Page 1 of 11 Unpublished Work

S TUDY T ITLE : Inhalation Toxicity Screen in Rats
A UTHOR :
S TUDY C OMPLETED ON : May 19, 2011
P ERFORMING L ABORATORY :

L ABORATORY P ROJECT ID:

W ORK R EQUEST N UMBER :

S ERVICE C ODE N UMBER :
S PONSOR :I n halation Toxicity Screen in Rats

- 2 - CERTIFICATION We, the undersigned, declare that this report prov ides an accurate eval uation of data obtained from this study.In h alation 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 appear ed 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, 2011I n halation Toxicity Screen in Rats

- 4 - SUMMARY One group of 2 male and 2 female Crl:CD(SD) ra ts 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 appropria te design concentration for an LC 50 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 cham ber, 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 ad verse 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 wit hout significant adverse effects during the 14-day recovery period.I n halation Toxicity Screen in Rats

- 5 - INTRODUCTION The objective of this study was originally to determine a 1-hour inhalation median lethal concentration (LC 50 ) of in male and female rats. The inhalation route was chosen based on the expected route of potential hum an 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 dir ector ensure that the study desc ribed in this report does not unnecessarily duplicate previous experiments, a nd 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 anim als. All methods are described in this study report or in written laboratory standard operating procedures.

• policy is that animals experien cing severe pain or di stress that cannot be relieved are painlessly euthanized, as deemed appropriate by the vete rinary staff and study director or appropriate designee. The spons or was advised by the study director of all circumstances that could lead to this ac tion 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.I n halation 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. Th e 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 mort ality 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, al l rats were observed each day for mortality. Rats were weighed and obse rved for clinical signs of toxicity on the day following exposure and 4 more times during the rec overy period. At the end of the recovery period, all rats were sa crificed by isoflurane anesthesia followed by carbon dioxide asphyxiation.

MATERIALS AND METHODS
A. Test Substance The test substance, was supplied by the s ponsor 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 ra ts were approximately 6 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 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.I n halation 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 identifie d after exposure and during the recovery period.
4. Housing Except during exposure, animals were housed in dividually 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 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.I n halation Toxicity Screen in Rats

- 8 - D. Duration of Exposure The animals were exposed for approximately 10 mi nutes. The starting time of the exposure was defined as the time when the generation system wa s 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 14 th day after exposure.
E. Inhalation Exposure System
1. Atmosphere Generation The chamber atmosphere was generated by dilution of the vapor test substanc e in air. The test substance vapor and chamber air were metered to a mixing flask using Brooks model 5850E mass flow controllers. The resul ting vapor and air was swept to a gl ass transfer tube that led to the top of the exposure chamber. The test atmosphere was exhausted through a dry-i ce cold trap and an MS A 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 19 L. A glass baffle inside the chamber promot ed uniform chamber distribution of the test atmosphere.
3. Exposure Mode During exposure, animals were placed in a stainl ess steel, wire-mesh module (sexes separate) and exposed, whole-body, inside the exposure ch amber. The module was placed on a stainless steel stand inside the exposure ch amber 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 deliv ered into the chamber), the atmospheric chamber concentration of the te st 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 concen tration of the test substance was determined from a standard curve derived from gas standards. Standards were prepared prior to exposure byI n halation 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 re sults were transferred to Camile Inhalation Reporting and Analysis System, whic h collated sample calculations.
2. Environmental Monitoring Chamber temperature was targeted at 20-24°C a nd chamber relative humidity was targeted at 30- 70%. Temperature and humidity were measured with a VWR dial-type Hygro-Thermometer and recorded twice during th e exposure. Chamber gas flows were set at the beginning of the exposure to achieve at least 10 atmosphere ch anges per hour. The gas flows were monitored continually with Brooks mode l 5850E mass flow controllers. Chamber oxygen concentration was targeted to be at least 19% and was measur ed with a Teledyne Analyt ical Instruments Model GB-300 oxygen analyzer. The chamber oxygen and ga s flows were also recorded twice during the exposure.I n halation Toxicity Screen in Rats

- 10 - RESULTS AND DISCUSSION
A. Exposure Conditions (Table 1) Animals were exposed to at a maximu m concentration of 33,000 ppm. The mean concentration of all samples taken from the tim e the exposure was started until 20 minutes after the vapor flow was s hut off was 11,000 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) 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 rela tive 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 th e 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-mes h module and injured its tail during the exposure.
When the animals were removed from the cham ber, 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 ad verse clinical signs except for some dried blood around the nose of the ma le rat that had gotten its teeth entrapped in the wire mesh while convulsing during the exposur e. 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.I n halation 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 wit hout significant adverse effects during the 14-day recovery period.

RECORDS AND SAMPLE STORAGE The raw data, the protocol, amendments (if any), a nd the final report will be retained at