Executive Summary:

Zinc Acetate Oral Solution 20 mg / 5 ml and

Zinc Acetate Tablets 50 mg

Application:
Request for Manufacturing and Marketing
permission of Zinc Acetate Oral Solution
20 mg / 5 ml and Zinc Acetate Tablets 50 mg
Applicant: Zuventus Healthcare Limited, India
Proposed Dosage form: Zinc Acetate Oral Solution 20 mg / 5 ml and
Zinc Acetate Tablets 50 mg

Name of the Zinc Acetate Oral Solution 20 mg / 5 ml and Zinc Acetate Tablets 50 mg
1. drug including
chemical name Zinc Acetate; Zn(CH3COO)2(H2O)2
2. Name of the Zuventus Healthcare Limited (A Joint Venture of Emcure Pharmaceuticals

Page 1 of 22
 applicant Limited)
3. Therapeutic Class:
Class/ A alimentary tract and metabolism product; Nutritional supplement.
Indication Indication:
Management of acute or persistent diarrhoeal episodes, acute respiratory
infections/pneumonia or nutritional deficiency in pediatric and adult population.
4. Brief Pre-
clinical Published Pre Clinical Study Summary attached as Appendix I
Information
5. Brief Animal
Toxicological Animal Toxicology Study Data attached as Appendix II
Information
6. Brief Clinical
Clinical Trials Summary attached as Appendix III
Information
7. Adverse Effect System organ class Adverse drug reactions
if any known to
Gastrointestinal Common
be associated Gastric irritation
disorders (≥ 1/100 To < 1/10):
with the drug
Common Blood amylase, lipase and
Investigations
(≥ 1/100 To < 1/10): alkaline phosphatase increased
Blood and lymphatic Uncommon Sideroblastic anaemia,
system disorders (≥ 1/1,000 To < 1/100): leukopenia
The most common undesirable effect is gastric irritation. Elevations of serum
alkaline phosphatase, amylase and lipase may occur after a few weeks of
treatment with zinc, with levels usually returning to normal within the first one
or two years of treatment.
Anaemia may be micro-, normo- or macrocytic and is often associated with
leukopenia. Bone marrow examination usually reveals characteristic "ringed
sideroblasts" (i.e. developing red blood cells containing iron-engorged
paranuclear mitochondria). They may be early manifestations of copper
deficiency and may recover rapidly following reduction of zinc dosage. 33
8. Regulatory Marketing Authorization
Country Brand / Trade Name(s)
status of Holder
individual USA ®
Galzin 25/50 capsules Teva Pharmaceuticals
components of
Europe Wilzin® 25/50 capsules Recordati Rare Diseases
Investigational
product (IP)
including
comparator, if
any, globally
9. Risk Vs. Benefit Zinc is an essential micronutrient required for the activity of more than 300
to the patients enzymes, 1000 transcription factors and for the control of genetic expression. It
plays an important role in nucleic acid and protein synthesis, cellular and tissue
replication, differentiation, and growth.34, 35 Zinc is involved in both non-specific
and specific immune system processes, including phagocytosis, maintenance of

Page 2 of 22
 gastrointestinal and respiratory tract linings, development and function of T and
B-cells.36, 37
High-quality evidence indicates that supplementation with zinc (5–50 mg/day)
can reduce the incidence of otitis media and reduce the incidence of lower
Respiratory Tract Infections (RTI) with zinc (20–140 mg/week) in children. An
analysis of mostly high-quality studies has demonstrated that the risk of RTI or
pneumonia and diarrhoea or dysentery may be reduced in children after zinc
administration. Analysis of low-to-moderate quality studies zinc found reduction
in mortality associated with RTI, diarrhoea, and malaria. The duration of the
common cold may be reduced in adults and children after administration of zinc
>75 mg/day. Zinc is an effective treatment for Wilson disease. Zinc supplements
are commonly used to alleviate a number of conditions, including zinc-deficient
states, age-related macular degeneration and wound healing. Greater benefits
with zinc acetate compared with other zinc salts has been found in high-quality
studies.37, 38
A meta-analysis was performed to quantify the effect of zinc supplementation in
prevention of childhood diarrhea and respiratory illnesses. 17 trials (3819
children: zinc, 3840 children: placebo; aged ≥ 3 months to ˂ 5 years) were
studied. Analysis revealed that zinc was associated with a significant reduction
in the occurrence of diarrheal episodes by 14% (ratio rate: 0.86), 8% reduction in
frequency of respiratory illness (ratio rate: 0.92), significantly fewer attacks of
severe diarrhea/dysentery (rate ratio: 0.85), persistent diarrhea (rate ratio: 0.75),
and lower respiratory tract infection/pneumonia (rate ratio: 0.80) compared to
placebo. Zinc supplementation significantly reduced the frequency and severity
of diarrhea and respiratory illnesses and the duration of diarrheal morbidity.39
In another meta-analysis of 22 studies [16 acute diarrhea studies (n = 15,231),
and 6 persistent diarrhea studies (n = 2,968)], the efficacy and safety of oral zinc
compared to placebo was studied. Total mean duration of acute and persistent
diarrhea was significantly lower for zinc compared with placebo. Children who
received zinc reported 18.8% and 12.5% reduction in average stool frequency,
15.0% and 15.5% shortening of diarrhea duration, and 17.9% and 18.0%
probability of reducing diarrhea over placebo in acute and persistent trials
respectively. It was found that zinc supplementation reduced the duration and
severity of acute and persistent diarrhea.40
A meta-analysis of 3 zinc acetate lozenge trials was conducted to estimate the
effect of zinc acetate lozenges (80–92 mg/day of elemental zinc) on the rate of
recovery in 199 common cold patients. Patients administered zinc lozenges
recovered faster by rate ratio 3.1. On the 5th day, 70% of the zinc patients had
recovered compared with 27% of the placebo patients. Accordingly, 2.6 times
more patients were cured in the zinc group. None of the studies observed serious
adverse effects of zinc. The 3-fold increase in the rate of recovery from the
common cold post zinc supplementation was found to be clinically important. 41
10. Innovation Vis- In India, Pneumonia and diarrhea accounts for 36.9% of the under five deaths.
à-vis existing Diarrhoea is more common in children with zinc deficiency. Preventive zinc
therapeutic supplementation alone can prevent nearly 5% of child deaths each year.

Page 3 of 22
 options According to the guidelines of World Health Organization, antibacterial,
antiamoebic and antidiarrhoeal agents have a little role in the management of
diarrhoea.42,43
Antibiotics rarely help in treatment and can make some people more sick in the
long term. Their indiscriminate use increase the resistance of some disease-
causing organisms and they are not cost-effective. Adsorbents (such as kaolin,
pectin, activated charcoal) are not effective in the treatment of acute diarrhea and
have been shown to induce only a slight change in stool consistency. However,
they do not reduce fluid and salt losses. Antimotility drugs (such as tincture of
opium or loperamide) is harmful, especially in children less than 5 years of age.
They temporarily reduce cramps and pain but delay elimination of organisms
causing the diarrhoea and prolonging the illness. These drugs can be dangerous
and even fatal if used improperly. 44,45
Zinc supplementation is a new addition to the diarrhoea treatment strategy and
being recommended by WHO, UNICEF, IAP and countries around the world for
the treatment of all diarrhoea episodes among children. The acceptability of Zinc
supplements is high; it has been tested in over 1,00,000 children. No serious side
effects are expected based on the multiple published literatures. Following are its
advantages: 44, 46, 47
 Decreases the length and severity of the diarrhoea. Less watery and
frequency of stools. 50% less children have diarrhea lasting for more than 5
days.
 Acts as a tonic after recovery from diarrhea. Improves appetite and growth.
Important for the child’s immune system and help child fight off new
episodes of diarrhea in 2-3 months of treatment.
 If given with ORS, promotes use and hence prevents serious and potentially
lethal outcomes.
 If used for 14 days, prevents diarrhea and pneumonia over the next 2
months.
 Reduce misuse and overuse of antibiotics, which leads to antibiotic
resistance.
 Also benefit other associated illnesses like acute respiratory infections/
pneumonia, septicemia and nutritional deficiency.
 Reduction in child mortality rate and hospitalizations.
Unmet medical 2.1 million Indian children aged <5 years die every year because of diarrhoea,
11. needs in the typhoid, malaria, measles, and pneumonia. 1,000 children die each day due to
country diarrhoea alone. Diarrhoea, respiratory infections, and malaria is associated with
zinc deficiency. Zinc deficiency is also an important cause of morbidity due to
infectious diseases and growth faltering among young children. Several studies
indicate that zinc deficiency results in poor growth, depressed appetite, impaired
motor development and can interfere with cognitive performance in infants and
children.48 Our immune system depends on a sufficient availability of this
essential trace element. Since there are no major body storage depots for zinc,
severe deficiency is produced easily and quickly, which can impair a variety of

Page 4 of 22
 immune functions and host defensive mechanisms.49
In India, inadequate intake of micronutrients are widespread because staple diets
are predominantly cereal based, and intakes of meat, fish, dairy products, fruits
and vegetables are low. Further, cereal-based diets are rich in phytate, which
reduce the bioavailability of minerals, implying a higher risk of zinc deficiencies
in Indian population.48
Amongst micronutrients, zinc is in the forefront due to the pervasive nature of
zinc-dependent enzymes in metabolic processes; its vital role in several body
functions such as vision, taste perception, cognition, cell reproduction, growth,
immunity; and beneficial effect of zinc supplementation in many disease states.
50

Due to the multitude of basic biochemical functions of zinc in the cells of human
body, there is a broad range of physiological signs of zinc deficiency. Organ
systems known to be affected clinically by zinc deficiency states include the
epidermal, gastrointestinal, central nervous, immune, skeletal, and reproductive
systems.49, 51
Clinical signs of zinc deficiency include acrodermatitis, suppressed immunity,
diarrhea, poor healing, stunted growth, hypogonadism, fetal growth failure,
teratogenesis and abortion. Zinc deficiency is also known to be associated with
various diseases such as malabsorption syndrome, chronic liver disease, chronic
renal disease, sickle cell disease, diabetes, malignancy, neurodevelopment
disorders and other chronic illnesses. Zinc deficiency also causes significant
impairment in adaptive and innate immune responses and promotes systemic
immune dysfunction in older populations.34,37 All age groups of the population
are at risk of zinc deficiency where infants and young children, pregnant and
lactating women are the most vulnerable.35
Numerous zinc supplementation trials have shown that a wide range of health
benefits can be realized by increasing the intake of zinc. Supplementation
programs are useful for targeting vulnerable population subgroups, which are at
a particular high-risk of zinc deficiency. When formulating supplements, it is
recommended that salts providing readily absorbable zinc like zinc acetate
should be used because they are absorbed more efficiently.52
11. Recommendatio N/A
n of NDAC/IND
Committee
12. Details of N/A
members of
NDAC/IND
Committee
recommended
for the clinical
trial

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 APPENDIX I: PRE-CLINICAL STUDY SUMMARY

NON-CLINICAL PHARMACOLOGY
The current study was conducted to study the experimentaly induced zinc deficiency and its
hematological consequences in 25 Iraqi goats. The animals were fed a high calcium content
ration (400-500 gm/head/day) to induce zinc deficiency, in addition to hay and water with ad
libitum during the study. The main clinical signs were retardation in hair growth, especially of
legs and head, swollen joint, poor growth, rough hair coat, loss of hair on head, limbs, and
scrotum, cracking of the hooves. The skin was rough, thickened, and cracked, alopecia, crusting,
and hyperkeratosis also occurred. Paleness of the mucous membranes of the eye, loss of appetite,
itching, and emaciation were observed. Body weight, heart rates, and body temperature were not
significantly changed, while respiratory rates significantly decreased in the 3 rd and 6th weeks in
comparison with the 1st week. The results of total red blood cells counts revealed significantly
decrease in the 2nd week compared with the 1st week, then increased in the 4th, 6th and 8th weeks in
comparison with the 1st week. Hemoglobin concentration significantly increased at the 2 nd, 4th
and 6th weeks in comparison with the 1st week. Packed cell volume increased at the 2nd, 4th, 6th
and 8th weeks compared with 1st week. Serum zinc was significantly decreased from the 2 nd
week till the end of the experiment. Significant decrease in lymphocytes was detected at the 4 th
week compared with the 1, 2, 6 and 8 weeks. Eosinophil count also significantly decreased in the
2nd and 6th weeks compared with the 1st week, and increased during the 4 th week and 8th week
compared with the 2nd and 6th respectively. It was concluded that zinc deficiency clearly affects
the overall health status.1
The carcinogenic effect of Zn supplementation in Zn-sufficient mice of wild-type and tumor
suppressor-deficient mouse strains was studied. All mice received N-nitrosomethylbenzylamine
and half the mice of each strain received Zn supplementation. At killing, mice without Zn
supplementation developed more tumors than Zn-supplemented mice: wild-type C57BL/6 mice
developed an average of 7.0 versus 5.0 tumors for Zn supplemented; Zn-supplemented Fhit -/-
mice averaged 5.7 versus 8.0 for control mice; Zn-supplemented Fhit-/-Nit1-/- mice averaged 5.4
versus 9.2 for control mice and Zn-supplemented Fhit-/-Rassf1a-/- mice averaged 5.9 versus 9.1
for control mice. Zn supplementation reduced tumor burdens by 28% (wild-type) to 42% (Fhit-/-
Nit1-/-). Histological analysis of forestomach tissues also showed significant decreases in
severity of preneoplastic and neoplastic lesions in Zn-supplemented cohorts of each mouse
strain. Zn supplementation significantly reduced tumor burdens in mice with multiple tumor
suppressor deficiencies. When Zn supplementation was begun at 7 weeks after the final
carcinogen dose, the reduction in tumor burden was the same as observed when supplementation
began immediately after carcinogen dosing, suggesting that Zn supplementation may affect
tumor progression rather than tumor initiation.2
A study was conducted to understand the role of dietary zinc supplementation on thymopoiesis
in aged mice. Groups of young mice were fed a zinc-adequate (30 mg/kg zinc) or zinc-
supplemented diet (300 mg/kg) for 25 days. Aged mice had impaired zinc status, with zinc
supplementation restoring plasma zinc to a concentration not different from those of young male
C57Bl/6 mice. Zinc supplementation in aged mice improved thymopoiesis, as assessed by
increased total thymocyte numbers. In addition, improved thymic output was mediated in part by
reducing the age-related accumulation of immature CD4-CD8-CD44+CD25- thymocytes, as well

Page 6 of 22
as by decreasing the expression of stem cell factor, a thymosuppressive cytokine. The study
results showed that in mice, zinc supplementation can reverse some age-related thymic defects
and may be of considerable benefit in improving immune function and overall health in elderly
populations.3
The effect of subacute zinc deficiency on immune responses and oxidant stress during bacterial
sepsis [i.e., cecal ligation and puncture (CLP)] was studied in C57BL/6 mice. The mice were
randomly placed on a zinc-deficient diet (Harlan Teklad, TD85419; 1 ppm) (n = 35) or a
matched control diet (TD85420; 50 ppm) (n = 35) for 3 weeks, a sufficient time to establish
subacute zinc deficiency. An additional group (n = 35) received a zinc-fortified diet (TD07129;
100 mg/kg) for 3 days following the 3-week zinc deficient regimen and before CLP to assess the
impact of zinc supplementation Mice were then assigned to receive either CLP or sham
operation (n = 15 each per diet). CLP and sham-operated treatment groups were further assigned
to a 7-day survival study (n = 10 per treatment per diet) or were evaluated at 24 hours (n = 5 per
treatment per diet) for signs of vital organ damage. Sepsis mortality was significantly increased
with zinc deficiency (90% vs. 30% on control diet). Zinc-deficient animals subject to CLP had
higher plasma cytokines, more severe organ injury, including increased oxidative tissue damage
and cell death, particularly in the lungs and spleen. None of the sham-operated animals died or
developed signs of organ damage. Zinc supplementation normalized the inflammatory response,
greatly diminished tissue damage, and significantly reduced mortality. The authors concluded
that the subacute zinc deficiency significantly increases systemic inflammation, organ damage,
and mortality in a murine polymicrobial sepsis model. Short-term zinc repletion provides
significant, but incomplete protection despite normalization of inflammatory and organ damage
indices.4
The effects of mild Zn deficiency and subsequent Zn supplementation during pregnancy and its
effect on immune function of the offspring was studied. Adult female rats were fed a Zn-
adequate diet (ZC, n = 8) or a Zn-deficient diet (ZD, n = 8) from preconception through
lactation. Pregnant rats were supplemented with either Zn (1.5 mg Zn in water) or placebo
(water) 3 times/wk throughout pregnancy. Pups were orally immunized with cholera toxin and
bovine serum albumin-dinitrophenol 3 times at weekly intervals and killed 1 wk after the last
dose. Zn supplementation of ZD dams led to enhanced lymphocyte proliferation and IFN-γ
responses in pups ZDZ+. In contrast, Zn supplementation of ZC dams suppressed these
responses in pups ZCZ+. Total and DNP-specific IgA responses were lower in pups of the Zn-
deficient group compared with the Zn-adequate group. Relative thymus weight was greater in
the pups (ZDZ-) of ZD placebo supplemented dams compared with the other groups at 31 d of
age. Prepregnancy and early in utero Zn deficiency affected IgA responses in pups that could not
be restored with Zn supplementation during pregnancy. Zn supplementation of ZC dams induced
immunosuppressive effects in utero that may also be mediated through milk and persist in the
offspring after weaning.5
The effect of zinc deficiency on the function of the intestine to absorb water and electrolytes was
studied in rats, stimulated by Vibrio cholerae enterotoxin. Sprague-Dawley rats were divided
into four groups: Zinc-deficient diet containing 0.8 μg/g of zinc for l6 days, ad libitum zinc-fed
control diet containing 55.0 μg/g of zinc for 16 days, zinc weight-matched control diet diet
containing 55.0 μg/g of zinc, and a group of zinc-deficient animals was acutely repleted by
feeding the zinc-containing diet (55.0 μg zinc per g) ad libitum for 48 hours. Significantly lower

Page 7 of 22
absorption of water and sodium per cm of the intestine was observed in the zinc-deficient
animals compared to the ad libitum zinc-fed control animals. An improved absorption capacity
was equally observed in the zinc deficient acutely-repleted animals and ad libitum zinc-fed
control group. The zinc-deficient animals showed four times greater cholera toxin-induced net
secretions of water and sodium compared to the ad libitum zinc-fed group, while a 40%
reduction was observed in the zinc-deficient acutely repleted group. The results suggest that zinc
deficiency is associated with reduced absorption of water and electrolytes and increased
secretion of the same stimulated by cholera toxin.6
An experiment was conducted to study the pathology of the lymphoid organs and peripheral
blood T lymphocytes in zinc (Zn)-deficient chickens. One hundred 1-day-old broiler chickens
were randomly divided into two groups and fed on diets with 100 mg/kg Zn (controls) or Zn-
deficient diets (Zn, 23.63 mg/kg) for 7 weeks. The G0/G1 phase of the cell cycle of the bursa,
thymus and spleen was much higher, and the S, G2+M phases and proliferating index lower in
Zn-deficient broilers than in the controls. The acid alpha-naphthyl acetate esterase-positive ratio
of the peripheral blood T lymphocytes and the CD4 and CD8 numbers were markedly reduced,
and the CD4/CD8 ratio increased. Histopathologically, lymphocytes of lymphoid organs were
depleted and the reticular cells of the thymus were also degenerate or necrotic in the Zn-deficient
birds. The results demonstrate that Zn deficiency seriously inhibited the development of
lymphoid organs, impaired the progression of lymphocytes from the G0/G1 phase to the S phase,
and caused pathological injury in the lymphoid organs. The results also showed that the effect of
Zn deficiency on the primary lymphoid organs occurred earlier than on the secondary lymphoid
organs. The effect of Zn deficiency was greatest on the bursa of Fabricius, followed by the
thymus, and then the spleen. 7
A study was conducted to assess the skeletal effects of alimentary zinc depletion and
supplementation in a growing rats. 36 male Wistar rats were divided into 3 groups: Group 1 (12
rats) was given a zinc-free diet containing 2 mg zinc/kg, group 2 (12 rats) was given a normal-
zinc diet containing 47 mg zinc/kg; and group 3 (12 rats) was given a zinc-supplemented diet
containing 60 mg zinc/kg. All animals were killed 4 weeks after initiation of the experiment and
the right femora were removed. Biomechanical testing revealed a significant zinc-induced
increase in bone strength at all sites investigated. It also showed that zinc influenced bone
strength in a dose dependent manner except at the distal metaphysis, where there was no
significant difference between the group fed normal-zinc diet and the group fed a hyper-zinc
diet. Zinc also improved the rates of growth in the rats. The body weights and length of femora
increased dose-dependently. Static histomorphometry showed that zinc exerted its main effect on
the periosteal envelope, thereby increasing bone area, tissue area, and axial moment of inertia. It
was concluded that the alimentary zinc supplementation in growing rats induces an increase of
bone strength in both the femoral neck and the femoral diaphysis. The study results further
supported the view that zinc has a positive effect on bone metabolism which mimicks that of
growth hormone (GH) or insulin-like growth factor 1 (IGF-1).8
A study was conducted to explore whether the mice fed with diet low in Zn (2:0mg Zn/kg diet)
for short period of time were more prone to severe Streptococcus pneumonia infection compared
to mice fed with normal diet (25 mg elemental Zn/kg). After 2 weeks of feeding, the mice were
infected intranasally with a suspension containing about 107 pneumococci. The survival time
and time before positive blood culture were significantly shorter in the Zn-depleted group than in
the pair-fed Zn-adequate group. At the end of the observation period, ten of the twelve mice in
Page 8 of 22
the Zn-deficient group were dead while one of twelve and two of twelve were dead in the two
Zn-adequate control groups. The study results showed that the acutely-induced Zn deficiency
dramatically increases the risk of serious pneumococcal infection in mice.9
A study was conducted to examine the effect on health and performance of feeding different
levels of zinc for one, two or three weeks after weaning to piglets weaned at 28 days of age. A
supplement of 2500 ppm zinc for two weeks after weaning reduced the incidence and severity of
non-specific post-weaning diarrhoea by up to 50%. The daily gain, but not feed intake and feed
efficiency, was significantly influenced by the dietary zinc level. Piglets fed 2500 ppm zinc
showed the best performance. The beneficial effect of a temporary supply of 2500 and 4000 ppm
zinc on post-weaning diarrhoea was found to be coincident with an increased alkaline
phosphatase activity and zinc concentration in serum.10
Eighty weanling beef calves were used to determine the effects of zinc and selenium
supplementation on performance, immune response, and blood characteristics during stress.
Treatments included: (1) control; (2) control diet with selenium injection [15 mg/hd (head)] (3)
zinc diet (25 mg Zn/hd/d); and (4) zinc diet with selenium injection. Feed intake and weight
gains were not affected by zinc throughout the 28-d study. However, selenium improved weight
gains in calves with low initial selenium status in the first 14 d of the study. Glutathione
peroxidase (GSH-Px) was increased by selenium and decreased by zinc on d 19. Zinc appeared
to interfere with the role of selenium in GSH-Px activity at the cellular level. Plasma zinc was
not affected by treatment. Zinc supplementation resulted in increased serum sodium and
decreased serum total protein and glutamic-oxaloacetic transaminase. Immune response was
measured via antibody titers to Infectious Bovine Rhinotracheitis (IBR) andPara-influenza3 (PIs)
viruses 19 d postvaccination. Levels of titers were not affected by either zinc or selenium,
however, titers did reflect a response to vaccination between sampling dates. On d 19, zinc
increased the percentage of leukocytes that were monocytes. Total leukocyte, neutrophil and
lymphocyte numbers did not differ across treatments. The study results suggested that selenium
or zinc supplementation may individually improve an animal's response to stress.11
NONCLINICAL PHARMACOKINETICS

Absorption, excretion and tissue distribution of 65Zn was determined following oral dosing in
zinc-deficient and normal calves and goats. In the livers, level of 65Zn was higher at 2 days than
at later times. In some tissues, including hair, bone and muscle, the 65Zn concentration increased
with time while in others the level reached a peak between 2 and 28 days after oral dosing. The
tissues of the zinc deficient animals held the 65Zn more tenaciously than those of the control
animals. Fecal excretion of 65Zn reached a peak the third day after dosing and declined there
after. 65Zn excretion via feces was higher for control than zinc-deficient animals. Apparent
absorption was very high exceeding 80% for some treatments. These high absorption values are
attributed to several factors including semipurified diets containing very low levels of zinc and
are a reflection of mechanisms resulting in homeostatic conservation of the zinc.12
Studies in rats and chicks show that the absorption of zinc is reduced when administered with
phytic acid, a soybean protein component. Some reports in rats show that calcium and
phosphorus reduce the absorption of oral zinc. Distribution studies in rats show that peak tissue
concentrations of 65Zn are reached within 5 days after oral dosing. Distribution studies following
oral long-term administrations in rats, cats and dogs, show that highest levels of zinc are attained

Page 9 of 22
in liver, gallbladder and bile, gastrointestinal tract, kidney, bone, bone marrow and pancreas.
Analysis of zinc contents in various organs and tissues showed no evidence of accumulation or
tissue specific storage. In rats chronically treated up to 53 weeks, distribution and elimination of
zinc salts (acetate, citrate, malate) are similar without any accumulation or tissue specific storage
in all cases. The elimination of 65Zn in mice, rats, dogs and humans is triphasic with half-life
values for the terminal phase of 75, 91, 94 and 154 days, respectively. In cats, tissue levels
returned to normal levels within 2 weeks of zinc dosing discontinuation, whereas in rats,
elimination from bone and pelt is delayed. Zinc salts were administered in drinking water to
male or female rats (n = 1 to 3/dose), for 35 to 53 weeks. Analysis of excreta (faeces, urine) from
control and zinc-treated rats indicated that faecal excretion was the main route of zinc
elimination. Increased doses produced only minimal increases in zinc urinary excretion. In all
species studied (mice, rats, dogs, humans), the majority of excretion occurs via the faeces, with
only a small fraction excreted in the urine. In rats, pharmacokinetic parameters of 65Zn was not
affected by gestation. It showed placental transfer and also appeared in milk of mother rats.13

Page 10 of 22
 APPENDIX II: ANIMAL TOXICOLOGY STUDY DATA

SUBACUTE/SUBCHRONIC/CHRONIC TOXICOLOGY

A total of 35 white rats including 13 control rats, were fed zinc orally as the oxide, acetate,
citrate, or maleate in gum acacia suspensions for periods of 35 to 53 weeks (2 male rats on zinc
acetate solution 1.9 and 3.6 mg [-7.6 and 14.4 mg/kg] of zinc daily for 53 and 48 weeks,
respectively). Treatment with zinc salts including zinc acetate produced no evidence of toxicity,
no treatment-related effects on body weight gain or food consumption. Two rats died during the
study as a result of a pneumonia infection of which one was treated with zinc acetate (4.4
mg/day). There were no treatment-related changes in red cells, white cells, or percent
hemoglobin in any group. Gross pathological findings were limited to the following: I) small
consolidated areas and pus sacs in the lungs of 7 zinc treated rats and 3 control rats; 2) a pus
pocket found in the bladder of one animals treated with zinc acetate (1.9 mg/day) and 3) parasitic
cysts found in the livers of three treated rats; of which 2 rats were treated with zinc acetate (1.9
mg/day). Histological correlates of bronchopneumonia or-its sequelae (i.e. patches of leukocytic
infiltration, atelectatic cavities and dilated bronchi filled with exudate, or encapsulated chronic
abscesses, apparently originating from pneumonic foci) were seen to varying degrees in all
animals which showed gross pulmonary lesions. In conclusion, the aforementioned studies in
rats indicate that daily oral doses of zinc (0.5 to 34.4 mg zinc; -2 to 137.6 mg zinc/kg body
weight), for periods of 35 to 53, weeks produced no evidence of toxicity at any level tested. 14
15 adult ferrets were fed 500, 1500 and 3000 ppm zinc in their diets for up to 6 months. The
controls (basal diet) composed of 5 animals; 3 animals were given the basal diet plus 3000 ppm
of zinc; 4 animals were given the basal diet plus 1500 ppm of zinc and the last group, composed
of 3 animals, was given the basal diet plus 500 ppm of zinc. The groups fed the two highest
concentrations of zinc showed severe signs of toxicity between 1 to 2 weeks and the ferrets on
the 3000 ppm diet died in less than 2 weeks. The lesions in this group were a diffuse nephrosis,
haemorrhages in the intestine and a severe macrocytic hypochromic anaemia. There was a more
severe diffuse nephrosis and some glomerular damage in the 1500 ppm group. These ferrets also
had a macrocytic hypochromic anacmia. In both 1500 and 3000 ppm groups there was an
increase in zinc and a depression of copper in the liver and kidney tissue. All the zinc-treated
ferrets showed decreased serum caeruloplasmin oxidase activity. The results indicated that the
anaemia was more the result of the haemorrhages than of the zinc induced copper deficiency.15
Four natural occurrences of zinc toxicity in sheep and one in calves were studied. To assist in the
confirmation of a diagnosis of zinc toxicity, 2 experiments (Exp. 1 Acute Intoxication: 3 Merino
wethers were each given 2 g of zinc on 13 consecutive day, Exp. 2 Chronic Intoxication: 2
Merino wethers was fed 08 g of zinc for 12 days, and then 1.2 g each day for the remaining trial
period. In the latter part of the experiment the ration contained approximately 2000 p.p.m. zinc).
Clinical manifestations included inappetance, loss of condition, diarrhea with dehydration or
subcutaneous oedema, profound weakness and jaundice. Significant rises in the concentration of
zinc were usually found in the liver, kidney and pancreas, but occasionally in only 1 or 2 of these
organs. Many affected sheep were anaemic. Pathological changes were found in the pancreas,
kidney, liver, rumen, abomasum, small intestine and adrenal gland. Lesions in the kidney and
abomasum apparently made the most significant contribution to the deterioration in health of

Page 11 of 22
affected animals, but the pancreas was the only organ consistently affected. The degenerative
changes in the pancreas were mainly restricted to the exocrine portion of the organ, and
regeneration of the damaged tissue was observed although exposure to toxic amounts of zinc
continued. 16
Levels of zinc from 3,000 to 12,000 ppm were fed to domestic mallard ducks in their diet. Food
intake and body weight both showed closely parallel decreases as the level of zinc in the diet
increased. In ducks fed zinc, the pancreas and gonads underwent reductions in relation to body
weight, where as ratios of adrenals and kidneys to body weights increased significantly. No
significant change occurred in liver : body weight ratios in ducks fed zinc-supplemented diets.
The gonads were so reduced in size that the reproductive function was probably lost. In birds fed
zinc-supplemented diets, partial paralysis of the legs, diarrhea, and weight loss were noted
within 10 days. Severe paralysis, with some ducks unable to walk, was noted after 20 days. Only
slight anemia was present in some ducks after 30 days, but by day 45, extreme anemia (5-13
grams Hb per 100 cc of blood) was evident in most ducks. Normal hemoglobin concentration
averaged 18.6 grams per 100 cc of blood. Significant increases in concentration of zinc were
found in the liver, kidneys, pancreas, gonads, and breast muscle. High mortality was observed in
all groups treated with zinc-supplemented diets; 2 of 24 ducks died within 60 days. 17
CARCINOGENECITY
In a study that examined both zinc deficiency and supplementation, Mathur et al. found that
animals with a deficient diet (5.9 mg/kg) and animals with a diet supplemented with excessively
high levels of zinc (200-260 mg/kg) had fully developed carcinomas of the palatial mucosa.
While the rats were on the specific diets, the palatial mucosa was painted with 4-nitroquinoline-
N-oxide, 3 times/week for 20 weeks. In the zinc-deficient group, 2/25 rats developed cancer of
the palatial mucosa; 2/25 rats in the excessive zinc group also developed this form of cancer.
Animals supplemented with moderate levels of zinc in the diet (50 mg/kg) developed only
moderate dysplasia. Thus, zinc’s modifying effect on carcinogenesis may be dose-dependent. 18
GENOTOXICITY
In a short-term in vivo assay, Stoner et al. injected strain A/Strong mice (20/sex/dose)
intraperitoneally with zinc acetate 3 times/week for a total of 24 injections (total doses were 72,
180, or 360 mg/kg). Controls (20/sex/group) consisted of an untreated group, a vehicle control
group administered 24 injections of saline, and a positive control group administered a single
injection of urethane (20 mg/mouse). Mice were sacrificed 30 weeks after the first injection;
survival was comparable for all groups. There was no increase in number of lung tumors per
mouse in treated animals relative to the pooled controls. While four thymomas were observed in
zinc acetate-treated groups and none in controls, the occurrence of these tumors was not
statistically significantly elevated.18

In the Salmonella assay (with or without hepatic homogenates), zinc acetate was not mutagenic
over a dose range of 50-7200 μg/plate. In the mouse lymphoma assay, zinc acetate gave a dose-
dependent positive response with or without metabolic activation; the mutation frequency
doubled at 10 μg/mL. In the Chinese hamster ovary cell in vitro cytogenetic assay, zinc acetate
gave a dose-dependent positive response with or without metabolic activation, but the presence
of hepatic homogenates decreased the clastogenic effect. Zinc acetate was not positive in the
unscheduled DNA synthesis assay in rat hepatocytes over a dose range of 10-1000 μg/mL.18

Page 12 of 22
Zinc was tested in several Ames tests. Zinc acetate did not show any mutagenic activity in an
assay with 5 strains of Salmonella, in the presence and absence of the S9 activation system.
However, in a study where zinc was complexed with an organic ligand, the results were positive
with 2 strains of Salmonella. In the in vitro cytogenetic CHO assay, dose-dependent positive
responses of zinc acetate were obtained in the presence and absence of the S9 activation system,
although the S9 reduces both the clastogenic response (≥ 34 to ≥ 45 μg/ml) and the cytotoxicity.
The unscheduled DNA synthesis in primary cultures of rat hepatocytes was not induced by zinc
acetate.13

REPRODUCTIVE AND DEVELOPMENTAL STUDIES

Reproduction studies performed during 2 or 3 generations showed that zinc acetate (∼25 mg
Zn++/kg) in mice and in rats, have no effects on fertility and reproductive performance of male
and female rats F0, F1, F2, if any. In addition, specific studies on the effects of excess dietary
zinc on the chemical composition and enzymatic activities of maternal and foetal tissues have
not revealed any serious adverse effects.13

Male (n=5) and female (n=5) albino rats were orally administered 1 of 3 organic Zinc salt
solutions (Zinc Acetate, Zinc maleate or Zinc Citrate), daily at doses of Zinc ranging from 4.4 to
12.4 mg (17.6 to 49.6 mg/kg) for a period of 29 weeks. A separate group of 3 males received
heavy suspensions of Zinc oxide (34.4 mg; ≈137.6 mg/kg) in Gum acacia for a period of 29
weeks prior to mating. The aforementioned study suggested that feeding of organic Zinc salts, or
Zinc oxide to rats, at doses from 2 to 38 mg (8 to 152 mg/kg) of zinc daily for many weeks prior
to mating, during pregnancy and lactation and no significant effects on the health of the parents,
fertility, nor upon the health and early growth of the F1 offspring. Finally, treatment of F1
offspring with organic zinc salts or zinc oxide (post delivery Day 23 throughout Day 60) had no
apparent effects on their survival growth or maturation.14

2-MONTH ORAL PANCREATIC TOXICITY STUDY

The potential for Zinc to produce pancreatic toxicity was investigated in rats. Briefly, groups of
male Sprague Dawley (9- 10/group) rats were orally administered Zinc Acetate aqueous
solution, by gavage at total daily doses of 5.7, 28.5, and 57.1 mg/kg, (administered as 2 divided
doses each in a total volume of 0.5 ml) for a period of 2 months. Two additional groups of rats,
groups A and B (6 rats/group), served as controls. All animals except Control Group A received
a diet supplemented with copper (100 ppm; -6 mg/kg) to insure that any observed effects were
due to Zinc treatment and not a zinc-induced copper deficiency. At the end of the two month
treatment period animals were sacrificed and the pancreas was removed. It was found that the
administration of Zinc Acetate to rats for 2 months at doses up to 57.1 mg/kg produced no
clinical signs of toxicity and no treatment-related histological effects on the pancreas.14

OTHER TOXICITY STUDIES

In copper overloaded rats, at doses up to 57.1 mg Zn++/kg/day, zinc acetate has no effects on the
pancreas. However, repeated administrations of excess oral zinc (in cats and chicks) result in the

Page 13 of 22
development of a pancreatic toxicity (fibrosis and reversible acinar cell vacuolation respectively)
which may be copper deficiency-mediated, or due to decreases in pancreatic enzymes secondary
to GI tract irritation. A prominent preclinical feature of excess oral zinc administration in rats
and dogs is the development of a microcytic hypochromic anaemia. 13

The rats were randomly divided into four groups (14 in each group), viz. one normal control
group (received saline), two zinc groups (Znlow: 4 mg/kg of zinc acetate; Znhigh: 8 mg/kg of
zinc acetate), and one cyclophosphamide group (50 mg/kg, as positive control of micronucleated
polychromatic erythrocytes (MPCEs)). Saline and zinc acetate were administered
intraperitoneally to the rats once every 2 days, seven times in total. Cyclophosphamide was
given intraperitoneally to the rats once. The concentration of blood zinc was determined and
accumulation of zinc was not observed in the experimental groups. The frequencies of basophilic
stippled erythrocyteand MPCEs in the Znhigh group were significantly higher than those in the
control group (P < 0.05). The levels of serum glutamic oxalacetic transaminase and serum
triiodothyronine in the Znhigh groups decreased significantly, compared with the control group
(P < 0.01 or 0.05). Moreover, we also observed that the level of serum cortisol, another adrenal
corticoid hormone in rats, was increased by zinc acetate in a dose-dependent manner. According
to the findings, exposure to zinc, especially at higher doses, may produce toxic effects on various
tissues and organs including the hematopoietic system, cytogenetics, biochemistry and endocrine
system function.19

APPENDIX III: CLINICAL TRIAL SUMMARY

A double blind controlled clinical trial was carried out to see the effect of zinc supplementation
in severe pneumonia. The study was conducted on 100 children with age group of “06 month to
05 years” diagnosed as case of severe pneumonia. They were randomly assigned (1:1) to receive
antibiotics and supplementation with 20mg of elemental zinc (5ml every 12 hourly) (group A)
and antibiotics (group B) alone. The antibiotics include inj. ceftrixone 50-75mg\kg in single dose
intravenous, inj. ampicillin 100-200mg\kg\day in three divided doses. The group who received
zinc and conventional treatment showed significant decrease in duration of symptoms and
duration of hospitalization. The study results showed that the administration of zinc along with
antibiotics can fasten the healing and recovery of pneumonia. It also decrease the incidence of
multiple antibiotic resistance caused by multiple antibiotic therapies. So it is recommended that
zinc should be added in treatment of lower respiratory tract infection on arrival of patient in
hospital. 20
A double-blind, randomised, placebo-controlled trial was conducted to investigate the efficacy of
zinc supplementation on the treatment outcomes of children admitted to hospital with acute
diarrhea. 86 eligible children with mean age 2.5 years (6 months to 9.3 years) were randomly

Page 14 of 22
allocated to receive either zinc supplementation (15 mg elemental zinc) or a placebo. The
median (IQR) number of hours to recovery from diarrhoea was significantly less in the zinc
group than in the controls [44 vs 52 hours, respectively, p < 0.01]. The median (IQR) number of
stools was significantly lower in the zinc group than in the controls [5 vs 7, p = 0.02]. The
median (IQR) duration of intravenous fluid therapy was 40 hours in the zinc group and 56 in the
control group (p < 0.01). The duration of hospitalisation was 60 hours in the zinc group and 84
hours in the controls (p < 0.01). There was good compliance by all participants in both groups.
Zinc supplementation can reduce the time to resolution of acute diarrhoea, the length of hospital
stay and the frequency of stools. 21
A study was conducted to evaluate the efficacy and safety of treatment with zinc acetate hydrate
in 97 patients aged 63-76 years diagnosed with liver cirrhosis complicated by hypozincemia. The
cumulative serum zinc normalization rates (defined as achievement of a serum zinc level ≥ 80
μg/dL), after 2, 4, and 6 months of treatment were 64.9%, 80.3%, and 82.5%, respectively.
Multivariate analysis identified an albumin level of ≥3.3 g/dL and branched-chain amino acid to
tyrosine (BTR) ratios of ≥ 3.46 as factors contributing to zinc normalization within 3 months of
treatment. Treatment resulted in a significant decrease in blood ammonia and serum copper
levels and significant increases in BTR and alkaline phosphatase levels. Seven patients
prematurely discontinued treatment due to hypocupremia. By the end of treatment, subjective
symptoms had resolved in 46.2% of patients. By 3 months post-treatment, serum zinc levels had
reverted to levels close to those at baseline. Treatment with zinc acetate hydrate resulted in
normalization of serum zinc levels at a high rate.22
The effectiveness of zinc, probiotic bacteria, and lactose-free formula and their different
combinations in the treatment of rotavirus diarrhoea in young children was evaluated by forming
eight different treatment groups, among which the time to resolution of vomiting was
significantly lower in group 4 (S. boulardii plus zinc) compared with groups 1 (S. boulardii) and
5 (S. boulardii plus lactose-free formula) and the duration of diarrhoea was significantly reduced
in groups 2 (zinc) and 4 (S. boulardii plus zinc) compared to control. A statistically significant
difference in the duration of hospitalization was observed for the groups 2 and 4 in comparison
to the control group. Hence it can be said that a different combination of adjunct therapies
brought additional value to rehydration therapy in children with rotavirus diarrhoea in those
receiving only zinc and zinc plus S. boulardii. 23
In a double-blind, controlled trial, 684 patients with acute watery diarrhoea of 3 days or less and
some dehydration, who were attending a hospital, were randomly assigned to 4 groups to
receive: (a) vitamin A 4500 mg retinol equivalent daily; (b) 14.2 mg elemental zinc as acetate for
the first 417 patients and 40 mg of the remaining 273 patients randomized to this group; (c) both
vitamin A and zinc at the above doses daily; or (d) placebo mixtures, the treatment duration was
for 15 days for all groups. Patients were observed in the hospital for 24 h and followed up at
home for 15 d. Zinc supplementation was associated with a reduced duration of diarrhoea (13%,
p = 0.03) and markedly reduced rate (43%, p = 0.017) of prolonged diarrhoea (>7 d). Vitamin A
supplementation was associated with a nonsignificant trend for reduced rate of prolonged
diarrhoea (p = 0.089). In conclusion, zinc supplementation as adjunct therapy had a substantial
impact on the rate of prolonged diarrhoea and some impact on duration.24

Page 15 of 22
The impact of zinc supplementation on clinical recovery, weight gain and subsequent growth
and morbidity in fifty-six moderately malnourished children, aged 12–59 months with culture-
proven shigellosis was studied through a randomized, double-blind, controlled trial by randomly
allocating them to receive zinc (20 mg/day elemental) in multivitamin syrup (intervention) or
multivitamin syrup without zinc (control) in two equally divided doses daily for 2 weeks and
pivmecillinam in a dose of 15 mg/kg every 6 h for 5 days with a follow up in every 2 weeks for
6 months. Children receiving zinc recovered from acute illness significantly faster with median
time (days) to recovery and disappearances of blood and mucous by 50% when compared to the
control group. Their mean body weight increased significantly from 8.8 kg on admission to 9.2
kg (P<0.01) at recovery with fewer mean episodes of diarrhoea observed during the 6-month
follow-up period. Zinc supplementation significantly shortens the duration of acute shigellosis,
promotes better weight gain during recovery and reduces diarrhoeal morbidity during the
subsequent 6 months. 25
With an aim to examine the ability of zinc to prevent clinical pneumonia and diarrhoea in
children younger than 2 years, 1665 children (aged 60 days to 12 months) were randomly
assigned 70 mg zinc (809 children) and placebos to 812 children, orally once weekly for 12
months and were assessed every week, 34 were excluded due to tuberculosis. A significant
decrease in the occurrence of pneumonia and diarrhoea, which were the primary outcomes of the
treatment regimen, was observed in the zinc group. Thus, it was concluded that 70 mg of zinc
weekly reduces pneumonia and mortality in young children. 26
The efficacy of zinc supplementation on growth and morbidity in infants aged 4–24 week was
studied by enrolling them at 4 weeks of age and randomly assigned them to receive 5 mg
elemental Zinc per day (n = 152) or placebo (n = 149) until 24 weeks of age. They were
followed weekly for information on compliance and morbidity; anthropometric measurements
were performed monthly. Serum zinc was assessed at baseline and at 24 weeks of age, at which,
serum zinc concentrations were higher in the zinc than in the placebo group (P < 0.001). Zinc-
deficient Bangladeshi infants showed improvements in growth rate and a reduced incidence of
acute lower respiratory infection after zinc supplementation. In infants with serum zinc
concentrations > 9.18 µmol/L, supplementation improved only biochemical zinc status.27
Moderately malnourished children 6 months to 2 years of age with diarrhoea for more than 14
days, recruited in a double-blind randomized controlled clinical trial, were randomly allocated
into 4 groups of 24 receiving a multivitamin syrup and (i) zinc (20 mg elemental), (ii) vitamin A,
(iii) both zinc and vitamin A, or (iv) neither, in 2 doses daily for 7 day to assess the effect of zinc
and/or vitamin A supplementation on the clinical outcome of persistent diarrhoea. The mean
daily stool outputs from days 2 to 7 of therapy were significantly less in the zinc and zinc plus
vitamin A groups, but not in the vitamin A group, in comparison with the control group. In
children receiving zinc, the cumulative stool weight in the 7 d was 39% less than in the control
group (p < 0.001) and 32% less than in the vitamin A group (p = 0.006). The cumulative stool
weight in the zinc plus vitamin A group was 24% less than in the control group (p < 0.001). The
change in body weight over the 7-d study period was significantly different between the group
receiving zinc and the control group (p = 0.045). The rate of clinical recovery of children within
7 d was significantly greater in the zinc group (88%) compared with the control group (46%, p =
0.002) or vitamin A group (50%, p = 0.005). The results indicate that zinc supplementation in
persistent diarrhoea reduces stool output, prevents weight loss and promotes earlier recovery. 28

Page 16 of 22
In order to test the efficacy of zinc acetate lozenges in reducing the duration of symptoms of the
common cold, a randomized, double-blind, placebo-controlled trial recruited 50 ambulatory
volunteers recruited within 24 hours of developing symptoms of the common cold and were
given one lozenge containing 12.8 mg of zinc acetate or placebo every 2 to 3 hours while awake
as long as they had cold symptoms. The treatment with zinc acetate lozenges was associated
with reduction in the duration and severity of symptoms of the common cold. In addition, in zinc
treated participants, plasma levels of zinc increased and plasma levels of proinflammatory
cytokines decreased. Improvements in clinical symptoms may be related to the effect of zinc on
immunomodulation of proinflammatory cytokines.29
A double blind randomized controlled clinical trial recruited sixty-five children aged 3±24
months with acute diarrhoea for less than 3 d to administer either elemental zinc (20 mg=d) in a
multivitamin syrup or multivitamin syrup alone divided in three divided daily doses for a period
of two weeks. Children were followed up weekly at home to assess subsequent growth and
morbidity for a period of eight weeks. Zinc supplemented children showed significantly greater
cumulative length gain (18.9mm vs 14.5 mm, P<0.03) and comparable body weight gain than
the children of the control group, which were the main outcomes of the intervention. Zinc-
supplemented children experienced significantly fewer episodes of diarrhoea and respiratory
illness compared to the control group. The underweight children receiving zinc-supplementation
also had fewer episodes of diarrhoea and shorter duration of diarrhoeal episodes (P<0.04)
compared to their counterparts in the control group. These results suggest that a short course of
zinc supplementation to malnourished children during acute diarrhoea reduces growth-faltering
and diarrhoeal and respiratory morbidity during subsequent two months.30
This report of a randomized, double-masked, placebo-controlled clinical study demonstrates the
effectiveness of zinc acetate lozenges on common cold symptoms in allergy-tested subjects.
Overall symptom duration was significantly less in the zinc group than in the placebo group
(mean, 3.8 days vs 6.1 days). The mean severity rating for all symptoms was lower in the zinc
group than in the placebo group. Allergy positive subjects who used zinc had a statistically
significant shorter duration of nasal symptoms than allergy-negative subjects (3.5 days vs 7.6
days). Zinc acetate lozenges may significantly shorten the duration of common cold symptoms
and relieve symptoms associated with allergies.31
A randomised double-blind controlled trial was conducted to evaluate the impact of zinc
supplementation (20 mg elemental zinc per day in three divided doses for two weeks) containing
multivitamins on the clinical course, stool weight, duration of diarrhoea, changes in serum zinc,
and body weight gain of children with acute diarrhoea. The results of which were compared to
control group (zinc-free multivitamins). Stool output was 28% less and duration 14% shorter in
the zinc supplemented group than placebo (p = 0.06). There were reductions in median total
diarrhoeal stool output among zinc supplemented subjects and better mean weight gain at the
time of discharge from hospital. Zinc supplementation is acceptable strategy which should be
considered in the management of acute diarrhoea and in prevention of growth faltering in
malnourished children. 32

Page 17 of 22
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