Claims:WE CLAIM
1. Method of monitoring acute toxicity of an inhalable composition of aqueous chlorine solution comprising administration through nebulizer and monitoring the subject, during and following the subjecting, at least one on - site parameter selected from the group consisting of :
mortality and morbidity studies,
biochemical,
hematological,
histopathological parameters and
Gross necropsy studies.
2. The Method ofmonitoring acute toxicity as claimed in claim 1; wherein subject is selected from rodent and non-rodent.
3. The Method of monitoring acute toxicity as claimed in claim 1; wherein subject is selected from rats and rabbits.
4. The Method of monitoring acute toxicity as claimed in claim 1; wherein monitoring the subject at least one on - site parameter selected from the group consisting of: a vital sign; and a pulmonary function.
5. The Method of monitoring acute toxicity as claimed in claim 1; wherein no deterioration is observed in at the at least one parameter during and following the subjecting.

Dated this on 29th day of June 2021

Asha P. Hole
(Patent Agent)
(IN/PA 2269)
, Description:FIELD OF THE INVENTION
The present invention relates to the pharmaceutical composition of chlorine administered by inhalation for treatment of respiratory viral infection. The present invention also provides a method for the management of respiratory viral infection associated with COVID and viral morbidities by administering chlorine through nebulizer. The invention further relates to acute toxicity and safety assessment of the formulated composition.
BACKGROUND OF THE INVENTION
The virus, technically named SARS-CoV-2, is a newly identified virus, but it is the seventh Coronavirus known to infect humans. Scientists are trying hard to find drugs to treat COVID-19. According to research there are more than 30 agents including Western medicines, natural products and traditional Chinese medicines that may have potential efficacy against COVID-19. However, several medicines have been suggested as potential investigational therapies, many of which are now being or will soon be studied in clinical trials, including the SOLIDARITY trial co-sponsored by WHO and participating countries. Several drugs such as chloroquine, arbidol, remdesivir, and favipiravir are currently undergoing clinical studies to test their efficacy and safety in the treatment of coronavirus disease 2019 (covid-19).
Crucially, enveloped viruses are easier to kill SARS-CoV-2, the virus responsible for the COVID-19 outbreak, is an enveloped virus and therefore the easiest to kill.
It is discovered that each disinfectant has totally different effects, attacking one or several of the virus’ functions. Even though the outcome is the same, the eradication methods are different. Ozone, ultraviolet irradiation, liquid chlorine, chlorine dioxide, and sodium hypochlorite disinfections are commonly used technologies for disinfection. Using the correct disinfectant is an important part of preventing and reducing the spread of illnesses. Isopropanol or Ethanol (Alcohol), Quaternary Ammonium Compounds, Sodium Hypochlorite (Bleach), Hydrogen Peroxide are some common disinfectants which kills viruses, bacteria as well as fungi. Chlorine is a kind of strong oxidizer, which is one of the most early used disinfection methods. WHO recommended use of chlorine to make inactive COVID-19 for treatment of drinking water and swimming pools.
US6333054 discloses topic hydrogel disinfectant composition comprising active chlorine.
Various non-patent literatures disclosed the Effects of a Low Concentration hypochlorous acid (HOCl). Hyun Jik Kim et al (Laryngoscope 118: October 2008) disclose Effects of a Low Concentration hypochlorous acid nasal irrigation solution on bacteria, fungi, and viruses. This literature disclosed the virucidal effects of HOCl and used the human influenza a virus to challenge the cells.
The treatment for COVID-19 is symptomatic because there is no specific drug or cure for it. It is essential to develop effective, economical, user friendly method of treating COVID-19.
Thus, the inventors of the present invention have successfully addressed the existing drawbacks and formulated pharmaceutical composition of chlorine for treatment of viral respiratory infection by inhalation. The inventors of the present invention have also worked on assessment of toxicity profile of the formulated composition.
OBJECTIVE OF THE INVENTION
An object of the present disclosure is to provide a pharmaceutical composition of aqueous chlorine for treatment of respiratory viral infection by inhalation.
Another object of the present disclosure is to provide an inhalational composition comprising aqueous solution of chlorine to treat COVID-19 through nebulizer.
Another object of the present invention is to provide acute toxicity assessment of the formulated composition.
SUMMARY OF THE INVENTION
The present disclosure provides a liquid composition of chlorine for treatment of respiratory viral infection and method thereof.
In one aspect the present invention provides acute toxicity potential of LifeViroTreat wherein LifeViroTreat is the formulated composition of aqueous chlorine.
In another aspect the present invention provides Method of monitoring acute toxicity of an inhalable composition of aqueous chlorine solution comprising administration through nebulizer and monitoring the subject, during and following the subjecting, at least one on - site parameter selected from the group consisting of :
-mortality and morbidity studies,
-biochemical,
-hematological,
-histopathological parameters and
-Gross necropsy studies.
According to an embodiment the present invention provides the Method of monitoring acute toxicity; wherein subject is selected from rodent and non-rodent.
According to an embodiment the present invention provides the Method of monitoring acute toxicity; wherein subject is selected from rats and rabbits.
According to an embodiment the present invention provides the Method of monitoring acute toxicity; wherein monitoring the subject at least one on - site parameter selected from the group consisting of: a vital sign; and a pulmonary function.
According to an embodiment the present invention provides the Method of monitoring acute toxicity; wherein no deterioration is observed in at the at least one parameter during and following the subjecting.
According to an embodiment the present invention provides acute toxicity assessment of formulated aqueous chlorine composition administered through inhalation.
According to another embodiment the present invention provides mortality and morbidity studies of the formulated aqueous chlorine composition.
According to another embodiment the present invention provides acute toxicity assessment of the formulated composition based on biochemical, hematological and histopathological parameters.
According to a further embodiment, the present invention provides acute toxicity assessment of the formulated composition based on Gross necropsy studies.
According to a further embodiment, the present invention provides acute toxicity assessment in rodent and non-rodent experimental animals.
According to a further embodiment, the experimental animals were selected from rats and rabbits.
According to another embodiment, the present invention provides acute toxicity assessment wherein the observation period was of 14 days.
According to an embodiment, the present invention provides acute toxicity assessment wherein 10 ml vials of LifeViroTreat were used.
According to a further embodiment, the test item was administered through a nebulizer.
According to another embodiment, the present invention provides acute toxicity assessment wherein the concentration of aqueous chlorine is 1mg/m3.
According to another embodiment, the present invention provides acute toxicity assessment wherein the experimental animals were exposed to the aqueous chlorine formulation for 15 minutes 6 and 12 times in a day.
According to a further embodiment, the experimental animals were observed for anxiety, motor activity, body weight and behavioral changes.
DESCRIPTION
In the description that follows, a number of terms are used, the following definitions are provided to facilitate understanding of various aspects of the disclosure. Use of examples in the specification, including examples of terms, is for illustrative purposes only and is not intended to limit the scope and meaning of the embodiments of the invention herein. Numeric ranges are inclusive of the numbers defining the range. In the specification, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to,” and the word “comprises” has a corresponding meaning.
The terms and words used in the following description are not limited to the bibliographical meanings, but are merely used to enable a clear and consistent understanding of the disclosure. Accordingly, it should be apparent to those skilled in the art that the following description of exemplary embodiments of the present disclosure are provided for illustration purpose only and not for the purpose of limiting the disclosure as defined by the appended claims and their equivalents.
Features that are described and/or illustrated with respect to one embodiment may be used in the same way or in a similar way in one or more other embodiments and/or in combination with or instead of the features of the other embodiments.
The therapeutically effective amount administered to the individual e.g., a mammal, particularly a human, in the context of the present invention should be sufficient to affect a therapeutic or prophylactic response in the individual over a reasonable time frame. The dose can be readily determined using methods that are well known in the art. One skilled in the art will recognize that the specific dosage level for any particular individual will depend upon a variety of potentially therapeutically relevant factors.
The term "subject" includes mammals, which can alternatively be used as ‘Individual’ (especially humans) and other animals, such as domestic animals (e.g., household pets including cats and dogs) and non-domestic animals (such as wildlife).
The term "Treating" or "treatment" of a disease includes (1) preventing the disease from occurring in subject that may be predisposed to the disease but does not yet experience or display symptoms of the disease, (2) inhibiting the disease, i.e. arresting its development, or (3) relieving the disease, i.e. causing regression of the disease.
Viral infections may include for example infections by respiratory viruses, including but not limited to, various types of coronavirus (COVID).
“Breathing air” is a term used to loosely describe the extent to which air in a local environment (ambient air) is sufficiently to be safe to breathe (Rajagopal Kannan et al, IJISET, Vol. 2 Issue 4, April 2015).
Figure 1: Illustrates LifeViroTreat pilot batch for clinical testing.
Figure 2 (A): Illustrates Gross necropsy of rat (Air Control).
Figure 2 (B): Illustrates Gross necropsy of rabbit (Air Control).
Figure 3 (A): Illustrates Gross necropsy of rat at 5 times (4.18 mg/kg) LD2 (Every 4 hours for 3 days) treatment group.
Figure 3 (B): Illustrates Gross necropsy of rat at 5 times (4.18 mg/kg) LD4 (Every 2hours for 3 days) treatment group.
Figure 4 (A): Illustrates Gross necropsy of rat at 10 Times (8.37mg/kg) HD2 (Every 4hours for 5 days) treatment group.
Figure 4 (B): Illustrates Gross necropsy of rat at 10 Times(8.37mg/kg) HD4 (Every 2 hour for 5 days) treatment group.
Figure 5 (A): Illustrates Gross necropsy of rabbit at 5 times (2.09mg/kg) LD2 (Every 4 hours for 3 days) treatment group.
Figure 5 (B): Illustrates Gross necropsy of rabbit at 5 times (2.09mg/kg) LD4 (Every 2 hours for 3 days) treatment group.
Figure 6 (A): Illustrates Gross necropsy of rabbit at 10 Times(4.19mg/kg) HD2 (Every 4 hours for 5 days) treatment group.
Figure 6 (B): Illustrates Gross necropsy of rabbit at 10 Times (4.19mg/kg) HD4 (Every 2 hour for 5 days) treatment group.
The present invention is further described by reference to the following examples, which are illustrative only and not limiting of the claimed invention.
A. Experimental Protocol:
The study was performed to determine the acute toxicity effect of LifeViroTreat (test item), when administered as multiple inhalations doses to rats and rabbits, followed by an observation period of 14 days. 3 female animals per dose level were used in this study to assess the acute toxicity of the test item as described in the OECD guidelines for testing of chemicals, 403.
1. Animals were kept in animal restrainer and connected with nebulizer.
2. Nebulizer chamber was filled before starting the experiment with aqueous chlorine formulation (1 mg/m3) for 15 minutes (LifeViroTreat, 10 ml glass vial).
3. Animals were exposed to the aqueous chlorine formulation for 15 minutes 6 and 12 times in a day.
Table 1: Characterization of test item:
Sr. no Characteristics Value
1. Name LifeViroTreat
2. Appearance Liquid
3. Colour Colorless
4. Batch No. SI-01/A-001/001
5. Formulation/Composition Dissolved chlorine
6. Manufacture Date May, 2020
7. Expiry Date April, 2021
8. Chemical Name Aqueous Chlorine
9. CAS No 7782-50-5
10. Molecular Formula -Cl
11. Molecular Weight 70.9 g/mol
12. Solubility Soluble in Water
13. pH 3-5

Table 2: Test method
AnimalModel Dose Frequency of dose Number of Animals Sex Route of administration

Rat Air Control - 3

Female

5Times
(4.18mg/kg) LD2
(Every4hour
for3days)
3

Inhalation through Nebulizer
LD4
Every2hour
for3days
3

10Times
(8.37mg/kg) HD2
Every4hour
for5days
3
HD4
(Every2hour
for5days)
3

Rabbit
Air Control
-
3

Female

Inhalation through Nebulizer

5 Times
(2.09mg/kg) LD2
Every4hour
for3days
3
LD4
Every 2 hour
For 3 days
3

10Times
(4.19mg/kg) HD2
Every 4 hour
For 5 days
3
HD4
Every 2 hour
For 5 days
3

B. Test Procedure:
1. Animals were kept in animal restrainer and connected with nebulizer.
2. Nebulizer chamber was filled before starting the experiment with aqueous chlorine formulation (1 mg/m3) for 15 minutes (LifeViroTreat, 10 ml glass vial).
3. Animals were exposed to the aqueous chlorine formulation for 15 minutes 6 and 12 times in a day.
4. Animals were assessed for their anxiety, motor activity and behavior changes after each exposure.
5. Moreover, after the exposure period on 14 day hematological and biochemical parameters were investigated.

C. Experimental details:

Animal Model Dose concentration Frequency of
dose Animal No. Clinical
Signs Mortality & Morbidity Gross Pathology

Rat
Air Control -
-
- 1
2
3
NAD
Nil
NAD

5 Times
(4.18mg/kg)
LD2
(Every 4 hour for 3 days) 1
2
3
NAD
Nil
NAD
LD4
Every 2 hour for 3days 1
2
3
NAD
Nil
NAD

10 Times
(8.37mg/kg) HD2
Every 4 hour for 5days 1
2
3 All animals were in somnolence condition with decreased motor activity following test item administration on day0. Nil NAD
HD4
(Every 2 hour for 5 days)

Rabbit
Air Control
- 1
2
3
NAD
Nil
NAD

5 Times
(2.09mg/kg) LD2
Every 4 hour for 3 days 1
2
3
NAD
Nil
NAD
LD4
Every 2 hour for 3 days 1
2
3
NAD
Nil
NAD

10 Times
(4.19mg/kg) HD2
Every 4 hour for 5 days 1
2
3 All animals were in somnolence condition with decreased motor activity following test item administration on day 0.

Nil

NAD
HD4
Every2 hourfor5days
All the animals were observed for mortality and morbidity for a period of fourteen days following the test item administration.
Table 3: Results of Mortality & Morbidity; Clinical signs and Gross pathology

Body weights of each animal were recorded prior to the test item administration (Day 0) and on Days 7and 14 of the particular experimental step.
Table 4: Individual animal’s body weights (g) (Rat)
AnimalModel Dose(mg/Kgb.w.) Frequency of dose Animal No. Day0 Day7 Day14 Increase in body weight at the end of the experiment

Rat Air Control - 1 164.8 176.2 184.5 19.7
- 2 171.4 179.8 187.9 16.4
- 3 173.7 181.3 190.1 16.5

5 Times
(4.18mg/kg)
LD2
(Every 4 hour
For 3 days) 1 168.4 175.2 182.9 14.5
2 170.2 177.8 186.8 16.6
3 169.0 176.2 184.7 15.7

LD4
Every 2 hour
For 3 days 1 167.9 173.4 181.7 13.8
2 173.3 179.9 186.5 13.2
3 174.8 181.1 187.2 12.4

10 Times
(8.37mg/kg)
HD2
Every 4 hour
For 5days 1 169.5 176.0 187.1 17.6
2 170.8 177.2 185.9 15.1
3 165.6 173.6 181.8 16.2

HD4
(Every 2 hour
For 5 days) 1 161.1 168.0 177.1 16.0
2 169.0 174.3 183.0 14.0
3 165.8 173.4 184.2 18.4

Table 5: Individual animal’s body weights (g) (Rabbit)
Animal Model Dose(mg/Kgb.w.) Frequency of
dose Animal No. Day0 Day7 Day14 Increase in body weight at the end of the experiment

Rabbit
Air Control - 1 2.29 2.49 2.68 0.39
- 2 2.31 2.59 2.8 0.49
- 3 2.20 2.40 2.61 0.41

5 Times
(2.09mg/kg) LD2
(Every 4 hour
For 3 days) 1 2.34 2.58 2.79 0.45
2 2.50 2.69 2.98 0.48
3 2.30 2.58 2.77 0.47

LD4
Every 2 hour
For 3 days 1 2.60 2.75 3.01 0.41
2 2.56 2.71 2.92 0.36
3 2.32 2.51 2.73 0.41

10 Times
(4.19mg/kg) HD2
Every 4 hour
For 5 days 1 2.68 2.88 3.00 0.32
2 2.54 2.72 2.90 0.36
3 2.36 2.53 2.75 0.39
HD4
(Every 2 hour
For 5 days) 1 2.67 2.79 3.01 0.34
2 2.58 2.78 2.99 0.41
3 2.60 2.82 3.19 0.59

Animals were fasted overnight prior to collect blood samples by retro-orbital technique on day14. The blood samples were analyzed for different hematological and biochemical parameters.

PARAMETERS Treatment Groups ReferenceValues
Control Group 5 Times
(4.18mg/kg) 10 Times
(8.37mg/kg)
Air Control LD2
(Every 4 hour for 3days) LD4
(Every 2 hour for 3 days) HD2
(Every 4 hour
For 5days) HD4
(Every 2 hour
for5days)
ALB(g/dL) 3.82±0.27 3.93±0.21 3.81±0.31 3.67±0.19 3.42±0.44 3.4–4.8
ALP(U/L) 217.33±18.15 230.00±50.74 209.33±14.72 178.33±33.61 162.67±7.02 62-237
ALT(U/L) 54.86±7.29 45.90±12.64 52.80±4.00 53.50±4.93 51.00±1.25 18-76
AST(U/L) 95.63±12.47 100.07±15.59 125.47±20.10 116.67±12.15 102.97±17.39 74-143
BIL(mg/dL) 0.10±0.00 0.10±0.00 0.10±0.00 0.10±0.00 0.10±0.00 0.05-0.15
CA(mg/dL) 10.38±0.61 10.93±0.67 10.00±1.75 9.23±1.10 9.17±2.11 9.5-11.5
CHO(mg/dL) 51.10±6.27 59.77±2.67 55.23±6.82 44.83±5.14 50.87±4.35 37-85
CREJ(mg/dL) 0.36±0.05 0.32±0.02 0.30±0.01 0.32±0.07 0.31±0.02 0.2-0.5
PHOS(mg/dL) 6.54±1.40 7.57±1.58 7.07±1.72 7.03±1.59 8.33±1.56 5.58-10.41
TP(g/dL) 6.30±0.54 6.64±0.84 7.04±0.29 6.81±0.45 6.67±0.06 5.2-7.1
UREA(mg/dL) 15.60±1.62 17.97±3.47 20.40±1.21 19.10±4.20 22.17±2.35 12.3-24.6
GLU(mg/dL) 144.33±15.28 133.00±10.58 132.33±14.64 120.00±12.77 142.33±17.24 70-208

Note: Each value represents the mean ± standard deviation(n=3)

Table 6: Effects of aqueous chlorine by inhalation route on some biochemical parameters in the acute toxicity study in rat.
Table 7: Effects of aqueous chlorine by inhalation route on some biochemical parameters in the acute toxicity study in rabbits.

PARAMETERS Treatment Groups

ReferenceValues
Control Group 5 Times
(2.04mg/kg) 10 Times
(4.19mg/kg)
Only Air LD2
(Every 4 hour for 3 days) LD4
(Every 2 hour
For 3 days) HD2
(Every 4 hour
For 5 days) HD4
(Every 2 hour
For 5 days)
ALB(g/dL) 3.40±.03 3.93±0.05 3.81±0.31 3.67±0.19 3.42±0.44 23-49
ALP(U/L) 204.44±17.64 196.67±10.02 259.33±32.52 278.33±33.61 229.33±51.47 44-402
ALT(U/L) 56.15±7.29 45.90±12.64 52.80±4.00 53.50±4.93 51.00±1.25 34-146
AST(U/L) 22.36±4.63 138.40±18.40 107.37±5.41 106.70±1.23 103.97±2.10 7.5-39.7
BIL(mg/dL) 0.10±0.00 0.10±0.00 0.10±0.00 0.10±0.00 0.10±0.00 0.019-0.2
CA(mg/dL) 10.90±1.99 12.00±1.91 11.00±2.14 9.99±0.91 10.47±0.91 9.22-13.63
CHO(mg/dL) 53.46±5.04 59.77±2.67 55.23±6.82 44.83±5.14 50.87±4.35 30-65
CREJ (mg/dL) 0.81±0.22 0.32±0.02 0.30±0.01 0.32±0.07 0.31±0.02 0.5-2.6
PHOS (mg/dL) 5.12±0.13 6.23±0.06 5.03±0.40 5.37±0.49 4.67±0.49 4-6
TP(g/dL) 60.62±8.64 7.09±0.21 7.04±0.29 6.81±0.45 6.67±0.06 49-79
UREA (mg/dL) 48.04±9.41 34.63±3.61 34.07±4.70 33.17±2.37 33.50±0.75 25.48-71.41
GLU(mg/dL) 150.67±11.06 136.67±9.28 143.33±13.01 132.33±11.59 140.00±11.00 89-150
Note: Each value represents the mean ± standard deviation(n=3)
ALB2:Albumin,ALP:Alkalinephosphatase,ALT:AlanineAminotransferase,AST:AspartateAminotransferase,BILT2:TotalBilirubin,CA2:
Calcium,CHOL:Cholesterol,CREJ:Creatinine,PHOS:Phosphorous,TP:Totalprotein,UREL:Urealevel,GLU:Glucose

Table 8: Effects of aqueous chlorine by inhalation route on some hematological markers in the acute toxicity study in rats (Fig 8, Fig 9, Fig 10, Fig 11)

PARAMETERS Treatment Groups

Reference Values
Control Group 5 Times
(4.18mg/kg) 10 Times
(8.37mg/kg)
Only Air LD2
(Every 4 hour
For 3 days) LD4
(Every 2 hour
For 3 days) HD2
(Every 4 hour
For 5 days) HD4
(Every 2 hour
For 5 days)
WBC (x10E03cells/µL) 6.62± 1.45 5.24± 1.26 7.30± 1.15 6.99± 1.71 6.14± 2.07 1.13-7.49
RBC (x10E06cells/µL) 7.72± 1.12 8.28± 0.40 7.63± 0.49 7.56± 0.18 7.15± 1.24 7.07-9.03
HGB (g/dL) 14.59±1.08 15.13±1.31 13.13±1.44 14.23±2.16 16.00±1.71 13.7-16.8
HCT (%) 42.00±3.61 42.17±3.70 39.03±0.75 36.07±0.05 37.90±2.62 35-52
MCV(fL) 55.89±4.06 50.83±2.23 50.43±2.50 54.33±4.71 54.67±5.11 49.90-60.1
MCH(pg) 18.83±3.55 18.40±2.16 18.49±1.22 17.90±2.15 17.80±1.37 17.8-20.9
MCHC(g/dL) 35.78±1.77 34.73±6.79 34.47±6.91 35.20±6.63 35.13±5.37 32.7-37.9
RDW(%) 12.60±0.64 13.43±1.11 13.70±1.15 13.43±1.96 14.30±1.50 10.5-14.9
PLT(x10E03cells/µL) 781.33± 105.64 710.33± 86.59 871.00± 24.98 891.67± 59.53 796.33± 79.43 680-1200
MPV(fL) 8.01± 1.07 9.57± 1.17 9.37± 0.61 8.40± 2.13 9.57± 1.46 6.2-9.8
%NEUT (%) 19.27±4.33 13.87±4.78 10.67±0.64 12.67±4.69 15.03±3.51 7.1-33.2
%LYM (%) 77.00±11.19 78.30±3.32 86.07±2.79 81.87±5.42 79.60±6.91 62.2-90
%MONO(%) 2.69± 0.51 4.47± 0.15 2.90± 0.72 3.07± 0.21 4.77± 0.21 0.8-5.9
%EOS(%) 2.42± 0.52 1.50± 0.36 1.57± 0.29 0.80± 0.10 1.57± 1.24 0.4-4.5
%BASO(%) 0.16± 0.06 0.43± 0.06 0.60± 0.35 0.10±00.00 0.43± 0.15 0-0.8
Note: Each value represents the mean ± standard deviation(n=3)

Table 9: Effects of aqueous chlorine by inhalation route on some hematological markers in the acute toxicity study in rabbits.

PARAMETERS Treatment Groups

Reference Values
Control Group 5 Times
(2.09mg/kg) 10 Times
(4.19mg/kg)
Only Air LD2
(Every 4 hour
For 3 days) LD4
(Every 2 hour
For 3 days) HD2
(Every 4 hour for 5 days) HD4
(Every 2 hour
For 5 days)
WBC (x10E03
cells/µL) 11.64±1.16 11.13± 0.80 9.87± 0.61 11.12± 1.39 11.33± 0.88 5.2-16.5
RBC(x10E06
cells/µL) 5.59± 0.88 5.18± 0.54 5.79± 0.69 5.03± 0.33 4.93± 0.21 3.7-7.5
HGB (g/dL) 8.56± 0.79 8.47± 1.27 6.07± 0.57 8.10± 1.65 8.7± 0.15 7.8-15.4
HCT (%) 30.14±1.98 28.03± 2.81 29.37± 0.87 27.30± 1.78 26.73± 1.05 26.7-47.2
MCV(fL) 53.60±5.50 54.13±0.25 53.57± 1.01 54.30± 0.00 57.60± 5.63 55.0-79.6
MCH(pg) 24.93±4.41 22.50±2.40 24.17 ±4092 23.53± 4.26 23.87± 6.80 19.2-29.5
MCHC(g/dL) 27.39±3.51 28.77± 4.58 25.13± 4.03 25.77± 3.20 26.50± 3.58 25.52-37
RDW(%) 13.78±1.25 15.80± 3.46 14.47 ±4.57 15.27± 0.70 13.10± 3.72 11.5-16.2
PLT (x10E03
cells/µL) 683.00± 95.77 609.33± 59.68 723.33± 87.51 764.67± 68.42 746.67± 83.27 112-795
MPV(fL) 6.60± 0.62 6.13± 0.95 7.37± 0.38 6.47± 1.36 6.83± 1.32 5.2-9.9
%NEUT (%) 50.16±8.12 46.63± 1.00 49.40± 1.32 53.20± 4.17 50.13± 2.87 21-73
%LYM (%) 45.88±5.45 42.40± 3.87 39.03± 0.72 44.93± 4.57 40.53± 8.12 9-64
%MONO(%) 9.19± 1.59 9.57± 1.50 8.73± 1.33 7.37± 1.90 2.60± 0.61 1-15
%EOS(%) 0.45± 0.25 0.57± 0.23 0.47± 0.29 0.27± 0.25 0.23± 0.23 0.0-0.7
%BASO(%) 8.27± 0.55 6.73± 2.40 6.33± 2.52 4.33± 1.55 6.33± 2.31 0.5-9
Note: Each value represents the mean ± standard deviation(n=3)
WBC:Whitebloodcells,LY:Lymphocyte,MON:Monocytes,GRAN:Granulocytes,HGB:Hemoglobin,HCT:Hematocrit,MCV:Meancorpuscularvolume,MCH:Meancorpuscularhemoglobin,MCHC:Meancorpuscularhemoglobinconcentration,RDW:Redbloodcelldistributionwidth,PLT:Platelets,MPV:Meanplatelet volume,

Group AnimalNo. Organ Weight(gm.)
Liver Lung Heart Kidney Brain Pancreas Spleen Ovaries
Right Left

Air Control 1 45.65 9.02 4.91 4.82 4.73 6.48 1.06 0.46 0.094
2 53.34 7.68 4.08 6.30 6.84 5.81 0.95 0.44 0.087
3 47.36 9.52 3.98 5.83 5.33 6.0 1.15 0.49 0.109
LD2
(Every 4 hour
For 3 days) 1 61.78 8.03 4.56 4.35 4.66 5.78 0.83 0.51 0.078
2 55.64 7.10 5.10 5.16 5.28 4.75 0.97 0.48 0.092
3 57.49 9.24 4.87 4.49 4.86 4.97 0.92 0.54 0.108
LD4
Every 2 hour
For 3 days 1 52.25 8.46 3.90 4.97 5.20 5.06 1.01 0.48 0.096
2 64.83 7.89 4.39 6.15 6.22 6.66 0.84 0.50 0.088
3 67.46 7.47 5.29 5.54 5.20 4.78 0.89 0.57 0.100
HD2
Every four hour for 5 days 1 61.61 8.73 6.02 5.89 6.02 5.77 0.90 0.51 0.074
2 51.63 9.09 5,40 5.17 5.90 4.36 0.84 0.44 0.080
3 50.90 7.04 3.90 4.94 4.81 5.02 1.01 0.47 0.078
HD4
(Every2hour
For 5 days) 1 52.56 9.02 4.47 5.96 6.11 4.78 0.91 0.49 0.089
2 61.61 7.6 5.14 5.12 5.06 6.18 1.06 0.57 0.075
3 53.9 8.7 4.98 6.14 6.04 5.10 0.98 0.41 0.083
Table 12: Organ weight of each rabbit

Gross pathology:
Gross pathology included a careful examination of the external surface of body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. Observations of the animals of various groups and doses were found to be normal in size, color and appearance. Gross necropsies for all animals are presented in Figures 2,3,4,5,6,7 and 8 for rat and rabbit respectively.