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Study Title

 One-Hour Inhalation Approximate Lethal Concentration (ALC) in Male Rats 

AUTHOR: 

STUDY COMPLETED ON: February 4, 2009 

PERFORMING LABORATORY: 

LABORATORY PROJECT ID: 

WORK REQUEST NUMBER: 

SERVICE CODE NUMBER: 

SPONSOR: 
One-Hour Inhalation Approximate Lethal Concentration (ALC) in Male Rats 

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CERTIFICATION 

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

from this study. 
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TABLE OF CONTENTS 

Page 

CERTIFICATION.........................................................................................................................2 

TABLE OF CONTENTS ..............................................................................................................3 

STUDY INFORMATION .............................................................................................................4

SUMMARY ....................................................................................................................................5 

INTRODUCTION..........................................................................................................................6 

ANIMAL WELFARE ACT COMPLIANCE .............................................................................6 

STUDY DESIGN............................................................................................................................6 

MATERIALS AND METHODS ..................................................................................................7 

A. Test Substance .....................................................................................................................7 

B. Test System..........................................................................................................................7 

C. Ani mal Husbandry...............................................................................................................7 

1. Qua rantine................................................................................................................................................7 

2. Anim al Selection......................................................................................................................................7 

3. Ide ntification ............................................................................................................................................7 

4. Housing ....................................................................................................................................................8 

5. Anim al Room Environment .....................................................................................................................8 

6. Feed and Water ........................................................................................................................................8 

7. Animal Health and Environmental Monitoring Program.........................................................................8

D. Duration of Exposure...........................................................................................................8 

E. Inhalation Exposure System ................................................................................................9 

1. Atm osphere Generation ...........................................................................................................................9 

2. Cham ber Construction and Design...........................................................................................................9 

3. Expos ure Mode ........................................................................................................................................9 

F. Characterization of Chamber Atmosphere...........................................................................9 

1. Test Substance Sampling and Analysis....................................................................................................9 

2. Environm ental Monitoring.......................................................................................................................9 

RESULTS AND DISCUSSION ..................................................................................................11 

A. Exposure Conditions..........................................................................................................11 

B. Mortality, Clinical Signs, and Body Weights ....................................................................11 

CONCLUSIONS ..........................................................................................................................11 

RECORDS AND SAMPLE STORAGE ....................................................................................12 
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STUDY INFORMATION 

Substance Tested: •

(CAS Number) 

•

 

 Number: 

 

Composition:

 

Purity: 99.6% 

 

Physical Characteristics: Liquid 

 

Stability: The test substance appeared to be stable under the conditions 

of the study; no evidence of instability was observed. 

 

Study Initiated/Completed: August 11, 2008 / (see report cover page) 

 

Experimental Start/Termination: August 11, 2008 / August 25, 2008 
One-Hour Inhalation Approximate Lethal Concentration (ALC) in Male Rats 

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SUMMARY 

One group of 5 male Crl:CD(SD) rats was exposed whole-body for a single, one-hour period to 

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

vapor in air. Concentrations of were measured by gas chromatography. Rats were 

weighed and observed for clinical signs of toxicity during a 14-day recovery period. 

Rats were exposed to an atmosphere of at a mean concentration of 24,000 ppm. No 

rats died following exposure to or during the recovery period. 

During exposure, the rats exhibited labored breathing and decreased activity levels. No clinical 

signs of toxicity were observed in any rat during the recovery period. 

One day after exposure, weight losses occurred in 3 of the 5 rats exposed; losses typically ranged 

from 1-2 grams of body weight. All rats subsequently resumed normal weight gains 

(approximately 5-10 g/day) on test day 2 and gained weight for the remainder of the recovery 

period. 

Under the conditions of this study, the one-hour approximate lethal concentration (ALC) for 

is greater than 24,000 ppm. 
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INTRODUCTION 

The objective of this study was to determine a one-hour inhalation approximate lethal 

concentration (ALC) of in male rats. The ALC is defined as the lowest atmospheric 

concentration tested that caused the death of one or more exposed rats either on the day of 

exposure or within at least 14 days following exposure. The inhalation route of exposure 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). The sponsor should make particular note of the following: 

• The signature of the sponsor and/or the study director ensures that the study described in this 

report does not unnecessarily duplicate previous experiments, and is in compliance with the 

 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. 

• Animals that experience severe or chronic 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. 

STUDY DESIGN 

One group of 5 male rats was exposed to a vapor atmosphere of in air. Rats were 

exposed whole-body for a single, one-hour period. Following exposure, rats were retained for a 

14-day recovery period. 
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Rats were approximately 8 weeks old and weighed between 259 and 288 grams at the time of 

exposure. 

Animals were observed for mortality and response to alerting stimuli 3 times 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 at least twice more during the recovery period. At the end of 

the recovery period, all rats were sacrificed by carbon dioxide asphyxiation and discarded. 

MATERIALS AND METHODS 

A. Test Substance 

The test substance, was supplied by the sponsor as a gas with a purity of 99.6%. 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 Crl:CD(SD) rats were received from Charles River Laboratories, Inc., 

Raleigh, North Carolina. The rats were approximately 7 weeks old on the day of arrival. 

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

Crl:CD(SD) 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, 5 male rats were selected for use on the study from the rats that were 

released from quarantine, had been gaining weight at a normal rate, had no overt signs of 

disease, and were the appropriate age and body weight. No attempt was made to randomly group 

animals. 

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. 
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4. Housing 

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

and nestlets as enrichment. 

5. Animal Room Environment 

Animals were housed in proximity to the inhalation chambers. Animal rooms were maintained 

at a temperature of 18-26oC and a relative humidity of 30-70%. Animal rooms were artificially 

illuminated (fluorescent light) on an approximate 12-hour light/dark cycle. Any 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. 

D. Duration of Exposure 

In general, the group of animals was exposed for one hour. 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, 
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animals were returned to their cages. 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 quantitative dilution of the vapor test substance in 

air. The test substance vapor was dispensed into an evacuated Tedlar® gas standard bag. Test 

substance vapor was metered from the Tedlar® bag into a 1000-mL, 3-neck mixing flask with 

dual Cole-Parmer Masterflex® console drive pumps equipped with Fluid Metering Inc. (FMI) 

pistons. High-pressure air, metered into the mixing flask by a Brooks model 5850E mass flow 

controller, carried the vapor and air mixture into the exposure chamber. Chamber concentrations 

of test substance were controlled by varying the test substance feed rates to the mixing flask. 

The test atmosphere was exhausted through a dry-ice cold trap followed by an MSA 

charcoal/HEPA 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 

17 L. A glass baffle at the chamber inlet promoted uniform chamber distribution of the test 

atmosphere. 

3. Exposure Mode 

During exposure, animals were placed in a stainless steel, wire-mesh module 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, the vapor concentration of the test substance was determined by gas 

chromatography 7 times. Volumes of chamber atmosphere were continually drawn from the 

breathing zone of the animals and were directly injected into an Agilent Technologies model 

6890N gas chromatograph equipped with an automated gas sample valve and a flame ionization 

detector. All samples were chromatographed isothermally at 80°C on a HP-5 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 the exposure by injecting 

known volumes of the vapor test substance into Tedlar® bags containing known volumes of air. 

2. Environmental Monitoring 

Chamber temperature was targeted at 20-24°C and recorded 3 times during the exposure. 

Chamber relative humidity was targeted at 30-70% and recorded 3 times during the exposure. 
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Temperature and humidity were measured with a VWR NIST Traceable Thermo-humidity 

meter. Chamber airflow was set at the beginning of the exposure to achieve at least 10 air 

changes per hour. The airflow was monitored continually with a Brooks model 5850E mass flow 

controller and recorded 3 times during the exposure. Chamber oxygen concentration was 

targeted to be at least 19%. The oxygen concentration was measured with a Biosystems model 

3100R oxygen analyzer and recorded 3 times during the exposure. 
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RESULTS AND DISCUSSION 

A. Exposure Conditions 

(Table 1) 

Animals were exposed to at a mean concentration of 24,000 ppm. 

Table 1

Characterization of Test Atmospheres and Associated Animal Mortality 

MEASURED VAPOR CONCENTRATION 

(ppm)a

MORTALITY 

(deaths / 

MEAN S.D. RANGE n exposed) 

24,000 790 23,000 - 25,000 7 0 / 5 

a Represents the mean, standard deviation (S.D.), and range for the exposure, based on n 

samples. Values are reported to 2 significant figures. Calculations were performed prior to 

rounding values. 

Chamber temperature was 23°C, chamber relative humidity ranged from 75 to 80%,a

 chamber 

airflow was 4.0 L/min, and the oxygen concentration was 20%. Although chamber relative 

humidity was outside the targeted parameters, this deviation was considered not to have 

adversely affected the validity of this study. 

B. Mortality, Clinical Signs, and Body Weights 

No deaths occurred during the study. 

During exposure, the rats exhibited labored breathing and decreased activity levels. No clinical 

signs of toxicity were observed in any rat during the recovery period. 

One day after exposure, weight losses occurred in 3 of the 5 rats exposed; losses typically ranged 

from 1-2 grams of body weight. All rats subsequently resumed normal weight gains 

(approximately 5-10 g/day) on test day 2 and gained weight for the remainder of the recovery 

period. 

CONCLUSIONS 

Under the conditions of this study, the one-hour approximate lethal concentration (ALC) for 

is greater than 24,000 ppm. 

 

a High chamber relative humidities were due to the presence of the animals and use of a small chamber with low 

airflows. 
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RECORDS AND SAMPLE STORAGE 

Specimens (if applicable), raw data, the protocol, amendments (if any), and the final report will 

be retained at