group 1 divided in wholegrain (BC-G1-whole­
production code, but were instead considered “paired” based on conti­
grain) and puffed (BC-G1-puffed).
nuity in brand identity and product label across both years. Thus, 34
paired samples, selected from a total of over 60 collected observations,
decrease in acrylamide levels has been observed: from a mean of 292 μg/
were included in the analysis. The paired sample size (n = 34) was
kg in 2006 (Rufián-Henares et al., 2006), to 118 μg/kg in 2019 (Mesías
adequate for statistical inference. However, given the presence of out­
et al., 2019), and 114 μg/kg in 2025 (Fig. 3). Moreover, the 95th
liers in both the 2018 and 2025 datasets, a more conservative and robust
percentile has decreased from 515 μg/kg in 2006 to 331 μg/kg in 2019
rank-based method was adopted. This approach is less sensitive to
and 262 μg/kg in 2025. It is important to highlight that although the
extreme values and well-suited for ordinal comparisons of paired
average values for 2019 and 2025 are similar, the current data set shows
differences.
a narrower range of variability, with values ranging from <15 μg/kg to
As illustrated in Fig. 4, the median acrylamide content in breakfast
337 μg/kg, compared to a wider range of 27–482 μg/kg in 2019. While
cereals was 83 μg/kg in 2018 and 86 μg/kg in 2025. Although this
85 % of breakfast cereals sampled in 2019 complied with the respective
overall shift is not statistically significant, it is noteworthy that cereals
benchmark levels, the present study shows an improvement, with 95 %
of samples falling below the established regulatory thresholds, indi­
cating increased compliance with European safety standards. Similar
results have been reported for breakfast cereals marketed in Turkey,
where only 15 % of the products exceeded the benchmark set by the
European Commission. (Basaran & Sadighara, 2024).
Mitigation strategies implemented in the cereal industry, including

Fig. 3. Overall mean acrylamide levels in breakfast cereal samples from 2006
to 2025, including standard deviations and trends in the 95th percentile. Fig. 4. Paired comparison of acrylamide content in breakfast cereal samples
Different letters indicate significant differences between years (p < 0.05). from 2018 and 2025 using the Wilcoxon Signed-Rank Test (n = 34).

6
M. Mesías et al. Food Chemistry: X 31 (2025) 103039

with higher acrylamide levels in 2018 tended to show substantial re­ exhibited relatively low initial acrylamide levels in 2018, possibly
ductions in 2025. In contrast, cereals with initially lower acrylamide indicating that mitigation efforts had been deployed earlier in response
levels exhibited modest changes. The Wilcoxon Signed-Rank Test re­ to previous regulatory pressure or internal quality standards.
ported a test statistic of 336.00, a p-value of 0.521, and an effect size (r)
of 0.11, indicating a small effect and confirming that the mean values 3.5. Evaluation of acrylamide exposure from Spanish commercialised
show an overall reduction although there is no statistically significant breakfast cereals
change in the median acrylamide concentration between 2018 and 2025
for breakfast cereals. Exposure to acrylamide from breakfast cereals in the Spanish popu­
A more detailed analysis of the results revealed that, among the 34 lation was estimated using the total per capita consumption of this food
paired observations, 15 exhibited positive ranks, indicating a net in­ category reported by the Spanish Ministry of Agriculture, Fishery and
crease in acrylamide content from 2018 to 2025, while 19 showed Food (1.51 kg/person/year) (MAPA, 2025) alongside the acrylamide
negative ranks, suggesting a reduction (Supplementary Fig. 2a). The content measured in the present samples. Considering the minimum and
sum of positive ranks was +721, whereas the sum of negative ranks was maximum acrylamide concentrations observed across all samples, esti­
− 1073, reflecting a stronger overall tendency toward decreased acryl­ mated exposure ranged from <0.06 to 2.35 μg/person/day, with a mean
amide levels across the dataset. Although this directional imbalance value of 0.47 μg/person/day. These results are slightly lower than pre­
suggests a downward trend, the effect was not statistically significant, as vious estimates from 2019, where the average contribution of breakfast
indicated by the overall p-value. The net positive differences in acryl­ cereals to dietary acrylamide exposure in Spain was calculated as 0.54
amide content ranged from +8 to +107 μg/kg, with a mean of +48 μg/ μg/person/day (range: 0.05–2.94 μg/person/day) (Mesías et al., 2019).
kg, while the negative differences ranged from − 182 to − 2 μg/kg, with a Assuming an average body weight (bw) of 70 kg (EFSA, 2012), the
mean of − 56 μg/kg. Interestingly, the five brands with the highest mean daily acrylamide intake from breakfast cereals for the general
acrylamide concentrations in 2018 experienced the greatest reductions population was estimated at 0.007 μg/kg bw, with a range of 0.001 to
by 2025. These particular samples had previously exceeded the bench­ 0.033 μg/kg bw. In comparison, higher exposure levels have been re­
mark threshold of 300 μg/kg, the highest reference value established for ported for breakfast cereal consumption in the Turkish population,
this food category. In contrast, three products showed acrylamide in­ ranging from 0.03 to 0.20 μg/kg bw/day (Basaran & Sadighara, 2024).
creases of approximately 100 μg/kg, which appeared to be associated Risk characterization for acrylamide in breakfast cereals was con­
with the incorporation of higher fibre content in their reformulated ducted taking MOE values of 125 and 10,000 as values indicating no
versions. Nevertheless, their final acrylamide concentrations remained concern for neurotoxicity and carcinogenicity in people, respectively.
below the benchmark threshold. MOE was calculated as the BMDL value divided by respective total
An evaluation of relative differences in acrylamide content between acrylamide exposure; 430 μg/kg bw/day was considered as the BMDL10
2025 and 2018 for each sample provided additional insights (Supple­ value for neurotoxicity, and 170 μg/kg bw/day as the value for carci­
mentary Fig. 2b). Among the positive rank subgroup (n = 15), relative nogenicity, as dictated in EFSA opinion on acrylamide (EFSA, 2005). In
increases ranged from 11.28 % to 73.47 %, with a mean increase of this respect, mean MOE value of 64,179 was obtained for neurotoxicity
38.40 % compared to the original acrylamide levels. In the negative rank (range: 53,750–13,030 for minimum and maximum exposure), indi­
subgroup (n = 19), relative decreases ranged from − 291.49 % to − 4.33 cating no health concern. When comparing with the BMDL10 for carci­
%, with a mean reduction of − 75.96 %. Overall, the paired samples nogenicity, a mean value of 25,373 was calculated (range:
exhibited a mean relative reduction of − 25.5 % in acrylamide content in 212,500–5151 for minimum and maximum exposure). In this case,
2025 compared to 2018. Although these changes were not statistically maximum exposure involves a MOE below the safety limit of 10,000,
significant, the observed downward trend, particularly among products suggesting that it should be considered from a public health point of
with initially high acrylamide concentrations, may reflect again the view. These findings underscore the critical importance of controlling
impact of mitigation strategies or modifications in processing practices both the formulation and, especially, the thermal processing conditions
implemented in recent years. of breakfast cereals to prevent the formation of high acrylamide levels.
Further analysis of the dataset revealed that, among the 34 matched Effective mitigation strategies focused on ingredient selection and
commercial cereal samples, 22 (64.7 %) were non–store-brand products, optimized processing are essential to minimize acrylamide content and
while 12 (35.3 %) corresponded to store-brand cereals. Notably, several thus reduce potential health risks associated with chronic exposure to
store-brand products exhibited substantial reductions in acrylamide this contaminant. Ensuring such control is vital for safeguarding con­
content, often exceeding 50 %, particularly when initial concentrations sumer health while maintaining the nutritional quality of breakfast
were high in 2018. This pattern may reflect proactive reformulation cereals.
efforts by retailers to comply with benchmark threshold or address
growing consumer health concerns. Statistical comparisons between the 4. Conclusions
two groups supported this trend: store-brand cereals showed a mean
absolute reduction of − 31.4 μg/kg and a mean relative reduction of This study provides a comprehensive evaluation of acrylamide levels
− 68.5 %, while non–store-brand products exhibited a smaller mean in breakfast cereals marketed in Spain over the last two decades,
absolute change of − 4.0 μg/kg and an average relative change of +1.2 demonstrating a significant overall reduction in acrylamide concentra­
%. These differences in relative acrylamide reduction were statistically tions, with 95 % of samples from 2025 complying with the benchmark
significant according to the Mann–Whitney U test (U = 70.0, p = levels established by European regulations. The data confirm that cereal
0.0113), suggesting that store-brand products may have implemented type and processing conditions critically influence acrylamide forma­
more effective or recent mitigation strategies during the 2018–2025 tion, as evidenced by the higher concentrations observed in puffed ce­
period. This trend may be partly explained by differences in reformu­ reals and those formulated with wheat and spelt. These results highlight
lation timelines. Large, well-established commercial brands likely the need to consider both raw material composition and thermal pro­
adopted acrylamide mitigation strategies prior to 2018, driven by cessing intensity when developing mitigation strategies.
greater technological capacity and reputational concerns, resulting in While reformulation efforts aimed at improving nutritional profiles,
relatively low acrylamide levels by that time. In contrast, store-brand such as increasing fibre content, reducing sugars and saturated fats, and
products, often produced by third-party manufacturers, may have incorporating more whole grains, were observed, their impact on
implemented such strategies more recently, aligning with evolving acrylamide levels was inconsistent, suggesting that nutritional im­
regulatory guidance or retail-driven quality standards (FDE, 2019). This provements alone may not directly translate to lower acrylamide for­
hypothesis is supported by the fact that many non–store-brand samples mation. The paired analysis of products from 2018 and 2025 further

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M. Mesías et al. Food Chemistry: X 31 (2025) 103039

confirms a general downward trend in acrylamide, especially notable in sectional survey examined in the context of the international breakfast research
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Marta Mesías: Writing – original draft, Validation, Supervision, Evaluation of Carcinogenic Risks to Humans (Volume 60). Retrieved from https
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Software, Resources, Project administration, Investigation, Funding fication-Of-Carcinogenic-Hazards-To-Humans/Some-Industrial-Chemicals-1994
acquisition, Data curation, Conceptualization. Alicia García: Formal Accessed June 2025.
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(2025). Comparison of physicochemical properties, volatile profiles, and 5-Hydrox­
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by “ERDF A way of making Europe”. This work is related to COST Action Applied Science, 15, 559. [Link]
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