Page 1 of 11 

Unpublished Work 

STUDY TITLE: Inhalation Toxicity Screen in Rats 

AUTHOR: 

STUDY COMPLETED ON: May 19, 2011 

PERFORMING LABORATORY: 

LABORATORY PROJECT ID: 

WORK REQUEST NUMBER: 

SERVICE CODE NUMBER: 

SPONSOR: 
Inhalation Toxicity Screen in Rats 

- 2 - 

CERTIFICATION 

We, the undersigned, declare that this report provides an accurate evaluation of data obtained 

from this study. 
Inhalation Toxicity Screen in Rats 

- 3 - 

STUDY INFORMATION 

Substance Tested:

 

Number: 

 

Composition 

 

Purity: 99.98% 

 

Physical Characteristics: colorless gas at room temperature 

 

Stability: The test substance appeared to be stable under the 

conditions of the study; no evidence of instability was 

observed. 

 

Study Initiated/Completed: March 1, 2011 / (see report cover page) 

 

Experimental Start/Termination: February 22, 2011 / March 8, 2011 

 

In-Life Initiated/Completed: February 22, 2011 / March 8, 2011 

 
Inhalation Toxicity Screen in Rats 

- 4 - 

SUMMARY 

One group of 2 male and 2 female Crl:CD(SD) rats was scheduled to be exposed whole-body for 

1 hour to a target concentration of 50,000 ppm vapor. This pilot exposure was 

conducted to determine an appropriate design concentration for an LC50 study. The test 

atmosphere was generated by diluting vapor in air, and concentrations of were 

measured via gas chromatography. 

During the first 5 minutes of the pilot exposure, the animals displayed decreased activity levels, 

but had normal startle response. As the exposure proceeded, the animals began to show tremors 

and convulsions. Approximately 10 minutes after the exposure began, the chamber 

concentration had reached 33,200 ppm, and the rats displayed muscle spasms and severe 

convulsions. The test substance vapor flow into the exposure chamber was shut off, and the 

exposure was terminated for animal welfare reasons. 

When the animals were removed from the chamber, the female rats quickly recovered and 

displayed no abnormalities, while the male rats were lethargic and displayed low posture and 

labored breathing. Two hours after the exposure, all rats displayed no adverse clinical signs 

except for an injury to the tail and some dried blood around the nose of a male rat that was 

injured during a convulsive episode. The injured rat displayed a body weight loss of 4.3% on the 

day after the exposure. There were no other body weight losses or abnormal clinical signs 

observed in any rats during the 14-day recovery period. 

Under the conditions of this study, 10 minutes of exposure to 33,000 ppm of vapor 

produced tremors, muscle spasms, convulsions and decreased activity levels during the exposure. 

Male and female rats survived the 10 minute exposure, and were without significant adverse 

effects during the 14-day recovery period. 
Inhalation Toxicity Screen in Rats 

- 5 - 

INTRODUCTION 

The objective of this study was originally to determine a 1-hour inhalation median lethal 

concentration (LC50) of in male and female rats. The inhalation route was chosen 

based on the expected route of potential human exposure and was requested by the sponsor. 

ANIMAL WELFARE ACT COMPLIANCE 

This study complied with all applicable sections of the Final Rules of the Animal Welfare Act 

regulations (9 CFR) and the Guidelines from the Guide for the Care and Use of Laboratory 

Animals (NRC 1996). All studies conducted by or for adhere to the following 

principles: 

• The sponsor and/or the study director ensure that the study described in this report does not 

unnecessarily duplicate previous experiments, and is in compliance with the Policy 

on Animal Testing. 

• Whenever possible, procedures used in this study have been designed to implement a 

reduction, replacement, and/or refinement in the use of animals in an effort to avoid or 

minimize discomfort, distress or pain to animals. All methods are described in this study 

report or in written laboratory standard operating procedures. 

• policy is that animals experiencing severe pain or distress that cannot be 

relieved are painlessly euthanized, as deemed appropriate by the veterinary staff and study 

director or appropriate designee. The sponsor was advised by the study director of all 

circumstances that could lead to this action in as timely a manner as possible. 

• Methods of euthanasia used during this study were in conformance with the above referenced 

regulation and the recommendations of the American Veterinary Medical Association 

(AVMA), 2007 Guidelines on Euthanasia. 

• Animals were provided with species-appropriate environmental enrichment. 

• is accredited by the Association for the Assessment and Accreditation of 

Laboratory Animal Care (AAALAC) International. 
Inhalation Toxicity Screen in Rats 

- 6 - 

STUDY DESIGN 

One group of 2 male and 2 female rats was exposed to a vapor atmosphere of in air. By 

design, the rats were to be exposed whole-body for a single, 1-hour period. The exposure period 

ended after 10 minutes due to animal welfare considerations. The rats were approximately 

9 weeks old at the time of the exposure. The male rats weighed 282 and 352 grams and the 

female rats weighed 199 and 237 grams the morning before the exposure. 

Animals were observed for mortality and response to alerting stimuli during the exposure and 

observed for mortality and clinical signs of toxicity immediately after they were removed from 

the exposure chamber following exposure. During the recovery period, all rats were observed 

each day for mortality. Rats were weighed and observed for clinical signs of toxicity on the day 

following exposure and 4 more times during the recovery period. At the end of the recovery 

period, all rats were sacrificed by isoflurane anesthesia followed by carbon dioxide asphyxiation. 

MATERIALS AND METHODS 

A. Test Substance 

The test substance, was supplied by the sponsor in a cylinder as a liquefied gas with a 

purity of 99.98%. is a colorless gas at room temperature. The test substance was 

assumed to be stable throughout the exposure phase of the study; no evidence of instability was 

observed. 

B. Test System 

Young adult, male and female (nulliparous and non-pregnant) Crl:CD rats were received from 

Charles River Laboratories, Inc., Raleigh, North Carolina. The rats were approximately 6 weeks 

old on the day of arrival. 

Rats have historically been used in safety evaluation studies for inhalation toxicity testing. The 

Crl:CD rat was selected based on consistently acceptable health status and on extensive 

experience with the strain at 

C. Animal Husbandry 

1. Quarantine 

Animals were quarantined after arrival for 6 days prior to testing. During the quarantine period, 

animals were weighed and observed for clinical signs of disease 3 times. 

2. Animal Selection 

Prior to the exposure, 2 male and 2 female rats were selected for use on the study from the rats 

that were released from quarantine, had no overt signs of disease, and were the appropriate age 

and body weight. 
Inhalation Toxicity Screen in Rats 

- 7 - 

3. Identification 

Each animal was assigned an animal number that was recorded on a card affixed to the cage. 

Prior to exposure, the tail of each animal and cage card were numbered with water-insoluble 

pens so that each animal could be identified after exposure and during the recovery period. 

4. Housing 

Except during exposure, animals were housed individually in solid bottom caging with bedding 

and nestlets as enrichment. 

5. Environmental Conditions 

Animal rooms were maintained at a temperature of 18-26oC (64-79oF) and a relative humidity of 

30-70%. Animal rooms were artificially illuminated (fluorescent light) on an approximate 

12-hour light/dark cycle. Excursions outside of these ranges were of insufficient magnitude 

and/or duration to have adversely affected the validity of the study. 

6. Feed and Water 

Except during exposure, PMI® Nutrition International, LLC Certified Rodent LabDiet® 5002 and 

tap water were available ad libitum. 

7. Animal Health and Environmental Monitoring Program 

As specified in the animal health and environmental monitoring program, the 

following procedures are performed periodically to ensure that contaminant levels are below 

those that would be expected to impact the scientific integrity of the study: 

• Water samples are analyzed for total bacterial counts, and the presence of coliforms, lead, 

and other contaminants. 

• Samples from freshly washed cages and cage racks are analyzed to ensure adequate 

sanitation by the cagewashers. 

Certified animal feed is used, guaranteed by the manufacturer to meet specified nutritional 

requirements and not to exceed stated maximum concentrations of key contaminants, including 

specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The 

presence of these contaminants below the maximum concentration stated by the manufacturer 

would not be expected to impact the integrity of the study. 

The animal health and environmental monitoring program is administered by the attending 

laboratory animal veterinarian. Evaluation of these data did not indicate any conditions that 

affected the validity of the study. 
Inhalation Toxicity Screen in Rats 

- 8 - 

D. Duration of Exposure 

The animals were exposed for approximately 10 minutes. The starting time of the exposure was 

defined as the time when the generation system was turned on. The ending time of the exposure 

was defined as the time when the generation system was turned off. Animals were exposed to 

the test substance during both the time it took for the chamber to reach concentration, and the 

time it took for the test substance to be purged from the chamber. After the exposure, animals 

were returned to their cages and observed for at least 14 days. The end of the recovery period 

was defined as the 14th day after exposure. 

E. Inhalation Exposure System 

1. Atmosphere Generation 

The chamber atmosphere was generated by dilution of the vapor test substance in air. The test 

substance vapor and chamber air were metered to a mixing flask using Brooks model 5850E 

mass flow controllers. The resulting vapor and air was swept to a glass transfer tube that led to 

the top of the exposure chamber. 

The test atmosphere was exhausted through a dry-ice cold trap and an MSA filter cartridge prior 

to discharge into the fume hood. 

2. Chamber Construction and Design 

The exposure chamber was constructed of glass (cylindrical) with a nominal internal volume of 

19 L. A glass baffle inside the chamber promoted uniform chamber distribution of the test 

atmosphere. 

3. Exposure Mode 

During exposure, animals were placed in a stainless steel, wire-mesh module (sexes separate) 

and exposed, whole-body, inside the exposure chamber. The module was placed on a stainless 

steel stand inside the exposure chamber so that the animals were elevated from the bottom of the 

exposure chamber. 

F. Characterization of Chamber Atmosphere 

1. Test Substance Sampling and Analysis 

During the exposure (when vapor was delivered into the chamber), the atmospheric 

chamber concentration of the test substance was determined by gas chromatography 4 times. 

The chamber concentration was also monitored for 20 minutes after the vapor flow was 

terminated resulting in additional 4 chamber samples. Chamber atmosphere was continually 

drawn from the exposure chamber and directly injected into an Agilent model 6890N gas 

chromatograph (GC) equipped with a gas sample valve and a flame ionization detector. All 

samples were chromatographed isothermally at 95°C on a HP-5 (5% Phenyl Methyl Siloxane) 

fused silica glass column. The atmospheric concentration of the test substance was determined 

from a standard curve derived from gas standards. Standards were prepared prior to exposure by 
Inhalation Toxicity Screen in Rats 

- 9 - 

quantitative dilution of the vapor test substance into Tedlar® bags containing known volumes of 

air. 

Upon completion of the exposure, GC sample results were transferred to Camile Inhalation 

Reporting and Analysis System, which collated sample calculations. 

2. Environmental Monitoring 

Chamber temperature was targeted at 20-24°C and chamber relative humidity was targeted at 30-

70%. Temperature and humidity were measured with a VWR dial-type Hygro-Thermometer and 

recorded twice during the exposure. Chamber gas flows were set at the beginning of the 

exposure to achieve at least 10 atmosphere changes per hour. The gas flows were monitored 

continually with Brooks model 5850E mass flow controllers. Chamber oxygen concentration 

was targeted to be at least 19% and was measured with a Teledyne Analytical Instruments Model 

GB-300 oxygen analyzer. The chamber oxygen and gas flows were also recorded twice during 

the exposure. 
Inhalation Toxicity Screen in Rats 

- 10 - 

RESULTS AND DISCUSSION 

A. Exposure Conditions 

(Table 1) 

Animals were exposed to at a maximum concentration of 33,000 ppm. The mean 

concentration of all samples taken from the time the exposure was started until 20 minutes after 

the vapor flow was shut off was 11,000 ppm. 

Table 1

Characterization of Test Atmospheres and Associated Animal Mortality 

MEASURED VAPOR CONCENTRATION 

(ppm)a

MORTALITY 

(deaths / 

MEAN S.D. RANGE n exposed) 

11,000 12,000 950 – 33,000 8 0 / 4 

a Represents the mean, standard deviation (S.D.), and range for the exposure and post-exposure 

period, based on n samples. Values are reported to 2 significant figures. Calculations were 

performed prior to rounding values. 

Chamber temperature was 21°C, chamber relative humidity ranged from 57 to 65%, chamber 

airflow was 4.0 L/min, and the oxygen concentration ranged from 19 to 20%. The 

environmental conditions were within the targeted parameters for this study. 

B. Mortality, Clinical Signs, and Body Weights 

No deaths occurred during the study. 

During the first 5 minutes of the pilot exposure, the animals displayed decreased activity levels, 

but had normal startle response. As the exposure proceeded, the animals began to show tremors 

and convulsions. Approximately 10 minutes after the exposure began, the chamber 

concentration had reached 33,200 ppm, and the rats displayed muscle spasms and severe 

convulsions. One male rat got its teeth entrapped on the wire-mesh module and injured its tail 

during the exposure. 

When the animals were removed from the chamber, the female rats quickly recovered and 

displayed no abnormalities, while the male rats were lethargic and displayed low posture and 

labored breathing. Two hours after the exposure, all rats displayed no adverse clinical signs 

except for some dried blood around the nose of the male rat that had gotten its teeth entrapped in 

the wire mesh while convulsing during the exposure. This male rat displayed a body weight loss 

of 4.3% on the day after the exposure. There were no other body weight losses or abnormal 

clinical signs observed in any rats during the 14-day recovery period. 
Inhalation Toxicity Screen in Rats 

- 11 - 

CONCLUSIONS 

Under the conditions of this study, 10 minutes of exposure to 33,000 ppm of vapor 

produced tremors, muscle spasms, convulsions and decreased activity levels during the exposure. 

Male and female rats survived the 10 minute exposure, and were without significant adverse 

effects during the 14-day recovery period. 

RECORDS AND SAMPLE STORAGE 

The raw data, the protocol, amendments (if any), and the final report will be retained at