Acute Inhalation Toxicity Study - Rats

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Institute for Industrial Research & Toxicology

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Registration No. 1303/C/CPCSEA (Ministry of Environment ft Forests, Government of India)
License No.: UP37370000001 (Food and Drug Administration, UP)
AN ISO 9001: 2015, ISO 14001: 2015, ISO 45001: 2018 Certified Organization
GLP Certified, NABL (1S0/IEC 17025) Accredited
ACUTE INHALATION TOXICITY OF

"ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER
IN WISTAR ALBINO RATS
(Guideline: OECD-403 Acute Inhalation Toxicity)
SPONSORED BY
HINDUSTAN AGRO
Haridwar Road Mandawar
District Bijnor U.P. (246721) (INDIA)
MANUFACTURED BY
HINDUSTAN AGRO
TESTING LABORATORY
INSTITUTE FOR INDUSTRIAL RESEARCH & TOXICOLOGY
F-209, UPSIDC, MG ROAD, GHAZIABAD-201302 (INDIA)

E-mail: info@toxicityindia.org
Website: www.toxicityindia.org
Tel No.: +91 9711623080
Fax: +91 11 22235111
Project No. 202109-125

Report No IIRTffOX/202109/AIT/116
URL No TC661221000000116P
Date 27-10-2021
Corporate Office: A-27. Street No. 2 Madhu Vihar, ( I. P. Extension), Delhi - 110092
Lab ft Works: F-209, UPSIDC, Phase-I, MG Road, Ghaziabad, Telefax: +91-11-22234111, 22235111, 9711623080/81/82/90
Email: iirtdelhi@gmail.com, info@toxicityindia.org, www.toxicityindia.org
"ZERO MAX"INSTITUTE FOR INDUSTRIAL RESEARCH & TOXICOLOGY, GHAZIABAD
TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

SPONSORED BY HINDUSTAN AGRO
STUDY ACUTE INHALATION TOXICITY IN WISTAR ALBINO RAT
PROJECT No 202109-125
REPORT No. IIRT/TOX/202109/AIT/116
CONTENTS
S.No. Particular Page No.
1 GLP COMPLIANCE STATEMENT 4
2. STATEMENT BY TEST FACILITY MANAGEMENT 5
3. QUALITY ASSURANCE STATEMENT 6
4. STUDY INFORMATION 7
5. STUDY PERSONNEL 8
6. SUMMARY 9-12
7. INTRODUCTION 13
Objective 13
Regulatory references 13
8. MATERIALS & METHODS 14-15
Test article 14
Test System 14-15
Husbandry 15
9. INHALATION EXPOSURE SYSTEM 16-18
Description ofinhalation chamber 16
Air compressor 16-17
2'' fluidized bed generator 17
Scrubber system 17
Monitoring ofexposure condition 18
Particle size distribution 18
Procedure 18
10. OBSERVATIONS 19
11. ARCIDVE 20
12. RESULTS 21-28
Exposure atmosphere 20
Body weight 20
Page 2 of33

STUDY ACUTE INHALATION TOXICITY IN WIST AR ALBINO RAT
REPORT No. IIRT/TOX/202109/AIT/l 16
Mortality 20
Clinical signs 20
LCso finding 20
Necropsy findings 20-21
Table-I Exposure atmosphere data 23
Table-2 Individual Body weight 24
Table- 3 Clinical signs & mortality 25-27
Table-4 Summary of necropsy findings 28
Table-5 Individual animal fate & necropsy findings 29-31
13. CONCLUSION 32
14. CERTIFICATE OF ANALYSIS 33
Page 3 of33
"ZERO MAX"INSTITUTE FOR INDUSTRIAL RESEARCH & TO�ICOLOGY, GHAZIABAD

REPORT No. IIRTffOX/202109/AIT/116
GLP COMPLIANCE STATEMENT

The study "Acute inhalation toxicity in wistar albino rats" with "ZERO MAX"
TRANSFLUTHRIN 1.60% LIQUID VAPORIZER was performed in accordance with the
standard with the standard operating procedures of Toxicology Department, Institute for
Industrial Research & Toxicology, as well as the approved study plan.
I hereby attest the authenticity of the study and guarantee that this report represents a true and
accurate record of results obtained and shall not be reproduced except in full, without the written
approval of the Sponsor.
The study was conducted to meet the requirements of the OECD Guideline No. 403 Acute
Inhalation Toxicity, Section-4: Health Effects (Adopted: 07th September, 2009) in
compliance with the principles of Good Laboratory Practices (G.L.P).
All original raw data including documentation, the draft report, a copy of the final report and the
representative test item are archived in the archives at TOXICOLOGY DEPARTMENT,
INSTITUTE FOR INDUSTRIAL RESEARCH & TOXICOLOGY. There were no known
circumstances that may have affected the quality or integrity of the study.
The sponsor is responsible for necessary evaluations of the test item concerning the chemicals
purity, identity, stability and other required data.
The chemical analysis of the test item "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID
VAPORIZER was carried out by the sponsor.
Mr.Mohseen 27-10-2021
Study Director Date
Page 4 of33

STATEMENT BY TEST FACILITY MANAGEMENT
Management of the test facility has been made available all the resources to the Study Director
which was necessary for conduct of the present study in compliance with the principles of GLP.
I, the undersigned, take overall responsibility for the reliability of the work described in the
report with the compliance of Good laboratory Practice.
27-10-2021
Date
Page 5 of33

QUALITY ASSURANCE REPORT

This Study Number: 202109-125/Report Number: IIRT/TOX/202109/AIT/116 entitled "Acute
Inhalation Toxicity of "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER
in Wistar Albino Rats" was perfmmed as per the OECD-403 Guideline for testing of
chemicals was subjected to inspections by the Quality Assurance Unit.
This report has been audited by the Quality Assurance Unit, and is considered to be an accurate
account of the data generated and of the procedures followed. In each case, the outcome of QA
evaluation is reported to the Study Director and Management on the day of evaluation. Audits of
study documentation, and process inspections appropriate to the type and as per scheduled of
this study.
Standard Test Method Compliance Audit 04-10-2021

Animal Preparation 05-10-2021
Test Material Preparation 06-10-2021
Application of test compound 06-10-2021
Assessment of Response 06-10-2020 to 21-10-2021
Draft Report Audit 25-10-2021
Signature Final Report Audit 27-10-2021
Ms. Shalini Mishra 27-10-2021
For Quality Assurance Unit

\:"·--=--
/'''
Signature,." Date
Page 6 of33

STUDY INFORMATION
PROJECT NUMBER 202109-125
REPORT NUMBER IIRT/TOX/202109/AIT/116
STUDY TITLE ACUTE INHALATION TOXICITY OF "ZERO MAX"

TRANSFLUTHRIN 1.60% LIQUID VAPORIZERIN
WISTAR ALBINO RAT
SPONSOR BY HINDUSTAN AGRO

HINDUSTAN AGRO
MANUFACTURED BY Haridwar Road Mandawar
INSTITUTE FOR INDUSTRIAL RESEARCH &

TESTING LABORATORY TOXICOLOGY
F-209, UPSIDC, MG ROAD, GHAZIABAD-201302
(INDIA)
E-mail: info@toxicityindia.org
Website: www.toxicityindia.org
Tel No.: +91 9711623080
Fax: +91 11 22235111
Page 7 of33

STUDY PERSONNEL
Study Director Mr. Mohseen (M.Phann)
Study Personnel Mr. Ankur Rana (B.Pharm)
Histopathology & Veterinarian Dr. Vandana Singh, M.V.Sc. Pathology
Page 8 of33

SUMMARY
The acute inhalation study of test compound "ZERO MAX" TRANSFLUTHRIN 1.60%
LIQUID VAPORIZER was conducted at Institute for Industrial Research & Toxicology in
Wistar albino rats as per OECD-Guideline -403 for testing of chemicals.
The experiment was carried out using inhalation equipment (nose only exposure) specially
designed and Supplied by Smit Fabricator, Va/sad, Gujarat, India. The inhalation equipment
consists of an inhalation chamber, air compressor, flow meter, 2' fluidized bed generator for dust
aerosols, cascade impactor, temperature and humidity, oxygen monitor, open phase sampler and
rat exposure tubes.
The test was carried out in the inhalation chamber (nose only exposure) at air flow rate of 30
LPM at 40 psi pressure. Minimum 10 to 15 air changes per hour were maintained and respirable
particle size was ranging between 2.30- 3.19 microns. Inside the chamber, slightly negative
pressure was maintained against the atmospheric to prevent leakage of the test substance.
The dynamic inhalation chamber capacity was of 18 liters. The heavy duty moisture separator
was used for separating from the compressed air. Filtration Regulation Unit(pre filter and micro
filter) used for separating moisture 25 and 5 micron particle from the compressed air. For
purification of air, activated charcoal filter column and silica gel column were used for the air
drying: The dry air passed into pressure regulator valve at 40 psi pressure was maintained and air
flow was regulated by rotameter. The 30 LPM air flow was maintained for fluidized bed in
fluidized bed generator. During this study the chamber temperature (22.20-22.24 °c), relative
humidity (51.24-50.84%), oxygen concentration (20.56-20.88 %) and carbon dioxide
concentration(< 1.0%) were recorded throughout the experiment.
Page 9 of33

STEP FIRST:
Six healthy wistar albino rats of body weight 200±20 gm were selected for study after
acclimatization. All the animals were divided into each consisting of 3 male and 3 female. The
rats of group-I were exposed to mean concentration 1.04 mg/I aerosol for period of4 hrs.
After exposure all the animals were closely observed for clinical signs of toxicity at various
intervals such as 1 hr, 2 hrs, 4 hrs, and 6 hrs on the day of test compound aerosol exposure and
later on twice a day throughout the experimentation period of 14 days. The body weight was
th
recorded on day 0 (pre exposure), 7th and 14 (post exposure). The necropsy was performed on
all the animals at termination of experiment.
RESULTS
Clinical signs:
The test compound "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER
elicits did not show any clinical signs of toxicity at the tested mean concentration of' 1.04 mg/I.
Mortality:
No mortality was observed in the tested dose level group throughout period of observation.
Body weight:
h th
The body weight of all the animals exposed to test compound, observed on day i and day 14
(post treatment) did not show significant change as compared to day 0 (pre treatment).
Necropsy findings
The necropsy was performed on all the animals at the termination of study did not show any
changes of gross examination.
STEP SECOND:
Six healthy wistar albino rats of body weight 200±20 gm were selected for this step. All the
animals were divided into each consisting of 3 male and 3 female. The rats of group-II were
exposed to mean concentration 5.02 mg/I aerosol for period of 4 hrs. After exposure all the
animals were closely observed for clinical signs of toxicity at various intervals such as 1 hr, 2
Page 10 of33

hrs, 4 hrs, and 6 hrs on the day of test compound aerosol exposure and later on twice a day
throughout the experimentation period of 14 days. The body weight was recorded on day 0 (pre
exposure), ?
1h
and 14 t (post exposure). The necropsy was perfo1med on all the animals at
h
termination of experiment.
RESULTS
Clinical signs:
The test compound "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER elicits
no clinical signs of toxicity at the tested mean concentration of 5.02 mg/I.
Mortality:
Body weight:
h th
(post treatment) did not show significant change as compared to day O (pre treatment).
Necropsy findings
The necropsy was perfonned on all the animals at the termination of sh1dy showed no sign of
toxicity in any internal organs.
STEP THIRD:
Six healthy wistar albino rats of body weight 200±20 gm were selected for this step. All the
animals were divided into each consisting of 3 male and 3 female. The rats of group-II were
exposed to mean concentration 10.04 mg/I aerosol for period of 4 hrs. After exposure all the
animals were closely observed for clinical signs of toxicity at various intervals such as 1 hr, 2
hrs, 4 hrs, and 6 hrs on the day of test compound aerosol exposure and later on twice a day
throughout the experimentation period of 14 days. The body weight was recorded on day O (pre
exposure), ?
1h
and 14th (post exposure). The necropsy was performed on all the animals at
termination of experiment.
Page 11 of 33

RESULTS
Clinical signs:
The test compound "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER
elicits no clinical signs oftoxicity at the tested mean concentration of 10.04 mg/I.
Mortality:
Body weight:
h th
(post treatment) did not show significant change as compared to day O (pre treatment).
Necropsy findings
The necropsy was performed on all the animals at the tennination of study showed no sign of
toxicity in any internal organs.
LCso Finding
The cut-off acute lethal concentration (LC5o) of test compound "ZERO MAX"
TRANSFLUTHRIN 1.60% LIQUID VAPORIZER is found to be greater than 10.04 mg/I
under the experimental conditions.
CONCLUSION
It can be concluded from the results of present investigation that the lethal concentration
(LCso) of test compound "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID
VAPORIZER is greater than 10.04 mg/I under the experimental conditions.
According to Globally Harmonized Classification (GHC) it comes under Category-III and
LC50 is greater than 10.04 mg/I as per the present test finding.
Page 12 of33

INTRODUCTION
OBJECTIVES OF THE STUDY:
The study was conducted with the following objectives to find out the clinical signs of toxicity
oftest compound "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER.
•!• To find out the LCso in albino rats and establish the safety levels of test compound
"ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER as per OECD
Guideline 403.
REGULATORY REFERENCES
Test Guidelines
•:• The study was conducted in compliance with the modification OECD Guidelines for
Testing of Chemicals (No. 403, Section 4: Health Effects) on conduct of "Acute
Inhalation Toxicity (Adopted: OJ1h September, 2009).
Page 13 of33

REPORT No. IffiTrfOX/202109/AIT/116
MATERIALS AND METHODS
TEST ARTICLE
Study title ACUTE INHALATION TOXICITY IN WISTAR ALBINO RATS
Test compound "ZERO MAX" TRANSFLUTHRlN 1.60% LIQUID VAPORIZER

Batch No. BN0080821
Characteristics CLEAR, COLORLESS VERY LIGHT-YELLOW LIQUID
Mfg. Date 25-08-2021
Exp. Date NA
Sponsored by HINDUSTAN AGRO
•:• The sponsor shall be responsible for the sample and its characterization.
TEST SYSTEM
1. Species Albino rats

2. Strain Wistar
3. Source Institute for Industrial Research & Toxicology
4. Age 8 to 12 weeks
5. Sex Male and Female
6. Body weight range 200 ±20g
7. Identification By cage tag and corresponding color body marking
8. No. of animals per 6 (3 males and 3 females)
dose group
9. No. of dose group Group I -step I (1.04 mg/1)
Group II -step II (5.02 mg/I)
Page 14 of33

REPORT No. IIRTtrOX/202109/AIT/116
Group III- step III (10.04 mg/1)

10 Acclimatization 18 healthy albino rats were selected and acclimatized
for standard laboratory condition for period of one week
in experimental room under veterinary examination.
11 Randomization After acclimatization and veterinary examination all the

selected rats randomly divided into two groups of 3
females and 3 males.
12 Nutritional conditions For feeding conventional Laboratory diets may be used
with an unlimited supply of drinking water.
Husbandry
1. Environmental conditions Air conditioned rooms with 10-15 air changes per hour,
temperature between 22 ± 3 °c, relative humidity 50-60%
and illumination cycle set to 12 hours artificial fluorescent
light and 12 hours dark.
2. Accommodation Groups of three animals of similar sex in polypropylene

cages with stainless steel grill top, facilities for food and
water bottle, and bedding of clean paddy husk.
3. Diet & Water UV sterilized filtered water through Aqua guard water
purifier and sterilized standard Pelleted feed supplied by
Krishna Valley Agrotech LLP., B7/8, first floor, double
storey, Ramesh Nagar, New Delhi-110015(India) were
freely available throughout experimentation. There were no
known contaminations in the feed and water at the levels
that would have interfered with the experimental results
obtained.
Page 15 of 33

INHALATION EXPOSURE SYSTEM
Nose only inhalation exposure chamber specially designed and fabricated was supplied by Smit
Fabricator, Va/sad, Gujarat, India. It also consist of inhalation chamber with cabinet, air
compressor, air filtration system, 2' fluidized bed generator, rat exposure tube, thermo
hygrometer, oxygen monitor, rotameter, cascade impactor, Open face filter holder and scrubber
system.
DESCRIPTION OF INHALATION CHAMBER
The dynamic inhalation equipment has three main parts namely upper, middle and lower
chamber. Each part is of 200 mm height and internal diameter. The total capacity of chamber is
18 liters. It was designed to sustain dynamic air flow to maintain 12 to 15 air changes per hour.
The inlet unit (upper) consists of glass cylinder with facility for the attachment of 2' fluidized
bed generator. The exposure unit (middle) was made up of stainless steel with 12 port holes to
accommodate transparent rat exposure tube to breath test items aerosol through the window
panel of the exposure tube. The outlet unit (lower) is made up of stainless steel with outlet
provision connected to the suction pump. The outgoing air from the chamber passes through
conical flask containing 1 % of sodium hydroxide and moisture trapped (by using cotton). This
is connected to suction pump before entering into the scrubber. The whole inhalation unit is
housed in an inhalation chamber cabinet. The cabinet is fitted with the conical dome at the top
which is connected to the cabinet exhaust. The cabinet has a primary panel attached at the side of
cabinet like pressure regulator with filter, rotameter, activated charcoal column and silica gel
column. The instrument is having provision for measuring chamber temperature, humidity and
oxygen concentration and is placed in cabinet.
Page 16 of33
TEST COMPOUND "ZERO MAX" TRANSFLUTIIlUN 1.60% LIQUID VAPORIZER

AIR COMPRESSOR
Before start of the compressor, drain out the reserve air by opening bottom valve. After drain out
of all the reserve air, close the bottom valve. Then switch ON the air compressor button situated
on the main control panel. The compressed air passes through the heavy duty moisture separator
and other filter system to inhalation chamber. The minimum pressure (60 to 80 psi) was
maintained in filtration unit pressure gauze so that the continuous air flow without moisture
could be obtained in inhalation chamber throughout the exposure.
AIR FILTRATION SYSTEM

It consists of pressure regulator and filter like heavy duty moisture separator. Filtration
Regulation Unit (Pre filter and micro filter) is used for separating 25 and 5 micron particle from
the compressed air and purification of air. For purification activated charcoal filter column and
silica gel column was used for the air drying.
2' FLUIDIZED BED GENERATOR (GENERATION OF DUST AEROSOLS)
2' fluidized bed generator used for dust aerosol generation. It is connected to the upper part of
inhalation chamber for generating dust aerosol. The test item was mixed 100 ml bed and place in
upper of the 2' fluidized bed generator with hopper valve closed. For generating dust aerosol at
the time of exposure, opened the hopper valve and allowed approximately 50 ml of pure bed
material to be flown into the bed chamber. The inlet air flow rate 30 LPM and O psi pressure
were maintained in 2' fluidized bed generator for constant aerosol generation and chamber outlet
flow was kept at 40 LMP.
SCRUBBER SYSTEM
The cabinet is connected to an individual suction pump and the pump is connected to the blower.
The toxic aerosol and gases coming out of the exhaust system finally enter inside the scrubber
packed with porcelain material pieces, move upward and the sprinkler is connected to the motor
Page 17 of33

pump that draw sodium hydroxide solution from the storage tank having the capacity of 500
liters for neutralization of the toxic aerosol and gases.
MONITORING OF EXPOSURE CONDITION

Chamber Air Flow:
The air flow rate of 30 LPM at 40 psi pressure was maintained in 2' fluidized bed generator
throughout the exposure.
Chamber Temperature, Relative Humidity and Oxygen Content:

The t�mperature (22.20-22.24 °c), relative humidity (51.24-50.84%) and oxygen concentration
(20.56-20.88 %) were measured five times during inhalation exposure.
Glass microfiber papers (Whatman Glass Microfiber filter, 25 mm diameter) were used by to
assess the breathing zone concentration. Whatman filter paper has particle retention capacity of
98% up to l .6µm of the particle size. A suitable measured volume of air was drawn from the
inhalation chamber at the level of the breathing zone at every one hour of exposure after
equilibrium of the chamber for 30 minutes. At the end of sampling glass microfiber papers with
the test substance were weighed to determine the concentration in aerosol at the breathing zone
of the rats.
PARTICLE SIZE DISTRIBUTION

A distribution curve of aerodynamic particle size was drawn based on the data obtained from the
cascade impactor. Mercer type seven stages cascade impactor (supplied by Intox products LLC,
P.O. Box 2070, and 101 Wilderness Ct. S., Moriarty, NH-87035 USA) was used for measuring
the aerodynamic particle size distribution of aerosol generated. The Mass Median Aerodynamic
Dian1eters (MMAD) 2.30-3.29 Micron respectively.
Page 18 of33

PROCEDURE:
In each group three male and three female healthy Wistar albino rats/dose level of body weight
200±20 gm selected for study after acclimatization to standard laboratory conditions. After
acclimatization all the animals divided into 3 males and 3· females in each study groups. Test
group of rats was subjected to a single 4 hour aerosol exposure of the test compound at the
concentration of 1.04 mg/I, 5.02 mg/I and 10.04 mg/I using inhalation exposure chamber
specially designed and fabricated was supplied by Smit Fabricator, Va/sad, Gujarat, India.
All animals were daily closely observed for clinical signs toxicity and mortality for a period of
14 days. The body weight and food consumption were recorded on day 0 (pretreatment), 7th, and
14th (post treatment).
OBSERVATIONS
Body weight:
The body weight of all the animals was observed weekly on day 0 (Before treatment), 7th and
14th (post treatment).
Clinical Signs:
The treated animals were observed for signs of intoxication, at various intervals for first six
hours after dosing and thereafter twice a day for 14 days. Any clinical signs in the treated group,
if observed, recorded and mentioned in the report. The clinical sign will be graded as 0 =
Normal, + = Mild, ++ = Moderate, +++ = High and ++++ = Severe.
Mortality:
All the animals were observed for mortality at various intervals for first six hours on the day of
dosing and thereafter twice a day for 14 days.
Page 19 of33

REPORT No. IIRT/TOX/202109/AJT/l 16
Necropsy:
Necropsy was carried out on all the animals at the end of the study to observe any gross
pathological changes
ARCHIVE
The raw data, sample of the test substance, study report and other material are being retained for
09 years at Institute for Industrial Research &Toxicology, Ghaziabad on completion of the
study.
Page 20 of33

RESULTS
EXPOSURE ATMOSPHERE
The mean exposure concentrations (measured in breathing zone of the animals), particle size and
temperature/relative humidity data are presented in (Table- 1).
BODYWEIGHT
The body weight of all the animals recorded individually on day ?

1h
and 14th (post treatment)
showed normal gain as compared to day 0 (Table-2).
MORTALITY
The test compound "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER did
not elicit any m011ality at test concentration 1.04 mg/1, 5.02 mg/1 and 10.04 mg/I (Table-3).
CLINICAL SIGNS
The test compound "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER did
not produce any sign oftoxicity at any dose level (Table-3).
LCso FINDING
The cut-off acute lethal Concentration (LC50) of test compound "ZERO MAX"
TRANSFLUTHRIN 1.60% LIQUID VAPORIZER was found to be greater than 10.04
mg/1.
NECROPSY FINDINGS
The test compound "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER at
dose level of 1.04 mg/1, 5.02 mg/1 and 10.04 mg/I showed no sign of toxicity in any organ. The
results of necropsy finding were presented in (Table 4).
Page 21 of33

NECROPSY FINDING
EXTERNAL
i. Skin- normal
ii. All external orifices- Normal
B. INTERNAL
i. Subcutaneous- No changes were observed.
ii. Superficial and deep lymph nodes- No change in mesenteric lymph node.
ABDOMINAL CAVITY
i. Opening and general examination- In the abdominal cavity all the organs were present
in normal position.
ii. Spleen- No changes were recorded.
iii. Digestive system- No gross changes were observed in stomach and intestine.
iv. Liver and biliary ducts- No gross pathological changes were observed
v. Excretory system- No gross pathological changes were observed.
vi. Adrenal- Observed normal.
vii. Male/female genital organs - Showed normal color, consistency and no inflammatory
changes.
2. THORACIC CAVITY
i. Opening and general examination- Thoracic cavity was found to be nonnal without
any fluid, mucous or blood etc.
ii. Lungs- No changes were recorded.
iii. Heart- No changes were observed in color and consistency. Heart found normal.
iv. Thyroid- Nonnal in shape, size and surface.
3. CRANIAL CAVITY
Brain- Normal in shape and size.
Page 22 of33

TABLE-1
Exposure atmosphere data
Group Time of Concentration Air flow Temperature Relative Particle size Oxygen
observation mg/L L/minute Humidity (µ) concentration
(minute) (%)
0 1.04 30 21.1 50.0 2.16-2.91 20.5
60 l.03 30 22.3 53.4 3.02-3.72 20.4
Group-I 120 0.99 30 21.5 52.8 2.38-3.00 21.0
1.04 mg/I 180 l.00 30 22.8 48.5 2.13-3.40 20.5
240 1.02 30 22.3 50.0 1.84-3.02 20.8
Mean 1.016 30 22.00 50.94 2.31-3.21 20.64
observation mg/L L/minute Humidity (µ) concentration
(minute) (%)
0 2.54 30 21.2 51.2 2.36-2.62 20.2
60 2.63 30 22.5 52.5 2.22-2.25 20.9
Group-II 120 2.59 30 21.4 53.6 2.18-3.00 22.26
5.02 mg/I 180 2.30 30 22.0 53.5 3.19-3.40 21.12
240 2.25 30 22.l 52.0 2.94-3.02 21.03
Mean 2.462 30 21.84 52.56 2.578-2.858 21.102
observation mg/L L/minute Humidity (µ ) concentration
(minute) (%)
0 4.98 30 22.0 50.1 2.51-2.98 20.8
60 5.03 30 22.6 49.8 2.12-2.68 20.5
Group-III 120 5.06 30 21.9 50.4 2.28-2.70 21.7
10.04 mg/I 180 5.99 30 22.5 52.7 2.14-2.66 20.4
240 5.04 30 22.2 51.2 2.45-3.15 21.0
Mean 5.22 30 22.24 50.84 2.30-2.834 20.88
The mean value was calculated as per formulae m = sum of terms / number of terms
Page 23 of33

TABLE-2
Individual Body Weight (gm)
Group Animal ID Sex Day 0 Day7 % Day 14 %
Gain/loss Gain/loss
202109-125-001 Male 209 212 1.41
215 2.79
Group-I 202109-125-002 Male 207 210 212
1.42 2.35
1.04mg/ I 202109-125-003 Male 212 215 218
1.39 2.75
202109-125-004 Female 206 209 1.43
212 2.83
202109-125-005 Female 208 211 1.42
214 2.80
202109-125-006 Female 204 207 1.44
210 2.85
Group Animal ID Sex DayO Day7 % Day 14 %

Gain/loss Gain/loss
202109-125-007 Male 218 2.75
212 215 1.39
Group -II 202109-125-008 Male 219
213 216 1.38 2.73
5.02mg/ l 202109-125-009 Male 217
211 214 1.40 2.76
202109-125-010 Female 1.38
219 2.73
213 216
202109-125-011 Female 1.38
220 2.72
214 217
202109-125-012 Female 0.93
218 2.29
213 215
Group Animal ID Sex DayO Day7 % Day 14 %

Gain/loss Gain/loss
202109-125-013 Male 207 210 1.42
213 2.81
Group-ID 202109-125-014 Male 211 214 217
1.40 2.76
10.04mg/l 202109-125-015 Male 215 218 220
1.37 2.27
202109-125-016 Female 216 219 222 2.70
1.36
202109-125-017 Female 210 213 1.40
216 2.77
202109-125-018 Female 214 217 1.38
220 2.72
Page 24 of33

TABLE-3
Clinical signs and mortality
Group: I Dose: 1.04 mg/I
WISTAR ALBNINO MALE RATS
Incidence of Clinical Signs Observed after Dosing on

Parameters Mortality
Day0 DAY
Min Hour
30 1 2 4 6 I 2 3 4 5 6 7 8 9 10 11 12 13 14 Total %
Mo1iality (total) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Clinical Signs 0 0 0 0 0/3 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
WISTAR ALBNINO FEMALE RATS

Parameters DayO Mortality
DAY
Min Hour
30 1 2 4 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Total %
Mortality (total) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Clinical Signs 0 0 0 0 0/3 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 = No clinical sign (Normal)
= Mild
= Moderate
+++ = High
= Severe
Page 25 of33

TABLE - 3 (Contd......
Group: II Dose: 5.02 mg/I

Day0 DAY
Min Hour
30 1 2 4 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Total ��
Mortality (total) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0/3 0
Clinical Signs 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Day0 DAY
Min Hour
30 1 2 4 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Total %
Mortality (total) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0/3 0
Clinical Signs 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
+ = Mild
++ = Moderate
+++ High
++++ Severe
++++ = Severe
Page 26 of33

TABLE - 3 (Contd ......
Group: III Dose: 10.04 mg/I

Day0 DAY
Min Hour
30 1 2 4 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Total �'o
Mortality (total) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0 0/3 0
Clinical Signs 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Day0 DAY
Min Hour
30 1 2 4 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Total %
Mortality (total) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0/3 0
Clinical Signs 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

+ = Mild
= Moderate
= High
++++ = Severe
Page 27 of33

REPORT No. IffiTffOX/202109/AIT/116
TABLE-4
Summary of necropsy finding
Fate: Terminal Sacrifice
s. Finding Wistar albino rats

No
Exposure level Exposure level Exposure level
(1.04 mg/I) (5.02 mg/I) (10.04 mg/I)
Male Female Male Female Male Female
1. Terminal 0/03 0/03 0/03 0/03 0/03 0/03

Sacrifice
Fate: Found dead
s. Finding Wistar albino rats

No
Exposure level Exposure level Exposure level
(1.04 mg/I) (5.02 mg/I) (10.04 mg/I)
Male Female Male Female Male Female
1. Found 0/03 0/03 0/03 0/03 0/03 0/03

Dead
Page 28 of33

STUDY ACUTE INHALATION TOXICITY IN WlSTAR ALBINO RAT
TABLE-5
Individual animal fate & necropsy findings
Group: I Dose: 1.05 mg/I
WIST AR ALBINO RATS- MALE
Animal ID Fate Time Gross Findings
202109-125-01 TS Day 15 NAD
202109-125-02 TS Day 15 NAD
202109-125-03 TS Day 15 NAD
WISTAR ALBINO RATS- FEMALE
202109-125-04 TS Day 15 NAD
202109-125-05 TS Day 15 NAD
202109-125-06 TS Day 15 NAD
Day 0 is the day of exposure

TS=Tenninal Sacrifice
NAD=No Abnom1ality Detected
FD=Found Dead
Page 29 of33

TABLE - 5 (Contd......
Group: IT Dose: 5.02 mg/I
WISTAR ALBINO RA TS- MALE
202109-125-07 TS Day 15 NAD
202109-125-08 TS Day 15 NAD
202109-125-09 TS Day 15 NAD
WIST AR ALBINO RATS- FEMALE
202109-125-010 TS Day 15 NAD
202109-125-011 TS Day 15 NAD
202109-125-012 TS Day 15 NAD
Day O is the day of exposure

TS=Terminal Sacrifice
NAD=No Abnormality Detected
FD=Found Dead
Page 30 of33

TABLE - 5 (Contd ......
Group: III Dose: 10.04 mg/I
WISTAR ALBINO RATS- MALE
202109-125-13 TS Day 15 NAD

202109-125-14 FD Day 15 NAD
202109-125-15 TS Day 15 NAD
WISTAR ALBINO RATS- FEMALE

TS Day 15
202109-125-16 NAD
TS Day 15
202109-125-17 NAD
Day 15
202109-125-18 FD NAD
Page 31 of33

REPORT No. IlRTffOX/202109/AIT/l 16
CONCLUSION
Based on the results obtained from present study, it is concluded that the test compound "ZERO
MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER sponsored by
HINDUSTAN AGRO is non toxic to wistar albino rat according to regulatory Guidelines of
"OECD-403" for Testing of Chemicals.
According to Globally Hannonized Classification (GHC) it comes under Category-III and LC50
is greater than 10.04 mg/I as per the present test finding.
Page 32 of33

CERTIFICATE OF ANALYSIS
-·,. Zobelc India Pl'ivntc Limited

Survey Nn. IR2, IR�/1, 11012, P,rnrhal Udyog Nngnr,
�-
ZOBELE Blli,npore, Dn111811 - 396210, India
OflOV>'
Tel: +91 260 6622800 L-R11 G .Rev.no
CERTIFICATE OF ANALYSIS
P,vJt!Cl - IC-f::l.[II-DUO-SET·(1!:NE:-lS MCiS-PLlJCK liK
S/\P ..:0Jc - 81107
Bat..:h Nu/ MFG -: n:,.,,,·,go�21
Qw,ntity :- 19.632 Pc,
Dut.:orM1'g :- 25-08-2021
Date or A;ial},is : . 25-08-2u2 I
Invoke no & dHIC : - 00(•4U0(1!"11)495/ll(, 09.2021
Sr. N,;. TEST SPECIFICATIONS RESULTS
I OJ,,ur Ch.11J�!,!ri,tic oJ,,vr Churad,ri�ri.: od,,"r
l A pf',:"r.u1..:e Cl".ir. c,,1,,urk·.,s to very light yellow liquid Ck:ir cn!,,urk,, liquiJ
J [),:n�ity "<1; 25°C (;/1111) 0.74 - 0.YO 0 RI I
Tr,111,tluthr'in .;.,,,,�nl (��11/w)

4 0 836-0.968 0.894%,
byOC
r '-�
L ....-
�-
StJlu, • Cnmrli,:,,/J�,>mt>IY spcc•itic:11i,,ns
........
/4�0IA,ol\
y-, I-
iii( o/W'N r;
--
;i
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� ob o � .
Anal) s.:J hy : Arr�•"' by, � ,
Ch<!mi.;1 QA i'vlanag�r
Page 33 of33

Acute Inhalation Toxicity Study - Rats

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ll�T

Institute for Industrial Research & Toxicology

ACUTE INHALATION TOXICITY OF

F-209, UPSIDC, MG ROAD, GHAZIABAD-201302 (INDIA)

Project No. 202109-125

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

GLP COMPLIANCE STATEMENT

Study Director Date

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

STATEMENT BY TEST FACILITY MANAGEMENT

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

QUALITY ASSURANCE REPORT

Standard Test Method Compliance Audit 04-10-2021

Ms. Shalini Mishra 27-10-2021

For Quality Assurance Unit

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

PROJECT NUMBER 202109-125

REPORT NUMBER IIRT/TOX/202109/AIT/116

STUDY TITLE ACUTE INHALATION TOXICITY OF "ZERO MAX"

SPONSOR BY HINDUSTAN AGRO

INSTITUTE FOR INDUSTRIAL RESEARCH &

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

Study Director Mr. Mohseen (M.Phann)

Study Personnel Mr. Ankur Rana (B.Pharm)

Histopathology & Veterinarian Dr. Vandana Singh, M.V.Sc. Pathology

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

MATERIALS AND METHODS

Study title ACUTE INHALATION TOXICITY IN WISTAR ALBINO RATS

Test compound "ZERO MAX" TRANSFLUTHRlN 1.60% LIQUID VAPORIZER

1. Species Albino rats

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

Group III- step III (10.04 mg/1)

11 Randomization After acclimatization and veterinary examination all the

2. Accommodation Groups of three animals of similar sex in polypropylene

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

INHALATION EXPOSURE SYSTEM

DESCRIPTION OF INHALATION CHAMBER

TEST COMPOUND "ZERO MAX" TRANSFLUTIIlUN 1.60% LIQUID VAPORIZER

AIR FILTRATION SYSTEM

2' FLUIDIZED BED GENERATOR (GENERATION OF DUST AEROSOLS)

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

MONITORING OF EXPOSURE CONDITION

Chamber Temperature, Relative Humidity and Oxygen Content:

PARTICLE SIZE DISTRIBUTION

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

The body weight of all the animals recorded individually on day ?

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

Group Animal ID Sex DayO Day7 % Day 14 %

Group Animal ID Sex DayO Day7 % Day 14 %

TEST COMPOUND "ZERO MAX" TRANSFLUTHRIN 1.60% LIQUID VAPORIZER

Group: I Dose: 1.04 mg/I

WISTAR ALBNINO MALE RATS