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Althaea
ocinalis
*
Althaea ocinalis
Althaea ocinalis
1Laboratory of Innovative Toxicological Research and Analyses, Institute of Medical Studies, MedicalCollege,
Rzeszo´w University, Al. mjr. W. Kopisto 2a, 35-959 Rzeszow, Poland. 2Department of Regulatory and Forensic
Toxicology, Institute of Medical Expertises, ul. Aleksandrowska 67/93, 91-205 Lodz, Poland. 3Department of Food
Chemistry and Nutrition, Medical College, Jagiellonian University, Medyczna 9, 30-688 Kraków, Poland. *email:
toksykologia@ur.edu.pl
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e oversight and regulation of elemental impurities (EI) within the pharmaceutical sector constitutes a notably
crucial and widely discussed subject. Nevertheless, it is oen observed that a scarcity of appropriate scholarly
literature is evident within this domain. In light of the fact that EI does not confer any discernible therapeutic
advantages to patients, meticulous control over their presence in nal pharmaceutical formulations is impera-
tive, aligning with established regulatory standards. A pivotal benchmark in this sphere is exemplied by the
ICH Q3D (R1) directive pertaining to elemental impurities1, which emphatically underscores the necessity
for judicious application of human health risk assessment (HHRA) to delineate an ultimate control strategy.
When evaluating the production lifecycle of a pharmaceutical product, a limited number of EI sources warrant
meticulous attention:
• e active pharmaceutical ingredient (API) embodies residual impurities arising from deliberately introduced
elements (e.g., catalyst).
• Excipients or supplementary constituents of the drug product components.
• e manufacturing apparatus (EI that may conceivably inltrate the drug substance and/or nal drug product
during the manufacturing process).
• Elemental impurities stemming from solvents (notably water), which are unintentional additions and could
potentially be present in the drug substance.
• Container closure systems, encompassing EI that possess the potential to leach into the API and pharmaceuti-
cal product from the enclosure system.
• e aforementioned sources of EI, encompassing those of higher likelihood (API, excipients) and lower risk
(manufacturing equipment, solvent, container closure system), are succinctly encapsulated in a schematic
depiction akin to a shbone diagram, as represented in Fig.1.
An illustrative instance of captivating elemental impurities (EI) within ultimate pharmaceutical formulations
is chromium, a constituent currently regarded as solely pharmacologically active and not classied as an indis-
pensable element2,3. e pharmaceutical sector exhibits a vested interest in comprehending not only the mecha-
nism of action (MoA) of active ingredient(s) but also their potential adverse eects or reactions4. Furthermore,
the process of making informed safety determinations, which encompasses the MoA of impurities, necessitates
integration into modern toxicological risk assessment protocols5. In this specic context, among the diverse array
of oxidation states, a handful of forms emerge as particularly signicant: Cr(0) (in its elemental form), Cr(II),
Cr(III), and Cr(VI)1. From a toxicological vantage point, Cr(VI) stands as the most pernicious variant. Delving
into the speciation of chromium, it becomes evident that a pivotal driver (primary MoA) behind the genotoxic
activity of Cr(VI) (as depicted in Fig.2) is its intracellular reduction from Cr(VI) to Cr(III). is reduction of
Cr(VI) to Cr(III) holds signicance even in an earlier phase of the MoA, given its substantial role in determin-
ing the bioavailability of Cr(VI) upon oral ingestion. is is particularly crucial due to the potential limitation
in the cellular entry of Cr(III) compared to Cr(VI), as the latter faces barriers in traversing cell membranes6.
It merits emphasis that following absorption, Cr(VI) undergoes reduction to Cr(III), culminating in the
generation of Cr DNA adducts and other forms of DNA damage that lead to mutagenesis7,8. is sequence of
events is regarded as the principal MoA (Mode of Action I; Fig.2). e secondary MoA involves the reduction
of Cr(VI), yielding Cr(V), which subsequently prompts the generation of reactive oxygen species (ROS) upon
reaction with H2O2. is process engenders the creation of hydroxyl radicals, ROS, and oxidative stress9,10, ulti-
mately causing DNA impairment and mutation (Mode of Action II; Fig.2). It is noteworthy that both modes of
action can manifest concurrently and contribute to the genotoxic ramications attributed to Cr(VI).
is aspect bears signicant relevance, particularly within the context of human health risk assessment
(HHRA) concerning traditional herbal medicinal products (THMPs). is pertinence is underscored due to the
prevalent presence of Cr(VI) in plants, which constitutes an unstable form subject to alteration under standard
Figure1. e shbone diagram for EI sources in nal pharmaceutical products.
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soil conditions. e availability of Cr(VI) to plants hinges on soil characteristics, primarily soil texture and pH
levels. Nonetheless, other forms, including Cr(III) and several intricate Cr anions (such as the well-documented
CrO42−), could also be readily assimilated by plants11. In the realm of pharmaceutical products, the primary
sources of overall chromium impurities encompass residual catalyst elements12, pigments employed as colorants13,
the leaching of substances from equipment or container closure systems, and environmental contamination (a
factor of pronounced signicance, particularly concerning THMP)1.
Of these sources, environmental contamination is relatively unexplored within scientic discourse regard-
ing the safety of THMP, particularly traditional herbal medicinal products. A notable exemplar among Europe’s
population is the utilization of extracts derived from Althaea ocinalis L., radix (Marshmallow root), a THMP
that nds widespread application as a demulcent remedy for addressing symptoms associated with oral or phar-
yngeal cough, including dry cough. Nevertheless, there exists a conspicuous dearth of studies concerning the
HHRA of elemental impurities in this category of THMP, readily available in pharmacies. It merits highlighting
that all ultimate pharmaceutical products are obligated to adhere to the criteria stipulated within the ICH Q3D
(R1) manual pertaining to elemental impurities. Moreover, given that Marshmallow Root cough syrups are tar-
geted towards distinct demographic subsets—children aged 3–6years, children aged 6–12years, and adults—an
appropriate exposure assessment is imperative.
Furthermore, the formulation of safety determinations encompassing the mode of action of chromium impu-
rities, particularly their neoplastic eects (notably Cr(VI)), necessitates a methodological approach grounded
in the Margin of Exposure (MoE). is approach is particularly pertinent due to the BMDL10 of 1.0mg Cr(VI)/
kgbw/day serving as the point of departure (PD) for the combined incidence of adenomas and carcinomas in
the murine small intestine14. Consequently, the objective of our original investigations resides in proposing
a dual regulatory strategy for evaluating the human health risk associated with total chromium impurities in
Marshmallow Root-based cough syrups available in Polish pharmacies. is approach encompasses:
1. Adherence to the principles outlined in the ICH Q3D (R1) guideline for elemental impurities.
2. e application of a Margin of Exposure (MoE) paradigm, tailored to both pediatric and adult populations,
with specic emphasis on the mode of action of Cr(VI) (utilizing BMDL10 for Cr(VI) as the Point of Depar-
ture (PoD)).
e fundamental workow of this bifurcated regulatory strategy, as applied to the HHRA of chromium within
THMP featuring Marshmallow Root (Althaea ocinalis), accessible through Polish pharmacies, is graphically
depicted in Fig.3.
e rst regulatory approach of our proposed HHRA strategy is based on the ICH Q3D (R1) guideline for
elemental impurities, which is described more clearly in separate steps (1–3).
Step 1: e measurement of total Cr impurities (raw results, determination of total Cr content in samples)
e rst step of our study was the measurement of total Cr impurities in all analyzed samples. In this situation
we decided to present results in raw format (Table1; ve independent replicates for each of three series for each
sample) and graphical form – impurity prole: Fig.4, simple column plot, Fig.5, violin plot with box and Fig.6,
box normal.
e data form Table1 and the Cr impurities prole (Fig.4) in all the samples analyzed of the Marshmallow
Root cough syrups (A–H) show that Cr was present in all the products analyzed products (minimum value to the
maximum value was 1: 8.5). e results obtained are not coherent (range: 1.12–9.61µg/L; median: 3.87µg/L).
Figure2. Possible mode of action for Cr(VI) impurities aer oral uptake.
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Figure3. e basic workow of the innovative double strategy in the Human Health Risk Assessment (HHRA)
of Cr in THMP with Marshmallow Root (Althaea ocinalis) available in Polish pharmacies.
Table 1. Raw results for the measurement of total Cr impurities in analyzed samples.
Sample
Raw results, µg/L
Series
123
A 1.12 1.11 1.13
B 1.60 1.63 1.64
C 6.21 6.19 6.16
D 5.42 5.39 5.43
E 1.67 1.70 1.68
F 2.64 2.61 2.66
G 9.62 9.59 9.63
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Figure4. e prole for Cr impurities in the analyzed samples (A–H) as a column plot.
Figure5. Prole of Cr impurities in analyzed samples (A–H) as a violin plot with box.
Figure6. e prole for Cr impurities in the analyzed samples (A–H) as a box normal plot.
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e lowest levels were observed for the samples: A, B, and E. However, the highest levels were observed for the
samples: H, C, and D. A similar level was observed for sample B (1.62µg/L) and E (1.68µg/L) and for sample F
(2.64µg/L) and G (2.70µg/L). However, all products considered meet the requirements of the ICH Q3D guideline
(1100µg/g1). e violin plot with box (Fig.5) and box normal plot conrms the symmetric distribution of the
data and the illustrated range of the total Cr concentration in investigated products.
It is dicult to compare our results because we analyzed nal pharmaceutical products. Only one article
described by Divrikli etal.17 pointed the level of total Cr content in Marshmallow ower available in Turkey as
4.1 ± 0.1µg/g. erefore, our results are extremely lower than the pointed result of18.
Step 2: Estimation of Cr content with single oral dose
e second required step is to estimate the total chromium content in a single oral dose. For this purpose, the
worst-case scenario should be applied based on doses of each analyzed product described by each manufacturer.
In this approach, we assume the highest amount (mL) of orally administered drug in a single dose (based on
TableS1) and additionally the results from the rst step (Table1). e estimation of Cr content in a single oral
dose to which the patient is exposed for a single dose of the analyzed samples of cough syrups with Marshmal-
low Root is shown in Table2.
It should be noted that the content of total Cr in a single dose, due to dierences in the maximum single dose
for each product, may signicantly aect the nal result.
Step 3: Estimation of Cr content in daily dose
e third step in our toxicological risk assessment of Cr is the estimation of Cr content in daily dose based on
the results of the second step (estimation of Cr content in a single oral dose) and, in comparison, with the oral
PDE value recommended by the ICH Q3D guideline (10,700µg/day). e estimated values of Cr content in
daily oral dose are presented in Table3.
e analysis of the results obtained for the Cr content in daily oral dose shows that the results are incoherent
(50.04–278.40ng/day). Furthermore, the results obtained in the nal steps are relatively low (< 300ng/day).
e comparison of the estimated results with the oral PDE value for Cr in the nal drugs suggested by the ICH
Table 2. e estimation of Cr content in single oral dose of the analyzed samples of cough syrups with
Marshmallow root. SD-standard deviation.
Cough syrups with Marshmallow Root Maximum single dose, mL
e estimation of Cr
content in a single
oral dose
ng/single dose SD
A 15 16.8 0.62
B 15 24.35 1.24
C 15 92.8 0.98
D 15 81.2 0.74
E 15 25.25 0.64
F 15 39.55 0.71
G 15 40.45 0.65
H 5 48.07 0.78
Table 3. Estimation of Cr content in daily oral dose of Marshmallow Root cough syrups (A–H). SD-standard
deviation.
cough syrups with Marshmallow Root Frequency of use, times/day
e estimation
of Cr content
in daily oral
dose
ng/day SD
A 3 50.40 0.62
B 3 73.05 1.24
C 3 278.40 0.98
D 4 243.60 0.74
E 4 101.00 0.64
F 4 158.20 0.71
G 3 121.35 0.65
H 5 240.33 0.78
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Q3D guideline (10,700µg/day) show that all the products analysed are below this value (the highest result was
278.40ng/day).
It should be underlined that we cannot compare the results obtained with any other because to our knowledge
we rst described estimation of Cr content in daily oral dose of cough syrups with Marshmallow Root.
e second regulatory approach based on Margin of Exposure (MoE) for children and adults
e second regulatory approach of our proposed HHRA strategy is the estimation of the margin of exposure
(MoE) for Cr in all investigated samples, which is described more clearly in separate steps (1–2). is approach
is a very universal and useful ‘toxicological tool’ that can be applied to impurities that are genotoxic and carci-
nogenic, regardless of their origin19. Hence, it can be applied for Cr assessment considering the genotoxicity of
Cr(VI) as the worst-case scenario (WCS) (see Fig.2).
Step 1: e estimation of Cr content in daily dose considering specic population groups: children (3–6years;
6–12years) and adults
First, the daily exposure values to a product (ng/kg bw/day) were estimated based on the amount applied and
the frequency of application and the average weight of the specic population groups: children (3–6years old;
6–12years old) and adults. For this purpose, the estimation of Cr in the daily dose was carried out, depending
on age and body weight for each population group (based on WHO growth standards20) was carry out. e
obtained results of a daily dose of Cr depending on specic population groups in analysed samples (ng/kg b.w./
day) are given in Table4.
Step 2: e Margin of exposure (MoE) calculated for Cr in daily dose for each cough syrups with Marshmallow Root
(A–H), depending on age and body weight for each specic population group
As mentioned above, MoE is a very universal and useful ’toxicological tool’ that can be applied to impurities
that are both genotoxic and carcinogenic, regardless of their origin19. MoE can be dened as the relationship
between a point of departure (PODsys; usually historical NOAEL or BMDL10 values from oral studies) and an
estimate of exposure—Eq.(1).
where PODsys—point of departure (mg/kg bw/day); Exposure—exposure (mg/kg bw/day).
In general, a MoE of 10,000 or more, if it is based on the BMDL10 of an animal carcinogenicity study, and tak-
ing into account general uncertainties in the interpretation, would be of low concern from a public health point
of view and might reasonably be considered as a low priority for risk management actions. It has been assumed
that the MoE value of 10,000 (or higher) is considered of low concern from a public health point of view with
respect to the carcinogenic eect19. In this context, the most suitable PoD should be BMDL10 for Cr(VI). e
justication is fact that Cr(VI) is most harmful for health—both MoA can occur and contribute to the genotoxic
eects of Cr(VI) (see Fig.2). Furthermore, since the sequence of adenoma-carcinoma is a well-recognised car-
cinogenesis pathway in the gastrointestinal tract, with a conservative approach, BMDL10 of 1.0mg Cr (VI)/kg
bw per day should be selected for combined adenomas or carcinomas of the small intestine in male and female
mice as PoD for the estimation of MoE for neoplastic changes19. e calculated values of MoE for Cr in daily
(1)
MoE
=
PODsys/Exposure
Table 4. e estimated of Cr in daily dose for each cough syrups with Marshmallow Root (A–H), depending
on age and body weight for each specic population group (ng/kg b.w./day).
Specic population
groups, age
Approximate body
weight
(kg)
THMP with Marshmallow Root
A B C D E F G H
Children, 3–6years old 15–23 3.36–2.191 4.870–3.176 18.560–12.104 16.240–10.591 6.733–4.391 10.547– 6.878 8.090–5.276 16.022–10.449
Children, 6–12years
of age 23–46 2.191–1.096 3.176–1.588 12.104–6.052 10.591–5.296 4.391– 2.196 6.878– 3.439 5.276–2.638 10.449–5.225
Adults 60 0.84 1.218 4.640 4.060 1.683 2.637 2.023 4.006
Table 5. Margin of exposure (MoE) calculated for Cr in daily dose for each cough syrup with Marshmallow
Root (A–H), depending on age and body weight for each specic population group.
Specic
population
groups, age
Approximate
body weight
(kg)
THMP with Marshmallow Root
A B C D E F G H
Children,
3–6years old 15 – 23 297,619.05–
456,349.21 205,338.809
–314,852.841 53,879.310–
82,614.943 61,576.35–
94,417.077 148,514.85–
227,722.77 94,816.68–
145,385.58 123,609.39–
189,534.40 62,413.31–
95,700.41
Children,
6–12years old 23 – 46 456,349.21–
912,698.41 314,852.841–
629,705.68 82,614.94–
165,229.91 94,417.077–
188,834.15 227,722.72–
455,445.54 145,385.58–
290,771.17 189,534.40–
379,068.80 95,700.41–
191,400.83
Adults 60 1,190,476.19 821,355.23 215,517.24 246,305.42 594,059.40 379,266.75 494,437.58 249,653.26
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dose for each cough syrups with Marshmallow Root (A–H), depending on age and body weight for each specic
population group are summarised in Table5.
Despite conservative assumptions, the MoE values obtained for Cr in daily dose for each cough syrup with
Marshmallow Root (A–H) are greater than 10,000, so exposure to Cr would not cause a health risk based on the
MoE-based strategy.
Chromium exhibits a range of oxidation states, with the trivalent (Cr(III)) and hexavalent (Cr(VI)) forms being
the primary impurities found in pharmaceutical samples. Evidently, total chromium impurities were detected
across all examined samples of Marshmallow Root cough syrups (A–H) available in Polish pharmacies. e
observed values display a lack of consistency (spanning from 1.12 to 9.61µg/L; median: 3.87µg/L), suggest-
ing variations in composition potentially stemming from distinct raw material sources employed by dierent
manufacturers. However, the initial phase of the regulatory approach, aligned with the ICH Q3D (R1) guideline
for elemental impurities, arms that all assessed products adhere to the stipulated requirements set forth by
the guideline (1100µg/g1).
e subsequent phase permits the estimation of Cr content within a singular oral dose (ranging from 16.8
to 92.8ng per single dose). While the aggregate Cr levels within a single dose appear relatively elevated in com-
parison to the raw data, they do not pose a threat to patients. e nal stage conclusively veries the safety of
the examined cough syrups containing Marshmallow Root. e comparison of the estimated outcomes with
the permissible daily exposure (PDE) value for chromium in nal medicinal products, as recommended by the
ICH Q3D (R1) guideline (10,700µg/day), underscores that all analyzed products remain below this threshold
(with the highest result recorded at 278.40ng/day). e second regulatory approach, hinging on the MoE con-
cept applicable to both pediatric and adult populations, rearms the safety of the scrutinized samples across all
scenarios (all instances displaying MoE values signicantly surpassing 10,000).
It is worth highlighting that this approach serves as a remarkably versatile and valuable ‘toxicological tool’,
apt for application to impurities characterized by both genotoxic and carcinogenic properties, regardless of
their origins. Given the potential occurrence of two distinct modes of action (MoAs) for orally administered Cr,
both contributing to the genotoxic eects of Cr(VI), the utilization of this approach is crucial for aligning with
contemporary regulatory requisites. Moreover, it is pertinent to emphasize that, while considering the speciation
of Cr, further investigations utilizing the HPLC-ICP-MS technique could provide valuable insights. However,
despite the unavailability of this costly and resource-intensive technique, we arm the safety of the analyzed
total Cr impurities, employing a MoE-based strategy for regulatory compliance, while accounting for worst-case
scenarios of Cr(VI) impurities.
e analyzed samples were cough syrups containing Marshmallow Root (Althaea ocinalis), which are readily
accessible within Polish pharmacies (n = 8). All eligible syrups featuring Althaea ocinalis as ocially registered
traditional herbal medicinal products in Poland were encompassed within the research. It merits acknowledg-
ment that each product was subjected to triple scrutiny. e majority of the examined products fell under the
category of over-the-counter (OTC) medications. e procured items were sourced from local pharmacies situ-
ated in the Małopolska Voivodeship (Kraków, Niepołomice) and Podkarpackie voivodeship (Rzeszów) during
the autumn period of 2022 (September–December). To ensure optimal conditions, the purchased products were
assigned random codes (A–H) and were stored in a light-shielded room at a temperature ranging from 18 to
24°C until the analytical procedures commenced. For comprehensive insight into the analyzed traditional herbal
medicinal products featuring Althaea ocinalis L., radix extracts (A–H), refer to TableS1.
e experimental protocol relied upon demineralized water and concentrated nitric acid. Concentrated nitric
acid (65%) of Merck SupraPur, Darmstadt, Germany, spectroscopic grade, was used for the preparation of solu-
tions. Working solutions of chromium were prepared by diluting 1mg/mL Cr(NO3)3 stock solutions (CertiPUR®)
with demineralized water containing 0.5mol/L nitric acid. e range of applied Cr working solutions spanned
from 0.0 to 100.0μg/L. A certied reference material (BCR-482; IRMM, Belgium) derived from lichen was
obtained and utilized. Purge gas in the form of Argon (Ar) of 5N purity was employed.
e microwave oven MDS 2000 (CEM USA) along with a microwave-assisted digestion methodology was instru-
mental in the acid digestion of samples. Prior to Cr determination, each sample of traditional herbal medicinal
products containing Althaea ocinalis L., radix, underwent homogenization. A measure of 0.5mL was extracted
from each sample, transferred into Teon vessels, and subjected to digestion using 5.0mL of concentrated nitric
acid (HNO3, 63%). e sealed vessels were subsequently subjected to microwave irradiation over a span of 2h.
Post-microwave treatment, the samples were allowed to cool to room temperature (25°C), aer which their
nal volume was adjusted to 20mL. ese cooled samples were then stored in plastic containers as stock sample
solutions until further analysis. A quintuple replication approach was maintained for all samples, augmenting
the precision of the outcomes.
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For the determination of total Cr content, the Perkin-Elmer 5100 ZL atomic absorption spectrometer (Perkin-
Elmer, Norwalk, CT, USA), equipped with Zeeman background correction and operating under the electro-
thermal atomization (ET AAS technique), was employed. e specic time–temperature programme employed
in the graphite furnace atomic absorption spectrometer is elucidated in Table6. e pertinent instrumental
parameters are consolidated in Table7.
e calibration function was constructed based on a series of working solutions of Cr (5.0, 10, 50.0, 100.0,
and 150.0μg/L). ese solutions were meticulously prepared from stock solutions of 1000μg/mL (Cr(NO3)3;
CertiPUR®), employing ultrapure demineralized water within a 0.5mol/L nitric acid matrix. e obtained cor-
relation coecient yielded a satisfactory value (R = 0.999). Recovery assessments were performed, resulting in
a recovery rate of 98.53% for BCR-482; IRMM, Belgium, and 98.2% for Corn Flour, INCT-CF-3. is recovery
rate was calculated as the ratio of the determined level to the known quantity of Cr, expressed as a percentage.
e certied Cr value stood at 0.134mg/kg, while the measured value amounted to 0.136mg/kg.
e limit of detection (LOD), as dened by (3 SD)/a, where SD represents the standard deviation correspond-
ing to ten blank injections and ‘a’ symbolizes the slope of the calibration function, was determined to be 1.67µg/L
for BCR-482; IRMM, Belgium, as well as for Corn Flour, INCT-CF-3. Correspondingly, the limit of quantica-
tion (LOQ), dened by (10 SD)/a, yielded a value of 4.95µg/L for both BCR-482; IRMM, Belgium, and Corn
Flour, INCT-CF-3. As mentioned in the “Chemicals and reagents” section, the certied reference material was
prepared from lichen (BCR-482; IRMM, Belgium) and Corn Flour, INCT-CF-3. Prior to analysis, the samples
were subjected to a drying process at 70°C for a duration of 24h. Post-drying, the samples were transformed
into a solution via microwave digestion, facilitated by a programmable microwave oven (MDS-2000; CEM Corp.,
Mattews, USA). e procedure involved introducing 5mL of nitric acid (65%) to 300mg of the certied refer-
ence material within Teon reaction vessels, allowing a 24-h pre-digestion phase. Subsequent to pre-digestion,
the samples underwent full digestion. Following cooling of the reaction vessels, the contents were quantitatively
transferred to Sarstedt vessels and supplemented with demineralized water to achieve a nal volume of 15mL.
Samples prepared according to this protocol were subjected to analysis using the Perkin-Elmer 5100 ZL atomic
absorption spectrometer, employing the graphite furnace mode.
e anticipated Cr value for BCR-482; IRMM, Belgium was 40.9 ± 1.4mg/kg, while the obtained value stood
at 38.05 ± 0.23mg/kg. For Corn Flour, INCT-CF-3, the projected Cr value was 0.052 ± 0.009mg/kg, and the
measured value coincided at 0.050 ± 0.009mg/kg. e analysis of the aforementioned certied reference materi-
als served as a crucial means of evaluating the traceability and accuracy of the results. e applied methodology
closely mirrored our preceding studies, employing the same analytical apparatus15–17.
Table 6. Time–temperature program in the graphite furnace atomic absorption spectrometer for total Cr
determination.
Step Temperature, °C Ramp, s Hold, s
1 110 5 15
2 180 30 10
3 450 1 5
4 500 5 5
5 1500 15 30
6 2450 0 5
7 2500 1 2
Table 7. e applied instrumental conditions for Cr determination.
Parameter Val u e
Emission source Cr hollow cathode lamp
Wavelength, nm 357.9
Lamp current, mA 0.5
Slit width, nm 0.7
Sample volume, µL 40
Integrated absorbance (peak area) Applied
Background correction Zeeman background correction
Atomization Electrothermal atomization (ETAAS technique)
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Althaea ocinalis
In our study we have two important regulatory issues: requirements of ICH Q3D (R1) guideline for elemen-
tal impurities and exposure for specic population groups. Hence, the double HHRA is needed. It should be
emphasized that the idea of this work is not determination of the total Cr content in the investigated THMP
samples (this is not analytical work). is is only the basis that is used for the comprehensive HHRA including
two approaches for regulatory purposes:
1. the requirements of ICH Q3D (R1) guideline for elemental impurities;
2. Margin of exposure (MoE) for children and adults with special emphasis on Cr(VI) mode of action (BMDL10
for Cr(VI) as PoD).
e basic workow of the applied double regulatory strategy in the HHRA of Cr in THMP with Marshmallow
Root (Althaea ocinalis) available in Polish pharmacies is shown schematically on Fig.3.
e results of ve independent replicates were expressed as the mean ± standard deviation. Obtained results were
analysed using statistical sowares: Excel 2010 (Microso Oce) and Origin 2021 Pro the Ultimate Soware for
Graphing and Analysis (OriginLab Corporation, One Roundhouse Plaza, Suite 303, Northampton, MA 01060,
USA) licensed by the Jagiellonian University in Krakow.
e datasets used and/or analyzed during the current study are available from the corresponding authors on
reasonable request.
Received: 1 August 2023; Accepted: 1 March 2024
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| (2024) 14:5293 | https://doi.org/10.1038/s41598-024-56057-7
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Conceptualisation, M.K. and K.J..; methodology, M.K. and K.J.; design experiments, M.K. and K.J..; soware,
M.K. and K.J.; validation of the experiments, M.K. and K.J.; analysis of the data, K.J.; writing—original dra
preparation, K.J.; writing—review and editing, K.J.; All authors have read and agreed to the published version
of the manuscript.
e authors declare no competing interests.
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 024- 56057-7.
Correspondence and requests for materials should be addressed to K.J.
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