Biochemical and Biophysical Research Communications 660 (2023) 88e95

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Biochemical and Biophysical Research Communications

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Acute and sub-acute dermal toxicity of meloxicam emulgel: Analysis

of biochemical, hematological, histopathological and
immunohistochemical expression
Vaskuri G.S Sainaga Jyothi a, 1, Harithasree Veerabomma a, 1,
Kamatham Pushpa Tryphena b, Dharmendra Kumar Khatri b, **, Shashi Bala Singh b,
Jitender Madan a, *
a
Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Hyderabad, Telangana, India
b
Department of Biological Sciences, National Institute of Pharmaceutical Education and Research, Hyderabad, Telangana, India

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

Article history: Meloxicam, a non-steroidal anti-inflammatory drug (NSAID) for the treatment of osteoarthritis. Despite
Received 2 March 2023 being more effective against pain mediated by inflammation, it is associated with gastrointestinal, car-
Received in revised form diovascular, and renal toxicity. In the current study, acute single-dose (2000 mg/kg) and subacute (500,
22 March 2023
1000, and 2000 mg kg
1 for 28 days) dermal toxicity analyses of meloxicam emulgel were conducted in
Accepted 2 April 2023
Wistar rats. Various biochemical, hematological, histopathological and immunohistochemical parame-
Available online 11 April 2023
ters were evaluated. The dermal LD50 (lethal dose) of meloxicam emulgel was found to be > 2000 mg/kg.
No significant adverse effects of meloxicam emulgel following topical administration in subacute toxicity
Keywords:
Meloxicam emulgel
studies were noticed. IL-1b was not expressed post treatment with meloxicam emulgel. IL-1b is an
Acute toxicity influential pro-inflammatory cytokine that is decisive for host-defence consequence to injury and
Subacute toxicity infection. Therefore, using data gleaned from the extant study, topical administration of meloxicam
Hematology emulgel may be regarded as safe as the “no observed adverse effect level” (NOAEL) was >2000 mg/kg in
Histopathology experimental animals.
Immunohistochemistry © 2023 Elsevier Inc. All rights reserved.

1. Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are generally
advised by the National and International guidelines for the man-
agement of pain and inflammation in osteoarthritis [1]. Although
NSAIDs are very effective in ameliorating pain, however; oral and
parenteral administration of NSAIDs is associated with specific
toxicities to visceral organs (Heart, Liver, Kidney, Lungs, and Brain)
and the gastrointestinal system [2]. Toxicity to the visceral and
gastrointestinal tract occurs due to the distribution of cyclo-
oxygenase (COX) enzyme in these tissues. And with few exclusions,
such as the heart, COX-1 is typically present in interstitial,
* Corresponding author. National Institute of Pharmaceutical Education and
Research, Hyderabad, Telangana, India.
** Corresponding author.
E-mail addresses: dkkhatri10@gmail.com (D.K. Khatri), jitenderpharmacy@
gmail.com (J. Madan).
1
Authors contributed equally.
mesothelial, and smooth muscle cells as well as platelets and blood
vessels with a predominance of several tissues (kidney, gut, and
brain) parenchymal cells [3].
Pathophysiologically, in the process of inflammation COX-2
uprises 20-folds by several stimuli in phagocytes, synoviocytes,
monocytes, fibroblasts, chondrocytes, and endothelial cells [4e9].
However, COX-1 activity is intact or elevated (2- to 4-folds) [10].
Owing to its affinity for COX-2 expression, meloxicam is approved
by US-FDA (United States-Food and Drug Administration) in the
management of rheumatoid, juvenile idiopathic, and osteo-
arthritis related pain and inflammation in humans. In addition,
meloxicam is also approved to control pain and inflammation
associated with mastitis [11] and osteoarthritis [12] in dairy cattle
and veterinary animals, respectively. However, administration of
meloxicam is associated with a risk of liver dysfunction, renal
failure (renal toxicity), cardiotoxicity, and arterial thrombotic ef-
fects, especially at high doses and when used for a prolonged period
of time. Oral consumption of meloxicam similar to other NSAIDs is
associated with gastrointestinal adverse effects, hypertension, and

https://doi.org/10.1016/j.bbrc.2023.04.001
0006-291X/© 2023 Elsevier Inc. All rights reserved.
V.G.SS. Jyothi, H. Veerabomma, K.P. Tryphena et al. Biochemical and Biophysical Research Communications 660 (2023) 88e95

peripheral edema [13]. On the other hand, meloxicam administra-
tion through the topical route may be advantageous as it escapes
the gastrointestinal environment and enhances the drug concen-
tration locally at the inflamed tissue (e.g., osteoarthritis, mastitis,
etc.) [14,15] Contemplating all these factors, we have formulated a
meloxicam emulgel for topical delivery to treat pain and inflam-
mation associated with osteoarthritis, and post-surgical pain [16].
As per OECD guidelines (Organization for Economic Co-operation
and Development), it is important to evaluate the acute and sub-
acute toxicity of emulgel in order to determine its safety to make
sure its safety prior to using it for any therapeutic purpose. In both
studies, it is essential to maintain a record of animals’ behavioral
and physical variations. Additionally, it is crucial to estimate fluc-
tuations in the blood constraints and histopathological examina-
tion of certain vital organs as well as skin. This is true for the sub-
acute toxicity report, in which an evaluation of toxic compounds is
conducted on a topical dermal formulation that has been used on
the skin for a long time [17].
Therefore, in the extant investigation, acute and sub-acute
toxicity studies for meloxicam emulgel [16] were conducted as per
OECD guidelines in Wistar rats. Moreover, alterations in biochemical,
hematological, histopathological and immunohistochemical features
were noticed as per standard and validated protocols.
2. Materials and methods
2.1. Ethical statement
All the experimental analyses were designed and conducted in
compliance with strategies of the Committee for the Purpose of
Control and Supervision of Experiments on Animals (CPCSEA),
Ministry of Fisheries, Animal Husbandry, and Dairying, Govern-
ment of India, New Delhi, India. The studies were permitted by the
Institutional Animal Ethics Committee (IAEC) at the National
Institute of Pharmaceutical Education and Research (NIPER),
Hyderabad, Telangana, India with protocol #NIP/12/2021/PE/443.
2.1. Animals
Healthy male and female Wistar rats, 8e10 weeks old, weighing
150e200g, were used in the toxicity studies. Before the start of the
study, the animals were monitored and acclimatized for a week to
adjust to their new habitat. All the animals were separated based on
their sex in groups of 5 individuals each and were accompanied for
a 12 h light/dark cycle with 22 ± 2  C temperature and 39e65% (RH)
relative humidity. The animals were all fed with commercially
available feed and water. The OECD guidelines were strictly

Table 1
Evaluation of hematological parameters in repeated dose dermal toxicity study of meloxicam emulgel.

Groups Parameters Normal control Vehicle control (Phosphate Low dose Moderate dose High dose Reversal control Reversal control of
saline buffer 10 mL kg
1) (500 mg kg
1) (1000 mg kg
1) (2000 mg kg
1) (water, high dose
10 mL kg
1) (2000 mg kg
1)

Male WBC (  103/mL) 18.5 ± 7.53 15.3 ± 3.37 18.72 ± 11.14 18.2 ± 4.73 11.9 ± 3.57 14.36 ± 5.15 15.76 ± 3.4
RBC (  106/mL) 7.66 ± 0.25 7.88 ± 0.23 7.87 ± 1.7 7.95 ± 0.74 6.89 ± 1.48 8.03 ± 0.21 8.63 ± 0.59
Haemoglobin (g/dL) 14.1 ± 0.76 14.34 ± 0.71 13.68 ± 2.74 13.52 ± 1.02 12.12 ± 2.78 13.96 ± 0.6 14.88 ± 0.45
Haematocrit (%) 40.42 ± 2.04 41.54 ± 1.19 39.58 ± 8.46 39.82 ± 3.02 35.66 ± 7.91 41.22 ± 1.78 43.12 ± 1.45
Mean cell volume 52.78 ± 1.93 52.68 ± 0.41 50.32 ± 0.97 50.14 ± 1.48 51.74 ± 2.04 51.28 ± 1.06 50.04 ± 2.1
(fL)
MCH (pg) 18.4 ± 0.69 18.18 ± 0.78 17.44 ± 0.59 17.02 ± 0.63 17.56 ± 0.81 17.36 ± 0.3 17.28 ± 0.79
MCHC (g/dL) 34.86 ± 0.67 34.52 ± 1.19 34.66 ± 0.77 33.96 ± 0.38 33.9 ± 0.53 33.88 ± 0.31 34.52 ± 0.29
Platelets (  103/mL) 871.8 ± 289.49 919.2 ± 163.96 1031.8 ± 337.64 921.4 ± 301.07 799.6 ± 221.51 951.2 ± 175.43 1032.8 ± 312.07
Lymphocytes (%) 66.66 ± 2.5 64.64 ± 1.83 66.04 ± 4.89 66.24 ± 2.19 64.78 ± 2.88 67.08 ± 5.63 68.2 ± 2.17
Monocytes (%) 2.3 ± 0.67 2.78 ± 0.76 2.86 ± 1.94 2.74 ± 0.75 2.94 ± 0.79 2.02 ± 1.58 2.84 ± 0.75
Granulocytes (%) 31.04 ± 2.77 32.58 ± 2.32 30.32 ± 2.77 31.02 ± 1.98 32.28 ± 2.67 30.9 ± 6.01 28.96 ± 2.5
Red cell distribution 14 ± 1.11 13.62 ± 1.02 12.88 ± 0.44 13.26 ± 0.21 12.92 ± 0.3 13.24 ± 0.57 12.98 ± 0.16
width (RDW)-CV (%)
Red cell distribution 29.56 ± 2.38* 28.68 ± 1.93 25.96 ± 1.37* 26.6 ± 0.99 26.78 ± 1.48 27.12 ± 0.63 26 ± 0.93*
width (RDW)-SD (fL)
PCT (%) 061 ± 018 0.64 ± 0.11 0.69 ± 0.24 0.63 ± 0.24 0.58 ± 0.19 0.65 ± 0.14 0.68 ± 0.21
MPV (fL) 7.1 ± 0.27 6.92 ± 0.36 6.7 ± 0.26 6.78 ± 0.53 7.12 ± 0.6 6.82 ± 0.36 6.66 ± 0.29
PDW (%) 15.66 ± 0.29 15.4 ± 0.48 15.54 ± 0.33 15.32 ± 0.48 15.26 ± 0.5 15.08 ± 0.37 15.06 ± 0.58

Female WBC (  103/mL) 14.32 ± 3.12 10.14 ± 3.23 12.2 ± 5.45 11.86 ± 2.92 11.42 ± 4.78 12.86 ± 4.24 11.94 ± 4.26
RBC (  106/mL) 8.29 ± 0.38 6 ± 1.57 6.53 ± 2.39 7.81 ± 0.2 7.316 ± 1.54 7.13 ± 1.24 8.09 ± 0.61
Hemoglobin (g/dL) 15.06 ± 0.47 10.5 ± 2.99 11.1 ± 4.1 13.68 ± 0.32 13.2 ± 2.62 12.36 ± 2.15 14.1 ± 0.64
Hematocrit (%) 41.84 ± 1.48 30.74 ± 8.1 31.98 ± 11.62 39.34 ± 1.17 37.56 ± 7.57 36.36 ± 6.27 41.76 ± 1.9
Mean cell volume 50.5 ± 0.66 51.2 ± 0.95 43.04 ± 12.96 50.38 ± 1.35 51.48 ± 1.54 51 ± 0.22 50.94 ± 0.99
(fL)
MCH (pg) 18.18 ± 0.5 17.42 ± 0.62 16.94 ± 0.42** 17.54 ± 0.49 18.08 ± 0.56 17.32 ± 0.13 17.16 ± 0.33*
MCHC (g/dL) 36 ± 0.62 34.02 ± 0.83*** 34.6 ± 0.55** 34.8 ± 0.48* 35.16 ± 0.38 33.96 ± 0.11*** 33.66 ± 0.34***
Platelets (  103/mL) 1113.4 ± 174.34 848.8 ± 229.19 921.4 ± 405.23 1189.6 ± 73.69 978.2 ± 211.52 832 ± 273.67 998.6 ± 230.55
Lymphocytes (%) 67.52 ± 2.58 62.1 ± 6.64 68.38 ± 3.19 68.34 ± 10.03 72.36 ± 6.65 59.7 ± 11.54 59.24 ± 13.27
Monocytes (%) 2.28 ± 0.66 3.26 ± 0.63 2.54 ± 0.68 2.08 ± 1.25 3.62 ± 1.09 3.26 ± 1.83 3.32 ± 1.35
Granulocytes (%) 30.2 ± 2.15 34.64 ± 6.4 29.08 ± 3.5 28.84 ± 10.27 24.02 ± 7.57 36.08 ± 9.73 37.94 ± 13.24
Red cell distribution 12.72 ± 0.34 13.56 ± 1.26 13.26 ± 0.59 13.24 ± 0.33 13.44 ± 0.84 12.48 ± 0.45 12.82 ± 0.55
width (RDW)-CV (%)
Red cell distribution 25.68 ± 0.94 27.8 ± 2.87 25.98 ± 1.29 26.68 ± 1.13 27.7 ± 2.29 25.46 ± 0.94 26.4 ± 1.53
width (RDW)-SD (fL)
PCT (%) 0.77 ± 0.13 0.47 ± 0.27 0.64 ± 0.29 0.78 ± 0.05 0.64 ± 0.23 0.52 ± 0.16 0.66 ± 0.15
MPV (fL) 6.92 ± 0.23 6.68 ± 0.48 6.84 ± 0.44 6.58 ± 0.4 7.08 ± 0.61 6.3 ± 0.23 6.58 ± 0.04
PDW (%) 15.48 ± 0.62 15.28 ± 0.83 15.42 ± 0.4 14.96 ± 0.5 15.26 ± 0.46 15.42 ± 0.56 14.76 ± 0.26

-vis’ normal control group.
Key: Data represented ***p < 0.001, **p < 0.01 and *p < 0.05 vis-a

89
V.G.SS. Jyothi, H. Veerabomma, K.P. Tryphena et al.
adhered to conduct acute and sub-acute (repeated dose) dermal
toxicity investigations. Hence, the present acute [18] and sub-acute
[19] dermal toxicity analyses were undertaken by conferring with
OECD 402 and 410 strategies, respectively.
2.2. Acute dermal toxicity analysis
In acute toxicity, a total of 3 female Wistar rats were selected
and the dorsal surface of the rats was clean-shaved by using an
electric clipper 24 h before the start of treatment. A 2000 mg kg
1
dose was applied to rats in a manner that the meloxicam emulgel
remains intact on the skin with a bandage gauze for 24 h exposure
time. Animals were monitored closely firstly every 4 h for any in-
dications of lacrimation, pupil size deviations, respiratory changes,
clonic or tonic seizures, stereotypes, or bizarre behavior and also
tracked once a day for a period of 14 days for toxicity and mortality.
Additionally, intake of food and water was recorded on alternate
days. Then again, body weight was observed every week. On the
14th day of the experiment, rats were scrutinized for food con-
sumption, body weight, gross behavioral vagaries, and death. After
the 14th day of the study, rats were sacrificed by intraperitoneal
administration of ketamine (0.35 mL/kg) and xylazine (0.10 mL/kg),
and any variation in size, color shade, and structure of organs was
noticed by gross necropsy.
2.3. Sub-acute toxicity analysis
Male and female Wistar rats were alienated into 7 groups of 5
animals per group and housed separately to analyze subacute
toxicity. According to the guidelines, 2000 mg/kg/day was ascer-
tained as a high dose, and the descending dosages of 1000 and
500 mg/kg/day were chosen as medium and low doses, respectively.
The back of the rat skin was shaved using an electric clipper 24 h
before the start of the study. Group-A was assigned as a control
group with no dermal application while Group B was vehicle control
given with PBS (Phosphate buffer saline, pH 7.4) applied dermally.
Fig. 1. Photomicrographs of heart of male and female rats in repeated dose dermal toxicity of meloxicam emulgel, (A) Normal control; (B) Vehicle control; (C) Low dose
(500 mg kg
1); (D) Medium dose (1000 mg kg
1); (E) High dose (2000 mg kg
1) (F) Reversal control (water, 10 mL kg
1); and (G) Reverse control of high dose (2000 mg kg
1)
captured at magnification of 10X and 40X.
90
Biochemical and Biophysical Research Communications 660 (2023) 88e95
Groups C, D, and E were designated as low (500 mg kg
1), medium
(1000 mg kg
1), and high doses (2000 mg kg
1) of meloxicam
emulgel, respectively for 28 days. Group F was the reversal control
group treated with water dermally and group G was the reversal
control of high dose treated with meloxicam emulgel at
2000 mg kg
1 dose for additional 14 days. The topical application of
meloxicam emulgel was covered with an aluminum foil patch held in
place by a bandage to prevent oral consumption and for a minimum
of 6 h/day with a 5-days/week schedule for a time frame of 28 days.
Animals of group A-E were noticed for 28 days and on the 28th
day, blood samples were collected through a retro-orbital vein to
perform hematological analysis and the animals were euthanized.
Necropsies were carried out and the organs were accurately
weighed. In addition, macroscopical changes were also recorded.
The skin, heart, kidneys, and liver tissues were fixed in 10% v/v
solution of formalin and stored at - 40  C until further analysis.
Similarly, animals of groups F and G were tested for toxicity pa-
rameters as specified on 42nd Day.
2.3.1. Clinical observations
Rats were assessed for signs of redness, erythema, and edema in
and around the treated skin twice daily before treatment followed
by frequently over the initial 8 h post-treatment. Next, rats were
examined thrice daily for the entire duration of the test. All rat's
body weights were recorded on 0, 7, 14, 21, 28, 35, and 42 days
[20e22].
2.3.2. Hematological analysis
The samples of blood were analyzed for a hematological profile
as per standard protocol. In brief, the heparinized blood samples
were undergone for an evaluation of white blood cells (WBC), red
blood cells (RBC), hemoglobin (Hb), hematocrit (HCT), mean
corpuscular volume (MCV), mean corpuscular hemoglobin (MCH),
mean cell hemoglobin concentration (MCHC), platelets, lympho-
cytes, monocytes, granulocytes, red cell distribution width (RDW)-
CV, red cell distribution width (RDW)-SD, procalcitonin (PCT),
V.G.SS. Jyothi, H. Veerabomma, K.P. Tryphena et al. Biochemical and Biophysical Research Communications 660 (2023) 88e95

mean platelet volume (MPV), and platelet distribution width (PDW)
using an automatic hematology analyzer [23,24].
2.3.3. Serum biochemical analysis
The blood samples were analyzed for biochemical tests as per
standard protocols. In brief, blood specimens were placed into
sterilized centrifuge tubes devoid of any anticoagulant and left to
sit for 30 min. Then the collected samples were centrifuged at 4  C
for 10 min at 1500 g. The serum from each sample was separately
transferred into a fresh tube and was stored at 4  C until it was
further processed. The serum samples were analyzed for
biochemical parameters like AST (Aspartate aminotransferase),
GGT (Gamma glutamyl transferase), ALP (Alkaline phosphatase),
ALT (Alanine transaminase), creatinine, bilirubin, albumin, total
protein, BUN (Blood urea nitrogen), potassium, phosphorus, cal-
cium, chloride, and sodium [25e27].
2.3.4. Histopathological examination
Histopathological analysis was performed as per the standard
procedure. In brief, the tissue of skin, liver, kidney, and heart was
separately fixed in 10% v/v formalin solution and the tissue frag-
ments were entrenched in paraffin wax. This was followed by
microtome sectioning and staining with hematoxylin and eosin (H
& E) dyes. The histological examination was carried out using a
light microscope (Leica microscope-LeicaICC50W-DF750) at 10x
and 40x magnification.
2.3.5. Immunohistochemistry analysis
Immunohistochemistry was used to assess the level of inflam-
mation [28]. Initially, skin samples were embedded in OCT tissue
freezing medium and sectioned to obtain 5e10 mm thick tissue
sections using cryotome. The sections were then mounted on the
poly-L-lysine coated slides. Following this, the tissue sections were
fixed in methanol for about 10 min, air-dried, and washed twice in

Table 2
Evaluation of biochemical parameters in the repeated dermal dose dermal toxicity study of meloxicam emulgel.

Groups Parameters Normal control Vehicle control (Phosphate Low dose Moderate dose High dose Reversal control Reversal control of high
saline buffer 10 mL kg
1) (500 mg kg
1) (1000 mg kg
1) (2000 mg kg
1) (water, 10 mL kg
1) dose (2000 mg kg
1)

Male AST (U/I) 89.53 ± 17.37 83.88 ± 5.97 91.09 ± 2.33 87.53 ± 8.02 78.81 ± 11.54 90.78 ± 5.77 90.24 ± 14.34
ALT (U/I) 33.75 ± 12.94 40.25 ± 15.35 28 ± 3.29 28.8 ± 3.12 31.39 ± 6.8 25.91 ± 3.99 27.69 ± 5.15
ALP (IU/I) 219.27 ± 19.37 190.22 ± 48.1 162.51 ± 35.34 225.76 ± 58.57 183.58 ± 25.06 162.51 ± 33.29 188.08 ± 27.96
GGT (IU/I) 9.23 ± 2.94 6.6 ± 3.47 10.36 ± 2.78 7.51 ± 2.24 13.97 ± 7.9 8.8 ± 1.54 9.27 ± 1.53
Creatinine 0.43 ± 0.18 0.76 ± 0.28 0.77 ± 0.1 0.66 ± 0.17 0.79 ± 0.12 0.54 ± 0.17 0.72 ± 0.41
(mg/dL)
Bilirubin 0.37 ± 0.25 0.38 ± 0.19 0.53 ± 0.09 0.61 ± 0.18 0.63 ± 0.3 0.48 ± 0.3 0.3 ± 0.2
(mg/dL)
Albumin (g/ 4.53 ± 0.6 4.79 ± 0.39 4.73 ± 0.44 4.61 ± 0.39 4.82 ± 0.38 4.82 ± 0.22 4.59 ± 0.69
dL)
Total 7.84 ± 0.44 7.49 ± 0.49 7.55 ± 0.47 7.21 ± 0.2 7.3 ± 0.31 7.82 ± 0.28 7.69 ± 0.29
protein (g/
dL)
BUN (mg/ 20.12 ± 4.3 17.74 ± 6.19 23.04 ± 3.61 21.02 ± 4.92 27.06 ± 7.5 18.57 ± 3.39 24.59 ± 7.7
dL)
Potassium 5.17 ± 1.99 5.46 ± 1.53 5.07 ± 1.87 5.32 ± 1.13 4.9 ± 1.22 4.47 ± 1.33 5.84 ± 1.6
(mEq/L)
Phosphorus 5.97 ± 1.41 7.03 ± 0.92 6.5 ± 1.17 6.84 ± 0.8 6.87 ± 1.09 6.75 ± 1.08 6.74 ± 1.18
(mg/dL)
Calcium 8.93 ± 1.84 9.26 ± 2.39 9.08 ± 2.6 8.6 ± 2.81 9 ± 1.84 9.34 ± 2.07 9.09 ± 1.82
(mg%)
Chloride 106.7 ± 3.5 105.64 ± 1.9 102.68 ± 3.54 104.32 ± 3.14 102.31 ± 2.29 102.37 ± 2.54 102.17 ± 1.89
(mEq/L)
Sodium 135.84 ± 3.12 141.75 ± 5 143 ± 5.3 139.35 ± 2.27 141.13 ± 3.87 136.71 ± 2.96 143.55 ± 1.41
(mEq/L)

Female AST (U/I) 93 ± 12.82 78 ± 6.33 80.27 ± 6.3 84.41 ± 10.07 88.42 ± 10.07 84.41 ± 11.76 82.63 ± 17.82
ALT (U/I) 32.63 ± 10.8 28.89 ± 5.4 32.46 ± 7 31.7 ± 4.8 32.86 ± 8.96 32.86 ± 5.85 32.9 ± 5.47
ALP (IU/I) 184.2 ± 23.46 219.67 ± 43.1 199.21 ± 23.1 186.16 ± 21.8 199 ± 25.19 214.44 ± 34.9 200.84 ± 26.21
GGT (IU/I) 10.1 ± 2.57 11.89 ± 3.02 11.01 ± 3.17 10.95 ± 2.75 14.17 ± 3.78 13.12 ± 6.21 15.44 ± 4
Creatinine 0.58 ± 0.22 0.63 ± 0.17 0.48 ± 0.07 0.72 ± 0.11 0.69 ± 0.18 0.68 ± 0.18 0.51 ± 0.17
(mg/dL)
Bilirubin 0.68 ± 0.33 0.64 ± 0.25 0.72 ± 0.31 0.65 ± 0.35 0.88 ± 0.19 0.7 ± 0.34 0.69 ± 0.33
(mg/dL)
Albumin (g/ 4.18 ± 0.98 4.81 ± 0.34 4.25 ± 0.79 4.42 ± 0.57 4.98 ± 0.22 4.02 ± 0.72 3.91 ± 0.43
dL)
Total 8.35 ± 1.79 7.93 ± 2.58 7.8 ± 0.85 6.98 ± 0.84 8.75 ± 1.76 9.57 ± 2.36 7.99 ± 1.26
protein (g/
dL)
BUN (mg/ 25.76 ± 8.9 23.98 ± 2.86 21.98 ± 6.61 17.2 ± 5.08 20.07 ± 7.45 19.88 ± 5.35 33.38 ± 10.42
dL)
Potassium 4.86 ± 1.35 4.94 ± 1.23 4.47 ± 0.7 4.91 ± 1.37 4.08 ± 0.87 4.3 ± 1.42 4.75 ± 0.31
(mEq/L)
Phosphorus 6.31 ± 3.44 7.27 ± 3.43 7.76 ± 2.53 4.92 ± 1.33 6.62 ± 2.97 6.32 ± 2.97 6.87 ± 3.53
(mg/dL)
Calcium 11.14 ± 2.25 9.72 ± 2.9 7.73 ± 2.01 10.07 ± 2.69 9.38 ± 4.66 11.24 ± 4.29 9 ± 2.98
(mg%)
Chloride 102.84 ± 7.95 108.28 ± 8.08 96.02 ± 8.24 107.32 ± 10.8 105.48 ± 12.49 102.33 ± 10.52 98.23 ± 7.25
(mEq/L)
Sodium 122.47 ± 15.28 136.89 ± 5.7 138.29 ± 19.01 131.11 ± 9.46 143.14 ± 11.32 131.45 ± 13.28 134.53 ± 8.51
(mEq/L)

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V.G.SS. Jyothi, H. Veerabomma, K.P. Tryphena et al. Biochemical and Biophysical Research Communications 660 (2023) 88e95

Fig. 2. Photomicrographs of liver of male and female rats in repeated dose dermal toxicity of meloxicam emulgel, (A) Normal control; (B) Vehicle control; (C) Low dose
(500 mg kg
1); (D) Medium dose (1000 mg kg
1); (E) High dose (2000 mg kg
1) (F) Reversal control (water, 10 mL kg
1); and (G) Reverse control of high dose (2000 mg kg
1)
captured at magnification of 10X and 40X.

PBS-T (Phosphate buffer saline-Tween™ 20). Post washing, tissue
sections were blocked with 3% bovine serum albumin blocking
buffer and incubated for 2 h. Slides were incubated with 1:500
diluted mouse primary antibody against IL-1b (Santa Cruz,
SC52012) in a humid chamber at 4  C overnight. The slides were
washed twice with PBS for 5 min and sections were incubated with
Alexa Fluor™ 546 goat anti-mouse IgG (H þ L) (Thermofischer
scientific, Alexa Fluor™ 546, A11003) and incubated for 2 h in dark.
Eventually, tissue sections were washed with PBS, dried, and
examined under a confocal laser scanning microscope (CLSM) at
20X magnification (Leica DMi8). The control group was included for
accurate interpretation of the results.
2.4. Statistical analysis
The outcomes from several studies were presented as
mean ± standard deviation (SD). The statistical analysis was per-
formed using GraphPad Prism 5.0 (Graph Pad Prism, San Diego,

Fig. 3. Photomicrographs of kidney of male and female rats in repeated dose dermal toxicity of meloxicam emulgel, (A) Normal control; (B) Vehicle control; (C) Low dose
(500 mg kg
1); (D) Medium dose (1000 mg kg
1); (E) High dose (2000 mg kg
1) (F) Reversal control (water, 10 mL kg
1); and (G) Reverse control of high dose (2000 mg kg
1)
captured at magnification of 10X and 40X.

92
V.G.SS. Jyothi, H. Veerabomma, K.P. Tryphena et al. Biochemical and Biophysical Research Communications 660 (2023) 88e95

Fig. 4. Photomicrographs of skin of male and female rats in repeated dose dermal toxicity of meloxicam emulgel, (A) Normal control; (B) Vehicle control; (C) Low dose
(500 mg kg
1); (D) Medium dose (1000 mg kg
1); (E) High dose (2000 mg kg
1) (F) Reversal control (water, 10 mL kg
1); and (G) Reverse control of high dose (2000 mg kg
1)
captured at magnification of 10X and 40X.

USA), and the one-way analysis of variance (ANOVA) led by Dun-
nett's test was used to evaluate the significant variations within the
groups. P  0.05 was regarded as statistically significant.
3. Results and discussion
3.1. Meloxicam emulgel did not exhibit any transience or adverse
reaction in acute toxicity
Nanotechnology has grown immensely in recent times of its
many potential benefits. Nanomedicine has revolutionized the
biomedicine industry, exclusively in the drug delivery arena owing to
its intriguing prospects such as high penetration, solubility, systemic
stability, and site-specific targeting [29]. Though, nanomedicines
propound both advantageous and detrimental effects based on their
nature in various environments, thus emphasizing the worth of
toxicology studies prior to use in humans. Toxicity testing is a pre-
requisite for drug development and delivery [30]. The toxicity risk of
an experimental product is divulged by a pre-clinical toxicology
assessment of diverse biological systems that examines the effects on
different doses, species, and organs. In the present investigation,
acute and sub-acute dermal toxicities testing of our meloxicam
emulgel was carried out according to OECD guidelines. The results of
acute dermal toxicity testing revealed that single-dose dermal
application of meloxicam emulgel at 2000 mg/kg dose didn't show
any transience or adverse reactions during 14 days of the observation
period (Suppl. Table 1 and Suppl. Figure 1). No discrepancy was
found in the rats gross pathology (Suppl. Figure 1). The lethal dose 50
(LD50) for the dermal pathway was considered to be > 2000 mg kg
1
as per the OECD 402 guiding principle. No Adverse-Effect Level
(NOAEL) was observed to be 2000 mg kg
1 body weight.
3.2. Meloxicam emulgel did not exhibit any expression for IL-1b, a
proinflammatory marker in subacute toxicity
Following acute toxicity testing, a subacute dermal toxicity study

Fig. 5. Immunohistochemical expression of IL-1 b in skin of male and female rats in repeated dose dermal toxicity of (A) Normal control (B) Vehicle control (C) Low dose
(500 mg kg
1) (D) Medium dose (1000 mg kg
1) (E) High dose (2000 mg kg
1) (F) Reversal control (water, 10 mL kg
1); and (G) Reversal control of high dose (2000 mg kg
1)
captured at 20X magnification using CLSM.

93
V.G.SS. Jyothi, H. Veerabomma, K.P. Tryphena et al.
was conducted for 28 days according to the OECD 410 guidelines. The
dosages applied dermally (2000 mg kg
1,1000 mg kg
1 and
500 mg kg
1) did not have an effect on food consumption
(Suppl. Table 2) and water intake (Suppl. Table 2) between the
treated and control groups and body weight (P < 0.05) was sub-
stantially increased over the course of treatment. Meloxicam emul-
gel did not produce any clinical symptoms like erythema, irritation
along with other toxic effects in rats. Macroscopically, rats that were
euthanized after dermal dosage and the control group did not exhibit
any noticeable pathological alterations at necropsy and in the mean
liver, heart, or kidney weights (Suppl. Table 3).
The presence of any illness, metabolic disorders, or damage to
the structure and functions of organs may be illustrated by alter-
ations in the blood profile [31]. In the hematological analysis
(Table 1), no significant (One-way ANOVA, P > 0.05) difference was
noticed during and at the end of the subacute toxicity study period
of 28 days except a remarkable (One way ANOVA, P < 0.05)
reduction in red cell distribution width (RDW)-SD (fL) of low dose
(25.96 ± 1.37) and reverse control of high dose (26 ± 0.93) in male
rats as compared to the control group (29.56 ± 2.38) (Table 1).
Besides, in female rats, a significant decrease in MCH (pg) was
noticed in the low dose group (16.94 ± 0.42) (One way ANOVA,
P < 0.01) and reverse control of high dose (17.16 ± 0.33) (One way
ANOVA, P < 0.05) in contrast to the healthy control (18.18 ± 0.5)
(Table 1). Moreover, a decrease in MCHC (g/dL) was noticed in the
vehicle control (34.02 ± 0.83; One way ANOVA, P < 0.001), low dose
(34.6 ± 0.55; One way ANOVA, P < 0.01), medium dose (34.8 ± 0.48;
One way ANOVA, p < 0.05), reversal control (33.96 ± 0.11; One way
ANOVA, P < 0.001), reverse control of high dose (33.66 ± 0.34; One
way ANOVA, P < 0.001) in contrast to the control group (36 ± 0.62)
(Table 1). The erythrocyte color index in the blood can be measured
in terms of mean corpuscular hemoglobin (MCHC) which is a
measure of Hb concentration in a packed red blood cell volume
[32]. A low MCHC indicates an iron deficiency, abnormal synthesis
of Hb, and alterations in osmolarity [33]. However, besides the
remarkable difference, the level of MCHC was found to be normal as
compared to the normal reference range level of MCHC
(31.9e38.5 g/dL). Additionally, the biochemical parameters
assessed against the control group (P > 0.05) didn't express any
significant difference (Table 2). Similar to other NSAIDs, meloxicam
affects kidney functions as it inhibits the urinary prostaglandin E2
pathway and furosemide-stimulated plasma rennin activity
[34,35]. Yet, there was no statistically significant (ANOVA One-way
test, P > 0.05) alteration in serum creatinine level (Table 2), a
marker for renal tubular impairment observed over the 28-day
treatment period. The normal reference range of serum creatinine
is found to be 0.4e0.8 mg/dL. Furthermore, no significant (One way
ANOVA, P > 0.05) increment in hepatic enzymes or blood urea ni-
trogen levels (Table 2) thus there were no dose-related adverse
events, and was found to be safe to use.
Furthermore, photomicrographs of histopathological analysis
also indicated somewhat similar results. A representative photo-
micrograph of the heart from each group of the sub-acute toxicity
study is presented in Fig. 1. Cardiomyocytes and cardiac muscle
fibers of heart tissue showed no derangement or dilation including
the absence of cellular infiltration. Congestion and hemorrhage of
the cardiac tissue and degeneration or necrotic changes of muscle
striations were also absent in all the groups. On other hand, the
histopathology of the liver of all the groups depicted the absence of
any vascular congestion in hepatic parenchyma (Fig. 2). The hepa-
tocyte cells are perceived to be intact with a deficiency of degen-
erative or necrotic changes and inflammatory alterations. Samples
of kidney tissue subjected to histopathology revealed the nonap-
pearance of congestion and hemorrhage. Moreover, no noticeable
degenerative or necrotic changes were detected showing the
94
Biochemical and Biophysical Research Communications 660 (2023) 88e95
intactness of glomeruli in addition to the absence of any inflam-
matory marks (Fig. 3). Fig. 4 represented the histopathology of skin
samples from all the groups which indicated no lesions or cellular
infiltration illustrating the absence of inflammation and edema.
The results obtained from the immunohistochemistry of skin sec-
tions indicated that IL-1b was not expressed post treatment with
meloxicam emulgel in all male and female rats (Fig. 5). IL-1b is an
influential pro-inflammatory cytokine that is decisive for host-
defence consequence to injury and infection [36]. This observa-
tion discloses the absence of any inflammation, non-irritability and
non-toxicity of meloxicam emulgel to the skin.
4. Conclusion
In conclusion, the acute and subacute dermal toxicity studies of
meloxicam emulgel did not show any signs of toxicity as evident
from hematological, biochemical, histological and immunohisto-
chemistry parameters at any of the dose levels that were investi-
gated. As a result, meloxicam emulgel was found safe to use
topically in the management of pain and inflammation.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgement
Authors are highly thankful to the Department of Pharmaceu-
ticals, Ministry of Chemicals and Fertilizers, Government of India,
New Delhi, India for providing the financial assistance.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2023.04.001.
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