Page 1 of 12 Study Title One-Hour Inhalation Approxim ate Le thal Concentration (ALC) in Male Rats

A UTHOR : S TUDY C OMPLETED ON : February 4, 2009
P ERFORMING L ABORATORY :

L ABORATORY P ROJECT ID:

W ORK R EQUEST N UMBER :

S ERVICE C ODE N UMBER : S PONSOR :O n e-Hour Inhalation Approximate Lethal Concentration (ALC) in Male Rats

- 2 - CERTIFICATION We, the undersigned, declare that this report prov ides an accurate eval uation of data obtained from this study.O n e-Hour Inhalation Approximate Lethal Concentration (ALC) in Male Rats

- 3 - 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 Environm ental Monitoring Program.........................................................................8 D. Duration of Exposure...........................................................................................................8 E. Inhalation Exposure System ................................................................................................9 1. Atm osphere Generation ....................................................................................................... ....................9 2. Cham ber Constr uction and Design............................................................................................. ..............9 3. Expos ur e M ode ............................................................................................................... .........................9 F. Characterization of Chamber Atmosphere...........................................................................9 1. Test Substance Sa mpling and Analysis........................................................................................ ............9 2. Environm enta l Monitoring.................................................................................................... ...................9 RESULTS AND DISCUSSION ..................................................................................................11 A. Exposure Conditions......................................................................................................... .11 B. Mortality, Clinical Signs, and Body Weights ....................................................................11 CONCLUSIONS ..........................................................................................................................11 RECORDS AND SAMPLE STORAGE ....................................................................................12O n e-Hour Inhalation Approximate Lethal Concentration (ALC) in Male Rats

- 4 - 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, 2008O n e-Hour Inhalation Approximate Lethal Concentration (ALC) in Male Rats

- 5 - SUMMARY One group of 5 male Crl:CD(SD) rats was expos ed whole-body for a singl e, one-hour period to in air. The test atmosphere was generate d 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 decrea sed activity levels. No clinical signs of toxicity were observed in any rat during th e 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.O n e-Hour Inhalation Approximate Lethal Concentration (ALC) in Male Rats

- 6 - INTRODUCTION The objective of this study wa s 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 expos ed rats either on the day of exposure or within at least 14 days following ex posure. The inhalation route of exposure was chosen based on the expected route of potential human expos ure 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 ma ke particular note of the following:

• The signature of the sponsor and/or the study dir ector ensures that the st udy described in this report does not unnecessarily duplic ate 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 anim als. All methods are described in this study report or in written laboratory standard operating procedures.

• Animals that experience severe or chronic pa in or distress that cannot be relieved are painlessly euthanized as deemed appropriate by the veterinary staff and study director or appropriate designee. The spons or was advised by the study di rector of all circumstances that could lead to this action in as timely a manner as possible.

• Methods of euthanasia used during this study we re in conformance with the above referenced regulation and the recommendati ons of the American Veterinary Medical Association (AVMA), 2007 Guidelines on Euthanasia.

• Animals were provided with species-a ppropriate environmental enrichment.

• is accredited by the Association for the Asse ssment 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.O n e-Hour Inhalation Approximate Lethal Concentration (ALC) in Male Rats

- 7 - Rats were approximately 8 weeks old and we ighed between 259 and 288 grams at the time of exposure. Animals were observed for mortal ity and response to alerting stim uli 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 weighe d and observed for clinic al signs of toxicity on the day following exposure and at least twice mo re during the recovery period. At the end of the recovery period, all rats were sacrificed by carbon dioxi de asphyxiation and discarded.

MATERIALS AND METHODS
A. Test Substance The test substance, was supplied by the s ponsor as a gas with a purity of 99.6%. The test substance was assumed to be stable throughou t the exposure phase of the study; no evidence of instability was observed.
B. Test System Young adult, male Crl:CD(SD) rats were receiv ed from Charles River Laboratories, Inc., Raleigh, North Carolina. The rats were appr oximately 7 weeks old on the day of arrival. Rats have historically been used in safety eval uation studies for inhalatio n toxicity testing. The Crl:CD(SD) rat was selected based on consiste ntly 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 select ed for use on the study from the rats that were released from quarantine, had been gaining we ight at a normal rate, had no overt signs of disease, and were the appropria te age and body weight. No atte mpt 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 identifie d after exposure and during the recovery period.O n e-Hour Inhalation Approximate Lethal Concentration (ALC) in Male Rats

- 8 - 4. Housing Except during exposure, animals were housed in dividually 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 durat ion 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 h ealth and environmental monitoring program, the following procedures are performe d 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 th e manufacturer to meet specified nutritional requirements and not to exceed stated maximum c oncentrations of key contaminants, including specified heavy metals, aflatoxin, chlorina ted 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 monitori ng 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 sy stem was turned on. The ending time of the exposure was defined as the time when the gene ration system was turned off. Animals were exposed to the test substance during both the tim e 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,O n e-Hour Inhalation Approximate Lethal Concentration (ALC) in Male Rats

- 9 - animals were returned to their cages. The e nd of the recovery period was defined as the 14 th day after exposure.
E. Inhalation Exposure System
1. Atmosphere Generation The chamber atmosphere was generated by quantita tive 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 w ith 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 cham ber. Chamber concentrations of test substance were controlled by varying th e test substance feed rates to the mixing flask. The test atmosphere was e xhausted 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 (c ylindrical) with a nominal internal volume of 17 L. A glass baffle at the chamber inlet prom oted uniform chamber distribution of the test atmosphere.
3. Exposure Mode During exposure, animals were placed in a stainless steel, wire-mes h module and exposed, whole-body, inside the exposure chamber. Th e 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 a tmosphere were continually drawn from the breathing zone of the animals and were directly injected into an Ag ilent Technologies model 6890N gas chromatograph equipped with an automa ted gas sample valve and a flame ionization detector. All samples were chromatographed is othermally at 80°C on a HP-5 fused silica glass column. The atmospheric concentration of the te st substance was determined from a standard curve derived from gas standards. Standards we re 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.O n e-Hour Inhalation Approximate Lethal Concentration (ALC) in Male Rats

- 10 - Temperature and humidity were measured with a VWR NIST Traceable Thermo-humidity meter. Chamber airflow was set at the beginn ing of the exposure to achieve at least 10 air changes per hour. The airflow was monitored co ntinually with a Brooks model 5850E mass flow controller and recorded 3 tim es during the exposure. Chamber oxygen concentration was targeted to be at least 19%. The oxygen concen tration was measured with a Biosystems model 3100R oxygen analyzer and recorded 3 times during the exposure.O n e-Hour Inhalation Approximate Lethal Concentration (ALC) in Male Rats

- 11 - 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 Atmosphere s 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 concentra tion 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 decrea sed activity levels. No clinical signs of toxicity were observed in any rat during th e 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 presen ce of the animals and use of a small chamber with low airflows.O n e-Hour Inhalation Approximate Lethal Concentration (ALC) in Male Rats

- 12 - RECORDS AND SAMPLE STORAGE Specimens (if applicable), raw data, the protocol , amendments (if any), and the final report will be retained at