FY99003 Final Report June 15, 1999 page I of8


### ACUTE INHALATION TOXICITY OF CANDIDATE HALON REPLACEMENT COMPOUNDS IN RATS Performed for:
Performed by:
Location: Co-Investigators:

Report Date : Quality Assurance New Mexico Engineering Research Institute (NMERI) Dr. J. Douglas Mather, Sponsor Representative Lovelace Respiratory Research Institute (LRRI) Dr. Gregory L. Finch, Principal Inve stigator Inhalation Toxicology Laboratory, LRRI Mr. E. Barr, Aerosol Scientist Dr. Kristen Nikula, Veterinary Pathologist Dr. Jennifer Krone, Chemist Dr. J. Douglas Mather, NMERI, Senior Research Chemist June 15, 1999
This study was not conducted 

### in full compliance with GLP regulations. It adhered to 

### the study protocol, pertinent LRRI SOPs, and high-quality standards ofquality checking and documentation I. 

### INTRODUCTION Work was preformed at 

### LRRI under LRRI research protocol FY99-003
. This protocol was approved by all institutionally-required reviewers including the LRRI Institutional Animal Care and Use Committee
. See copy ofthe protocol attached to this report (Attachment #1) for rationale for this study and the study design. 

### II. MATERIALS AND METHODS A. Compounds:: Compounds were supplied by NMERl. The compounds studied carry a NMERI CCOD identifier. The CCOD #s were 707,872,873, 903, 1116, 1358 , 1359, and 1360. Specific compound names and formulas for these identifiers are listed below. 707 1-Bromo-3 , 3 , 3-trifluoropropene, CF3 CH = CHBr 872 3-Bromo-3,3-difluoropropene, CFzBrCH = CHz 873 2-Bromo-3 , 3,3-trifluoropropene, CF3 CBr=CHz 903 4-Bromo-3 , 3,4,4-tetrafluorobutene, CFzBrCFzCH=CHz 1116 2-Bromo-3,3,4,4,4-pentafluorobutene, CF3 CF2CBr=CHz 1358 2-Bromo-3,3,4,4,5,5,5-heptafluoropentene, CF3CFzCFzCBr=CHz 1359 2-Bromo-3-trifluoromethyl-3,4,4,4-tetrafluorobutene, (CFJ)zCFCBr=CHz 1360 2-Bromo-3,4,4,4-tetrafluoro-3-trifluoromethoxy-butene, CF3CF(CF30)CBr=CH2FY99003 Final Report June15 , 1999 pag e 2 of8


### B. Experimental Design/Order of Exposures: 

### Group Treatment 1 Air controls, 30 min sham exposure 2 873, 5%, 30 min exposure 

### 3 707 , 

## ? % , 30 min exposure 

### 4 872, 5%, 30 min exposure 5 903, 5%, 30 min exposure 

### 6 1116, 5% , 30 min exposure 

### 7 1360, 5%, 30 min exposure 

### 8 1358 , 5% , 30 min exposure 

### 9 1359, 5%, 

### 30 min exposure


### C. Animals: 

### Number 5M/5F 5MI5F 5M /5F 5M/5F 5M /5F 5M/5F 5M/5F 5M /5F 5M /5F Total = 90 Exposure Date 04/19/99 04119/99 04/ 19/99 04/20/99 04/20/99 04/20/99 04/21199 04/ 21 / 99 04/ 21199



### This study used rats , male and fema le , 

### F344/Crl (Charles River Laboratories) , 

### 10- 1 2 wk of age at time ofexposures. Animals were received and quarantine d for

- 2 wk prior to use. Animals were weighed 5 days before the 

### first exposure and randomly a s signed to groups by weight (using Excel)
. Identification was by tail markings. Animals were housed in polycarbonate shoeb o x-type cages over hardwood bedding, 12-hr light cycle, food and water available 

### ad libitum except during exposure . Animals were conditioned to the exposure tubes twice during the week prior to exposures . 

### D. Exposures: A schematic ofthe exposure system is shown as Figure 1
. Room barometric pres sure and chamber temperatures were checked daily and used to calculate the quantity o f compound required for a 5% air c oncentr ation, using the following equation : 

### Mass = ((90L*(Pres sure/760)(273.15!femp
. ))/22.41409 L/ mole)*MW*O.OS Test compounds were loaded into a 50ml syringe and metered from the syringe using a calibrated syringe pump . Syringe weights were recorded after filling and ag ain aft er each exposure to establish the t otal quantity ofcompound inj ected during the 30 minute exposures . The chamber flow rate of 3 L/minute was verified each day of
. exposures as was the pressure integri ty ofthe system. Material was metered into a heated J-tube device partially filled with glass bead s . J-tube temperature was approximately 80 degrees C for the first five compounds, and 110 degrees C for the last 3 compounds
. Vapor was passed to a 24-port nose-only exposure chamber with a flow rate of approximately 3 Llmin. The chamber was monitored for oxygen c ontent, tempera ture, chamber pressure, and flow
. A piston pump whose output was regulated by a rotometer supplied a flow of approximately 50 to 75 cc /min from the chamber into sample bags supplied by NMERI. Samples were transported to NNIERI for analysis ofvapor concentration
. The order oftesting ofthe compounds was from lowest to highe st boiling point.FY99003 Final Report June 15, 1999



### E. Animal Procedures: page 3 of8

Rats were loaded into exposure tubes and transported to the expo s ure room. Tubes were loaded into the chamber and the exposure begun
. Animals were observed throughout the 30-min exposure period
. Following exposure, rats were removed from the exposure tubes and placed into polycarbonate shoebox-type cages . Rats were observed continuously until recovery from exposure . At approximately 2 hours after exposure, rats were sacrificed using an overdo se of sodium pentobarbital. A gross necropsy was conducted , including collection ofterminal body weight, weight ofthe tissues lung, liver, heart, spleen, and kidneys, and gross observation ofthese tissues
. 

## m. RESULTS A. Exposure Atmospheres The following 

### table lists exposure atmosphere compo sitions
. Targeted weights refer to the calculated amo unts ofeach compound required
. Weights delivered are in close agreement except where temporary plugging occurred for CCOD 707. Exposure levels in each case were at 5% based on the vol u metric flow rate ofthe exposure line (verified at the beginning ofeach day oftesting) and the total weights ofcompound delivered (determined by weighing the syringe before and after each exposure), and the duration ofeach exposure . Sam p ling ofthe exposure chamber air was intended to provide additio n al means ofverifying the 5% exposure level failed to perform as expected. The sampling performed empl o yed a piston pump whose output was regulated at between 50 to 75 cc ' s per minute by a rotameter. The output ofthe rotameter was collected in sample bags
. Analytical testing by gas chromatography did not provide the air concentration verification expected the subsequent testing demonstrated that compounds were condensing in the pump head due to high compressor head pressures . CCOD Compound Target \\' eight %of Wei g ht Delivered Target (g) (g) co ne . 873 2-Bromo-3 , 3) -trifluorcpropene 26 .9 5 26 .9 6 100 707 1-Bromo-3 ,3, 3-trifluoropropene 26.95 23
. 73 1 100 1 872 3-Bromo-3 , 3-difluoropropene 24.02 24
. 68 102 903 4-Bromo- 3 ,3,4 ,4-tetrafluorobutene 31.67 32.00 101 1116 2-B rorno-3 3,4, 4,4-pentafluorobutene 34.43 34 .3 0 100 1360 2-Bromo-3 , 4, 4 , 4-tetrafluoro-3- 44
. 10 44
. 0 1 100 trifluoromethoxy-butene 1358 2-Bromo-3,3 , 4,4,5 , 5 , 5-heptafluoropentene 41
. 68 42
. 19 101 1359 2-Bromo-3-trifluoromethyl- 3,4,4,4- 41
. 68 39.15 99 tetrafluorobutene (39
. 6i I Synnge needle pluggtng occurred dunng the first 10 mmutes ofthe exposure. The plugging was eliminated and the exposure was extended 5 minutes to compensate for lost Air Sample Vol. % 4:4 4.5 3.8 3
. 9 3.8 3.4 2
. 9 1.7FY99003 Final Report June 15, 1999 page 4 of8
exposure time. Concentration is based on the amount ofcompound delivered and a r,rojected exposure duration of26
.4 minutes.

Exposure was terminated after 28
. 5 minutes due to deaths of all test animals, the value in parenthesis reflects the shorter exposure.
B. Observations During and After Exposure This section describes observations of the rats during exposure and during the 2- hour post-exposure peri o d. Survival during this period is also summarized. Controls: Normal during 30
.min exposure . Occasional movement in tubes
. Good pink color ofextremities with normal respiration. Upon being unloaded rats appeared normal and began grooming themselves
. Compound 873 : Relaxed breathing began shortly after exposure . Little movement within tubes within first few minutes then none thereafter. Animals appeared to be anesthesized
. After exposure animals were anesthesized for a few minutes then began moving about although with impaired motor activity. Back to normal after about 10 min postexposure, and normal throughout to sacrifice. Compound 707 : First 10 min rats appear ed normal (correlated with period! oftime syringe pump had apparent partial clog)
. After 15 min tremors developed; most noticeable in hind limbs. By 20 min tremors had increased a nd respiration was more rapid. Color remained OK. By 26 min several rats had their tremors lessen in severity. Several rats had fast respiration (e.g
. C028) , while others had slowed respiratio n (e
. g . C025). By 28 min, some rats had contractions ofhind paws . By 32 min tremors had markedly decreased except for C028. Upon removal from tubes the rats were awake but had moderate ataxia. Hair coats were somewhat rough. Rats resumed normal grooming behavior and had good limb strength within a few min
. A few still had labored breathing 1 0-15 min post
- exposure with ears a bit pale indicating incomplete oxygenation
. By 45-min post exposure all rats appeared normal. Compound 872 : At 12 min extremities were pale blue and! rats began gasping. Breathing rate decreased and became labored. At 17 m in breathing rate was very slow. Rats were not moving in tubes and appeared to be anesthesized. At 22 min rats continued having l abored breathing and were pale. D037 was barely breathing at 27 min ofexposure and was dead at end ofexposure. Upon removal from tubes the rats appeared deeply anesthesized and then began moving within 3-5 min. 

### 0034 died 

### 10 min post- · exposure. 

### D038 died 15 min postexposure . The live rats had rough hair coats and continued labored breathing
. 

### D03 

### 5 diedFY99003 Final Report June 15, 1999 page 5 of8

at 75 min post- · exposure, all others continued to have labored breathing and were lethargic. 0033 died 90 min post-exposure. 0032 died 100 min post-exposure. D031 had foam on nose and died 106 min post-exposure. Thus a total of7/IO rats died before sacrifice. Compound 903 : At 12 min of exposure color was go od, breathing relaxed, and there was no movement indicating the rats were anesthesized
. Animals remained this way throughout exposure . After exposure rats remained anesthesized for 2-3 min then began waking
. Rats were stumbling around by 5 min post-exposure and were very wobbly
. By 20 min postexposure all rats appeared normal and continued normally until sacrifice
. Compound 1116 : At 3 min into exposure respiration was irregular, and became labored with gasping by 8 min
. At 13 min respiration was s lowed and animals ap peared to be anesthesized
. By 20 min F058 was breathing very slowly , paws were pale purple indicating insufficient oxygenation
. By 5 min po st -expo sure rats were waking and had labored breathing; labored breathing continued to 20 min po st
- exposure . Respiration continued in this manner until sacrifice
. Co mpound 1360 : Excitement and agitation was observed at 3 min of exposure, slight agitation c ontinued to 10 min ofexposure . By 12 min there was still movement, and rats had slow deep breathing. At 15 min G061 w as turned in the tube and dead. Additional deaths were G066 at 20 min ofexposure, G064 at 21 

### min ofexposure, G069 at 22 min of exposure, G068 at 24 min ofexposure ; also , G067 , G062 , and G070 all appeared dead at this time
. G063 appeared dead at 28 min ofexposure . G065 survived exposure and was foaming at the nose 5 min post-exposure and had agonal breathing
. This rat died at 13 min post
- exposure. Thus all rats died before the 2-hr sacrifice. Compound 1358 : Excitement and agitation was observed at 3 min ofexposure . At 6 min breathing rate decreased and became deeper; H075 had labored breathing
. Color was good at 13 min and there was still some movement and slow deep breathing
. At 21 min there was still good color and slow deep breathing. At 22 min a few rats began somewhat labored respiration
. Post

- exposure rats had cool body temperatures and were anesthesized
. H075 continued gasping. Recovery from anesthesia began at 4-5 min post
- exposure; rats were still very groggy, had little movement, and some gasping
. By 10 min post-exposure grip strength was returning but the rats were still cool to the touch, hunched , and very lethar g ic. By 20 min post-exposure H075 was moving about. H075 died 36 min afterFY99003 Final Report June 15, 1999 page 6 of8
exposure. 

### All other rats survived to sacrifice but continued to be very lethargic, hunched, and huddled together. Compound 1359: Animals were somewhat agitated by 2 min ofexposure . Rats appeared to be mostly anesthesized by 6 min
. During the 6-10 min period respiration was very slow with sporadic labored breathing. I087 began gasping by 11 min. Rats had purple paws indicating poor oxyge nation
. By 13 min most animal s had very labored breathing with gasping ; respiration continued to deteriorate
. AH 10 rats died, and the following exposure times to death were noted : 1081 dead at 18 min, I082 dead at 20 min, I088 dead at 21 min, 1086 dead at 23 min, I083 , 1085 , 1087 and 1089 dead at 24 min, 1090 dead at 25 min, and 1084 dead at 26

- min
. Thus all rats died during the 30-min exposure . ' Table 1
. Mortality Observed During and After Exposure (No
. de ad/No
. observed) Compound Males Females Sexes combined Controls 0 /5 0/5 0/ 10 873 0 /5 0/5 0/10 707 0 /5 015 0/10 872 5/ 5 2/5 7/10 903 0/5 015 0/10 1116 0 / 5 0/5 0/10 1360 5/5 5/5 10 / 10 1 3 58 115 0/5 1/ 10 1359 5/5 5/5 10/10 Survival is shown in figure 2
. C. Observations at Necropsy: Controls : 

### All rats normal. Compound 873 : All rats normal. Compound 707: All animals in this group had unusually pronounced postmortem muscular contractions. The contractions were noted immediately upon opening the pelt and involved the entire body. They persisted for approximately one minute after beginning the necropsy . The contractions were evident as very rapid tremors which were more intense than and persisted longer than the occasional muscle contraction often seen at necropsy . Lungs appeared normal.FY99003 Final Report June 15 , 19 99 Compound 872: page 7 of8

Several ofthe rats had foam/frothy discharge at the nose . Lungs 

### did not fully collapse upon opening the thorax. Lungs for several ofthe rats were mottled red. Several ofthe rats had air in the stomach consistent with gasping
. Livers were generally darker than normal. Rats in this group had eleva t ed lung-to-body weight ratios compared with controls, and slightly elevated liver weights. Compound 903 : All rats normal. Compound 1116: Lungs were generall y mottled and patchy red to dark red in appearance . Lungs did not fully coliapse upon opening the thorax
. · Several ofthe animals had air in the stomach
. Lung-to-body weig ht ratios were elevated compar ed with controls
. Livers appeared normal. Compound 1360: Lungs were moderately to markedly mottled dark red
. Lungs from only one rat did not fully collapse up o n opening the thorax
. Livers were darker red than is normal. Lung

-to
-body weig ht ratios and mean liver weights were increased compared to c ontrols
. Compound 1358 : Some rats had froth in the**trache~** and occasionally lungs did not fully collapse
. Lungs were mottled moderately to markedly
. Lung-to

- body weight ratios were increased compared to controls. Compound 1359 : Lungs did not fully collapse upon opening the thorax
. Lungs were markedly mottled dark red. Livers were also darker red than normal. Lung-to-body weight ratios and liver weights were elevated compared with controls
. 

### D. Organ Weights and Lung-to-Body weight ratios Body and Organ weights (weights in grams) are included in Table 2 , and summaries of mean body and organ weights by sex, and overall lung

-to-body weight ratios are presented in Table 3
. 

### IV. CONCLUSIONS A. Test Apparatus and Air Concentrations Exposures of 5 male and 5 female rats were conducted and exposure atmospheres were approximately 5% based on weights of com p ound delivered , the chamber air flow , and the duration ofeach exposure .FY99003 Fin al Report June 15, 1999


### B. C ompound Toxicities page 8 of8

There were no noteworthy differences in the responses between male and female rats except as noted below. Compounds 873 and 903 appeared to be the most inn ocuous in this test. Both compounds induced anesthesia during exposure, and animal awoke from anesthesia without any apparent toxicity
. Post-exposure sleeping time was slightly greater for compound 903 compared with 873
. Altho ugh not lethal under these conditions, compound 707 had apparent nervous system effects as evidenced by tremors during exposure and abnormal muscular cont ractions postmortem. Compound 1116 was also nonlethal, but animals did not achieve a normal clinica l appearance after exposure, and there was some lung toxicity associated with exposure. . The remaining four comp 0 unds induced some let hality and were associate d with lung toxicity. Lung toxic ity was typified by increased lung-to-body weight ratios at necropsy, and appearance of mottling and/or failure to fully collapse when the tho rax was opened. The appearance of frothy material at the nares and fai lure ofthe lung to fully collapse are consistent with pulmonary edema. Both pulmonary edema and congestion will contribute to an increased lung-to-body weight ratio. An increased incidence of abnormally darkened livers and increased liver weights are indicative ofhepatic congestion. One of 10 rats exposed to compound 1358 died**~6** min after exposure, and at necropsy lu ngs were moderately to markedly affected
. Seven of 10 rats ( 5 of 5 males and 2 of 5 fema l es) exposed to compound 872 died eith..:r at the end ofexposure or after exposure, and at necropsy both lungs and livers exhibited ab normal appearance . The other two compounds, 1359 and 1360, resulted in 100% lethality upon exposure (either during or immediately after exposure), and at necropsy lungs had marked mottling and livers were abnormal in co l or.