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Effects of zinc supplementation on duration and action of botulinum toxin

applied to face muscles: A systematic review of randomized clinical trials

Article in Journal of Trace Elements and Minerals · June 2023

DOI: 10.1016/j.jtemin.2023.100080

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 Journal of Trace Elements and Minerals 5 (2023) 100080

Contents lists available at ScienceDirect

Journal of Trace Elements and Minerals

journal homepage: www.elsevier.com/locate/jtemin

Effects of zinc supplementation on duration and action of botulinum toxin

applied to face muscles: A systematic review of randomized clinical trials
Hélio Trindade Junior a, Caroline dos Santos Melo b, Renata Rabello Mendes a,
Ramara Kadija Fonseca Santos c,∗
a
Nutrition Department. Federal University of Sergipe, São Cristóvão, Sergipe, Brazil
b
Nutrition Sciences Post-Graduate Program, Department of Nutrition, Federal University of Sergipe, Sergipe, Brazil
c
Graduate Program in Health Sciences, Federal University of Sergipe, Aracaju, Sergipe, Brazil

a r t i c l e i n f o a b s t r a c t

Keywords: Background: Zinc supplementation has been used to improve the effects of botulinum toxin (BT) on facial muscles.
Botulinum toxins However, a scientific consensus regarding this has not been reached. We aimed to discuss the effects of zinc
Zinc supplementation, isolated or phytase-associated, on the duration and action of BT in the face muscles of adults
Zinc compounds
and the elderly.
Esthetics
Methods: According to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guide-
lines PRISMA and registered in the International Prospective Register of Systematic Reviews PROSPERO nº
CRD420222319473, database searches were performed in PubMed, Embase, Lilacs, Bireme, Scope, Open The-
sis, Cochrane Library, Clinical Trial, Web of Science, and Google Scholar using the Medical Subject Headings
“botulinum toxin”, “zinc”, and “supplementation” in May 2023. Exclusion criteria: Studies involving children or
teenagers. Inclusion criteria: Randomized clinical trials (RCTs), published as full-text versions developed with
females and males at least 18 years old, that compared the effect of zinc supplementation on the duration and
action of BT in facial muscles with a placebo. We are not delimited language and year of publication. All studies
included as evaluated about risk of bias using the Revised Cochrane risk-of-bias tool for RCTs.
Main findings: Two RCTs were eligible, involving 123 individuals of both genders living in the United States of
America and Egypt. In individual analysis of the studies the administration of mg.day−50 of zinc citrate supple-
mented with phytase increased the duration of BT treatment by 29.6% for the treatment of hemifacial spasms,
blepharospasms, and cosmetic facial rhytids, according to the evaluators; 88% of the individuals treated for ble-
pharospasms were “hard to treat” and 47% of those treated for blepharospasms were “easy to treat”, affirming
improvements after BT intervention. Supplementation with mg.day−50 zinc gluconate without phytase increased
the smiling-type commissure by 76.9%.
Principal conclusions: The botulinum toxin is zinc-dependent metalloprotein but, was not scientific evidence to
clinical practice of zinc supplementation for improve BT action in face muscle.

Introduction for the treatment of blepharospasm, hemifacial spasm, and headache

Botulinum toxin (BT) is an active compound of the botulinum neu-
rotoxin produced by anaerobic bacteria and gram-positive Clostridium
botulinum [1,2]. BT types A, B, C, D, E, F, and G are the most extensively
evaluated [1–3]. BT has been used for esthetic purposes and the treat-
ment of clinical neurological conditions [2,3]. Aesthetically, the type
A botulinum toxin (BTA) is normally applied for the treatment of fa-
cial asymmetries, hyperhidrosis, frown lines, gummy smile, periorbital,
periorbital, glabellar, and nasal rhytids, and eyebrow elevation [2,3],
whilst, for therapy of neurological conditions BTA has been applied
[2,4,5].
The BTA effect occurs through synaptosome-associated protein 25
(SNAP-25) cleavage through zinc-dependent proteolytic activity [6,7].
SNAP-25 is a protein of the presynaptic membranes and is responsible
for binding vesicles loaded with acetylcholine to cellular membrane re-
lease of the neurotransmitter [8]. Cleavage of SNAP-25 by BTA prevents
the release of acetylcholine into the synaptic cleft, preventing the trans-
mission of nerve impulses and leading to muscle relaxation [9].
The increased use of BTA for esthetic purposes has drawn the atten-
tion of researchers, especially considering the toxic potential of excess

∗
Corresponding author.
E-mail address: rkadijanutri@gmail.com (R.K.F. Santos).

https://doi.org/10.1016/j.jtemin.2023.100080
Received 15 February 2023; Received in revised form 23 May 2023; Accepted 15 June 2023
Available online 16 June 2023
2773-0506/© 2023 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)
H.T. Junior, C.d.S. Melo, R.R. Mendes et al.
BTA [10,11]. The BTA application technique and dosage seem to be the
main factors causing adverse effects [12]. Studies showed that repeated
applications of BTA in less than 12 weeks or doses exceeding 300 units
contribute to increased production of antibodies that neutralize the ac-
tion of BTA [10,11]. A single dose of 3500 units can lead to human
poisoning and 30,000 units is considered a lethal dose [10,13]. Simple
adverse reactions to BTA application include nausea, fatigue, malaise,
flu-like symptoms, and rashes at sites distant from the injections, and the
most common significant adverse effect is unwanted weakness, which
can impair routine activities [11].
The effects of BTA in terms of aesthetics begin to be observed after
24 h of application, and the maximum effect occurs after 15 days but,
changes are observed after six weeks and six months [2,14]. The action
time of BTA can be dependent on both the dose [15,16] and the applica-
tion objective [17]. Furthermore, the action of BTA is zinc-dependent,
due to the activity of zinc in BT activation and subsequent cleavage of
SNAP-25 [18].
Although the application of BTA is widely used for esthetic purposes,
few clinical trials evaluated the effect of the zinc supplementation in the
action of BTA in face muscles, in addition, is observed disagree between
studies published. A pioneering study showed that BT action improves
after zinc and phytase supplementation [5]; however, this result was
considered a methodological failure that was impaired in the study [19].
In fact, another study showed that zinc supplementation without phy-
tase increased the time and effects of BTA in face muscles [20].
Thus, is observed the lack of scientific consensus regarding the ef-
fect of zinc supplementation in adjuvant therapy on the effects of BTA
on facial muscles. The common practice of this conduct in medical of-
fices shows the need to establish scientific evidence on the subject, in
order to guide the clinical practice of the supplemental use of zinc for
this purpose, as well as to justify the need to develop more clinical trials
that evaluate this therapy. Therefore, to answer the question: “Does zinc
supplementation, alone or associated with phytase, improve the effect
of BT applied to face muscles in adults?”, we are proposed this system-
atic review with main objective of the effect of zinc supplementation,
with or without phytase, on the duration and action of BTA on facial
muscles.
Methods
The study protocol was registered with the International Prospec-
tive Register of Systematic Reviews (PROSPERO) database (registration
number: CRD420222319473). This systematic review was performed
according to the Preferred Reporting Items for Systematic Reviews and
Meta-Analyses (PRISMA) guidelines supplemented by the Cochrane Col-
laboration Handbook for Systematic Reviews of Interventions [21].
The search question and eligibility criteria were defined according to
Population Intervention, Comparators, Outcomes, Study types (PICOS).
a) Population: adults, both genders, who were administered BT into
face muscles;
b) Intervention: zinc supplementation with or without phytase;
c) Comparator: adults, both genders, who were administered BT into
face muscles but did not receive zinc supplementation;
d) Outcome: time and effect of BT action in face muscle;
e) Study type: Randomized Clinical trials (RCTs);
Searches of PUBMED, EMBASE, LILACS, BIREME, SCOPUS, OPEN
THESIS, Cochrane Library, Clinical Trials, databases and gray literature:
Web of Science and Google Scholar were performed in May 2023 us-
ing Medical Subject Heading (MESH) terms “botulinum toxin”, “zinc”,
“supplementation” combined with booleans markers “OR” and “AND”.
We checking the list of references in the included studies. The search
strategies according each database are presented in supplementary
material 1.
2
Journal of Trace Elements and Minerals 5 (2023) 100080
Study selection and data extraction
The inclusion criteria were RCTs published as full-text versions de-
veloped with females and males at least 18 years old. The studies will
show the dose of zinc supplementation, methods of outcome measure-
ments, and the effects of zinc supplementation and placebo on the du-
ration and action of BT in facial muscles. Studies involving children or
teenagers were excluded. Studies published in non-English languages
were translated using professional translation services if necessary.
Selection of studies was performed independently by two evaluators
(H.T.J. and C.S.M) following two steps: reading the title and abstract,
and reading the full text. Disagreements in any step were resolved by
a third evaluator (R.K.F.S). The reference lists of the articles included
were checked to identify studies that could answer the search ques-
tion.
The concordance level between authors in the selection steps was
evaluated using the coefficient Kappa, which exhibited a range of < 0 to
1, with < 0 = no agreement, 0–0.19 = slight agreement, 0.20–0.39 = fair
agreement, 0.40–0.59 = moderate agreement, 0.60–0.79 = substantial
agreement, and 0.80–1 = near-perfect agreement, according to Landis
and Koch [22].
Data extraction was performed following the recommendations of
Consolidated Standards of Reporting Trials tools for RCTs [23]. Informa-
tion regarding the characteristics of the studies (author, year of publica-
tion, title, objective, randomised type, and blinding), included popula-
tion (age, gender, race), intervention (time, type, and dose), statistical
analysis, main results, discussion, and limitations.
Risk of bias assessment
The risk of bias assessment of each RCT included in this systematic
review was performed using the Revised Cochrane risk-of-bias tool for
RCTs (RoB 2) [24]. The risk of bias assessment of crossover RCT was
performed using the Revised Cochrane risk of bias (RoB 2) tool for RCTs
with additional concerns for crossover [25], which includes in domain
1 the S domain: bias due to period and transition effects.
Five domains were evaluated: (1) bias arising from randomization,
(2) bias due to deviations from intended intervention, (3) bias due to
missing outcome data, (4) bias in the measurement of outcomes, and
(5) bias in the selection of the reported results.
A study was considered low risk when all domains were considered
low risk, high risk when at least one domain was considered high risk,
and of some concerns when it did not show high risk in any domain
evaluated and showed some concerns in all domains [24].
Results
After database searching, 165 reports were identified of which 117
had their titles and abstracts read in the first step. The evaluators shared
substantial agreement during this phase (Cohen’s Kappa: 0.79). Five re-
ports were fully read; in this phase, the evaluators presented almost per-
fect agreement (Cohen’s Kappa: 1.00). After two phases of selection, one
RCT and one crossover were included in this systematic review. We did
not include reports after checking the list of references in the included
studies. The steps involved in the study selection phase are shown in
Fig. 1.
Study characteristics
A total of 123 individuals were evaluated in the two RCTs included in
this systematic review. The studies were conducted in Houston, United
States of America [5], and Cairo, Egypt [[20]]. Only one study reported
the gender and age of the population, with 23 (92%) females and 2 (8%)
males [20], with an average age of 25 ± 4.5 years in the test group and
25 ± 4.0 years old in the placebo group [20].
H.T. Junior, C.d.S. Melo, R.R. Mendes et al. Journal of Trace Elements and Minerals 5 (2023) 100080

Fig. 1. Flowchart PRISMA - Phases of study selections in-

cluded in this systematic review.

Table 1
Main characteristics, population and intervention of studies included in this systematic review.

Study Design/ Blinding Total (n) Sex (F/M) Age (years) Type / dose Intervention

Shemais et al. RCT / double blind 25 (23/2) TG: 25 ± 4.5 BTA 50 mg of zinc gluconate
(2021) [19 ] CG: 25 ± 4.0 5 UNI
Koshy et al. (2012) RCT / double blind / 98 NP All groups: BT A/B 10 mg of zinc gluconate
[5 ] crossover > 18 NP 50 mg of zinc citrate + 3000
UNI of phytase
Legend: TG: test group; CG: control group; NP: not presented; UNI: units. BTA: type A botulinum toxin; BT A/B: types A and B botulinum
toxin.

In one study, BTA and botulinum toxin type B (BTB) were used to
decrease blepharospasm, hemifacial spasm, and cosmetic facial rhytids,
and the outcomes were measured using view analysis [5].
In another study [20], BTA was applied to decrease gummy smile,
which is characterized by the excessive gingival display when smiling,
and outcomes were assessed through patient self-reporting associated
with the assessment of photographs taken before and after the proce-
dure.
Daily doses of zinc gluconate supplementation were administered
from four days before BTA application [20] or mg.day−50 of citrate of
zinc associated with 3000 units of phytase and mg.day−10 of zinc glu-
conate without phytase from the fifth day, the day of BTA application
[5]. The studies did not evaluate dietary zinc intake or body zinc re-
serves [5,20].
Characteristics of the studies included in this systematic review are
summarised in Table 1.
Risk of bias assessment
Of the two RCTs included in this systematic review, one was consid-
ered to be of some concern because it showed a low risk of bias in most
domains (D1, D2, D4, and D5) but presented some concerns in domain
3 (D3) that evaluated the risk of bias due to missing outcome data [20].
Another study considered a high risk of bias [5] and presented a high
risk of bias in D2 because it was not clarified if washout ensured that
the first intervention did not interfere with the second, and certain con-
cerns in D5 evaluated bias in the selection of the reported intervention
results [5]. The results of the risk of bias assessment for each study are
presented in Fig. 2.
Effect of zinc supplementation in duration and effects of BT in facial
muscles
In a study with a high risk of bias, mg.day−50 of zinc citrate supple-
mentation, independent of phytase, improved the effects of BT in face
muscles but was dependent on the objective of BT application and clin-
ical severity [5]. The duration of BT in the treatment of blepharospasm,
hemifacial spasm, and cosmetic facial rhytids were higher (29.6%) in
the group that received mg.day−50 of zinc citrate with phytase than in
the group that received 10 mg of zinc gluconate without phytase (p <
0,001). In addition, 84% of patients who received mg.day−50 of zinc cit-
rate with phytase exhibited a better effect of BT when compared with
a placebo. There was no significant difference between the groups that
received 10 mg of zinc gluconate and the placebo (p = 0,88) [5].
Supplementation with mg.day−50 of zinc citrate with phytase
showed a greater effect in individuals treated for hemifacial spasm than

3
H.T. Junior, C.d.S. Melo, R.R. Mendes et al.
Fig. 2. Summary of risk of bias assessment of each study included in this systematic review.
in those treated for blepharospasm and facial rhytids [5]. According to
clinical severity, the superior effect was observed after administration
of mg.day−50 of zinc citrate with phytase and applied BT for treatment
of hard-to-treat blepharospasm; following easy-to-treat blepharospasm,
and facial rhytids (88%, 47% and 27%, respectively) (p < 0,001) (88%,
47% and 27%, respectively) (p < 0,001) [5].
In the evaluation of smile type prevalence, another study with some
concerns in terms of risk of bias assessment and included in this sys-
tematic review showed that supplementation with mg.day−50 of zinc
gluconate was associated with a higher prevalence of commissure smile
(76.9%) complex (15.4%) and cusp (7.7%) when compared to placebo
(p < 0,01); however, no difference was observed in lip competence, av-
erage lip length at rest and smiling, and in measures of smile [20].
The main results of the studies included in this systematic review are
presented in Table 2.
Discussion and conclusion
Two studies with low size population were included in this system-
atic review, showing the necessity of development of most RCTs that
scientifically agree the clinical practice of the supplement zinc with ob-
jective improve BTA actions. Supplementation with 50 mg of zinc citrate
and 3000 units of phytase increased the duration, efficacy, and action
of BT in the treatment of blepharospasm, hemifacial spasm, and cos-
metic facial rhytids (RCT whit higher risk of bias). Supplementation with
mg.day−50 zinc gluconate without phytase increased the prevalence of
commissure smiles (RCT whit some concerns about risk of bias).
The gummy smile is characterized by exposition of four mm or more
of gum during smiling [26]. In clinical practice, the smile can also be
classified into three types: commissure or “Mona Lisa” smile (occurs
4
Journal of Trace Elements and Minerals 5 (2023) 100080
when there is greater exposure of the upper teeth with a greater dis-
placement of the labial commissure upwards), cusp or canine smile (the
upper lip moves upwards with a greater participation of the incisors and
canines in the smile) and complex smile (when upper and lower teeth
are exposed) [27,28].
Other studies have reported that BT decreases gummy smile [29,30]
blepharospasm [31,32], hemifacial spasm [31,33], and facial rhytids
[34,35], independent of zinc. This is explained because BT breaks trans-
mission of the nerve impulse in the presynaptic cleft, by cleaving SNAP-
25, preventing the passage of acetylcholine to the synaptic cleft, thus
interrupting motor excitation of the postsynaptic neuron [9].
The effect of BT is dependent on body zinc reserves, considering that
BT is a zinc-dependent metalloprotease [18]. However, in the studies in-
cluded in this systematic review, body zinc reserves were not assessed,
which limited the extrapolation of results regarding nutrient supplemen-
tation.
Phytase inhibits phytates and organic compounds that decrease zinc
absorption and bioavailability; thus, phytase is associated with zinc sup-
plementation with increased zinc bioavailability [36–38]. Nonetheless,
in the studies included in this systematic review, phytase did not con-
tribute to an increase in the effect of zinc on the duration and activity
of BT in face muscles.
In the studies included in this systematic review, the interventions
were performed using zinc gluconate without phytase daily for four
days [20] and zinc citrate with phytase for five days [5]. The quan-
tity of zinc available in the different types of supplements can modify
outcomes. According to the calculation of the equivalence factor pro-
posed by the Federal Council of Pharmacy [39], mg.day−50 and 10 mg
of zinc gluconate offers 7 mg and 1.4 mg of zinc as being bioavailable,
respectively, falling below the daily needs of this mineral [36,40] (8 mg
H.T. Junior, C.d.S. Melo, R.R. Mendes et al.
Table 2
The main findings about the effect of zinc supplementation on botulinum toxin effect in facial muscle showed in studies included in this systematic review.
Author (year) Intervention Variable
Shemais et al. Zinc gluconate 50 mg/day (4 Smile type
(2021) [19] days before BT application)
Placebo
Koshy et al. Group 1: 10 mg zinc citrate/day Blepharospasm
(2012) [5] (5 days being 4 days before hemifacial spasms
application and one in facial rhytids
application day)
Group 2: 50 mg of zinc
citrate + phytase 3000 uni/day
(5 days being 4 days before
application and one in
application day)
Placebo
Legend: BT: botulinum toxin; UNI: units.
for women and mg.day−11 for men [41]); this may contribute to no ef-
fect after supplementation with 10 mg of zinc gluconate [5]. However,
50 mg of zinc citrate makes available 17 mg of zinc, a greater dose
when compared with the daily recommended intake, but a less tolera-
ble upper intake level (mg.day−40 ) [41], which justified the absence of
collateral effects after interventions in the included studies. In addition,
studies have shown that zinc citrate and gluconate are equally bioavail-
able [42,43], which explains the similarity in terms of outcomes when
administered the same doses of different types of zinc.
These studies did not evaluate the zinc body reserves of individu-
als. Zinc deficiency is associated with lower metalloprotease activity
and contributes to lower BT activity [44]. The zinc body reserve may
be evaluated by serum concentrations of metallothionein, an important
predictor of dietary zinc intake [36], as well as by leukocyte, platelet,
and erythrocyte zinc levels [45]. Erythrocyte zinc is considered a bet-
ter predictor of nutritional condition relative to zinc, reflecting a body
reserve of three months, erythrocyte half-life, and a 21% decrease after
90 days of inadequate intake [45].
In humans, 2–3 g of zinc is distributed around the body, with 80% in
bone, muscle, liver, and skin, and the greatest absorption occurs in the
jejunum, followed by the liver via the portal circulation, where it is dis-
tributed throughout the body [46]. During inadequate zinc intake, zinc
binds to cysteine-rich intestinal proteins (CRIP) to be absorbed; when
supply increases, absorption occurs via a diffusion mechanism [36].
Zinc is naturally present in shellfish, fish, liver, eggs, red meat, nuts,
and chestnuts, among other foods. Brazilian adults present adequate
daily zinc intake (mg.day−12.7 and mg.day−9.6 , for men and women,
respectively) [47]. Individuals 40 years old living in the USA intake an
average of mg.day−10.5 ± mg.day−0.1 of zinc [48]. In Egypt, the aver-
age zinc intake of adults is mg.day−8.5 [49]. The studies included in this
systematic review did not evaluate dietary zinc intake. It was not possi-
ble to confirm the relationship between dietary zinc intake and effects
attributed to minerals.
The effect of zinc on BTA can be affected by the clinical severity of
the patient [15]. Koshy et al. [5] classified participants according to ble-
pharospasm severity as “hard-to-treat” or “easy-to-treat”, and showed
better effects of BTA in the treatment of “hard-to-treat” blepharospasms
after delivery of 50 mg of citrate of zinc associated with 3000 units of
phytase. This is explained by the cleavage of SNAP-25 by BTA [18].
The studies did not present a standardized method for evaluating out-
comes. The effects of zinc supplementation in use of BT for the treatment
of blepharospasms were reported through self-reporting by patients and
by professional clinical perception; the decrease in muscle contraction
is more noticeable in more complex cases [5].
5
Journal of Trace Elements and Minerals 5 (2023) 100080
Outcome evaluation methods Main outcome
Patient’s self-perception. 76.9% of prevalence of commissure smile in the
Photographic records made group that received 50 mg of zinc gluconate
by the researchers. compared to placebo (p<0,05)
7.7% of prevalence of smile type cusp and 15.4%
of complex type in the group that received 50 mg
of zinc gluconate compared to placebo (p<0,05)
Perception of the researchers ↑ time of action of BT in group 2 (29.6%) when
compared to group 1 (1.15%) (p<0,001)
84% of group 2 related better efficacy of BT when
compared to group 1 and placebo (p<0,001)
50 mg of zinc citrate increased BT effect in the
treatment of blepharospasms “hard to treat” and
“easy to treat”, but had no effect on the treatment
of facial rhytids
88% treated to blepharospasms “hard to treat”,
47%% treated to blepharospasms “easy to treat”,
and 27% treated to of facial rhytids relates better
effect of BT after supplementation with 50 mg of
citrate zinc (p<0,001)
Although zinc has a promising effect on BT in facial muscles, was not
possible affirm that zinc supplementation increases the duration of BT
action due the low number of RCTs, as well as important methodological
errors observed in certain studies, and the different objectives in the
application of BT, as well as, was not possible to define the best type
and dose of supplemental zinc for this outcome.
This study has important limitations. Only two studies were included
in this systematic review, which were developed with low size popula-
tion, and significant differences in methods of measured of the BT effect
in face muscle. Despite supplementation with mg.day-50 of zinc showing
a positive effect on the action of BTA according to the applied purpose,
the main findings of this systematic review do not support the recom-
mendation of zinc supplementation to improve the effect of botulinum
toxin action on facial muscles, considering the presence of important
risk of bias of the studies and the limitations showed.
Thus, despite to BTA be considerate a zinc dependent metallopro-
tein, is not scientific evidence that backed the clinical practice of zinc
supplementation for improve BTA action in face muscle, being neces-
sary to perform additional RCTs to close the gaps in science regarding
the effects of zinc supplementation on the durability of BT action, which
has been used for various therapeutic purposes in clinical practice.
Funding source declaration
This research did not receive any specific grant from funding agen-
cies in the public, commercial, or not-for-profit sectors.
Declaration of Competing Interest
The authors declare the following financial interests/personal rela-
tionships which may be considered as potential competing interests: his
research did not receive any specific grant from funding agencies in the
public, commercial, or not-for-profit sectors.
CRediT authorship contribution statement
Hélio Trindade Junior: Conceptualization, Methodology, Valida-
tion, Investigation, Writing – original draft, Methodology, Valida-
tion, Investigation, Writing – review & editing. Caroline dos Santos
Melo: Methodology, Validation, Investigation, Writing – review
& editing. Renata Rabello Mendes: Writing – review & edit-
ing. Ramara Kadija Fonseca Santos: Conceptualization, Methodology,
Validation, Investigation, Writing – original draft, Writing – review &
editing.
H.T. Junior, C.d.S. Melo, R.R. Mendes et al.
Acknowledgments
This research did not receive any specific grant from funding agen-
cies in the public, commercial, or not-for-profit sectors.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.jtemin.2023.100080.
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