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 Investigator 

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 of quality 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 of the 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, CF3CH=CHBr 

872 3-Bromo-3,3-difluoropropene, CFzBrCH=CHz 

873 2-Bromo-3,3,3-trifluoropropene, CF3CBr=CHz 

903 4-Bromo-3,3,4,4-tetrafluorobutene, CFzBrCFzCH=CHz 

1116 2-Bromo-3,3,4,4,4-pentafluorobutene, CF3CF2CBr=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=CH2 
FY99003 Final Report 

June15, 1999 page 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 female, F344/Crl (Charles River Laboratories), 10-

12 wk of age at time of exposures. Animals were received and quarantined for - 2 wk 

prior to use. Animals were weighed 5 days before the first exposure and randomly 

assigned to groups by weight (using Excel). Identification was by tail markings. 

Animals were housed in polycarbonate shoebox-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 of the exposure system is shown as Figure 1. Room barometric 

pressure and chamber temperatures were checked daily and used to calculate the quantity 

of compound required for a 5% air concentration, using the following equation: 

Mass =((90L*(Pressure/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 again 

after each exposure to establish the total quantity of compound injected during the 30 

minute exposures. The chamber flow rate of 3 L/minute was verified each day of . 

exposures as was the pressure integrity of the system. 

Material was metered into a heated J-tube device partially filled with glass beads. 

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 content, temperature, 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 of vapor concentration. The order of testing of the compounds was from lowest 

to highest boiling point. 
FY99003 Final Report 

June 15, 1999 

E. Animal Procedures: 

page 3 of8 

Rats were loaded into exposure tubes and transported to the exposure 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 overdose of sodium pentobarbital. A gross necropsy was 

conducted, including collection of terminal 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 compositions. Targeted weights 

refer to the calculated amounts of each 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 volumetric flow rate of the exposure line 

(verified at the beginning of each day of testing) and the total weights of compound 

delivered (determined by weighing the syringe before and after each exposure), and the 

duration of each exposure. Sampling of the exposure chamber air was intended to 

provide additional means of verifying the 5% exposure level failed to perform as 

expected. The sampling performed employed a piston pump whose output was regulated 

at between 50 to 75 cc's per minute by a rotameter. The output of the 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 

Weight Delivered Target 

(g) (g) cone. 

873 2-Bromo-3 ,3) -trifluorcpropene 26.95 26.96 100 

707 1-Bromo-3 ,3,3-trifluoropropene 26.95 23.73 1 1001 

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-Brorno-3 3,4,4,4-pentafluorobutene 34.43 34.30 100 

1360 2-Bromo-3,4,4,4-tetrafluoro-3- 44.10 44.01 100 

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 of the 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 

1.7 
FY99003 Final Report 

June 15, 1999 page 4 of8 

exposure time. Concentration is based on the amount of compound 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 period. Survival during this period is also summarized. 

Controls: 

Normal during 30.min exposure. Occasional movement in tubes. Good pink 

color of extremities 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 appeared normal (correlated with period! of time syringe pump 

had apparent partial clog). After 15 min tremors developed; most noticeable in hind 

limbs. By 20 min tremors had increased and 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 respiration (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 min breathing rate was very slow. Rats were not 

moving in tubes and appeared to be anesthesized. At 22 min rats continued having 

labored breathing and were pale. D037 was barely breathing at 27 min of exposure and 

was dead at end of exposure. 

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 died 
FY99003 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 good, 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 slowed and animals appeared to be 

anesthesized. By 20 min F058 was breathing very slowly, paws were pale purple 

indicating insufficient oxygenation. 

By 5 min post-exposure rats were waking and had labored breathing; labored 

breathing continued to 20 min post-exposure. Respiration continued in this manner until 

sacrifice. 

Compound 1360: 

Excitement and agitation was observed at 3 min of exposure, slight agitation 

continued to 10 min of exposure. By 12 min there was still movement, and rats had slow 

deep breathing. At 15 min G061 was turned in the tube and dead. Additional deaths 

were G066 at 20 min of exposure, G064 at 21 min of exposure, G069 at 22 min of 

exposure, G068 at 24 min of exposure; also, G067, G062, and G070 all appeared dead at 

this time. G063 appeared dead at 28 min of exposure. 

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 of exposure. 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 

lethargic. By 20 min post-exposure H075 was moving about. H075 died 36 min after 
FY99003 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 of exposure. 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 oxygenation. By 13 min most animals 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. dead/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 

1358 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, 1999 

Compound 872: 

page 7 of8 

Several of the rats had foam/frothy discharge at the nose. Lungs did not fully 

collapse upon opening the thorax. Lungs for several of the rats were mottled red. 

Several of the rats had air in the stomach consistent with gasping. Livers were generally 

darker than normal. Rats in this group had elevat ed lung-to-body weight ratios compared 

with controls, and slightly elevated liver weights. 

Compound 903: 

All rats normal. 

Compound 1116: 

Lungs were generally mottled and patchy red to dark red in appearance. Lungs 

did not fully coliapse upon opening the thorax. ·Several of the animals had air in the 

stomach. Lung-to-body weight ratios were elevated compared with controls. Livers 

appeared normal. 

Compound 1360: 

Lungs were moderately to markedly mottled dark red. Lungs from only one rat 

did not fully collapse upon opening the thorax. Livers were darker red than is normal. 

Lung-to-body weight ratios and mean liver weights were increased compared to controls. 

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 compound delivered, the chamber air flow, 

and the duration of each exposure. 
FY99003 Final Report 

June 15, 1999 

B. Compound 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 innocuous 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. 

Although not lethal under these conditions, compound 707 had apparent nervous 

system effects as evidenced by tremors during exposure and abnormal muscular 

contractions postmortem. Compound 1116 was also nonlethal, but animals did not 

achieve a normal clinical appearance after exposure, and there was some lung toxicity 

associated with exposure. . 

The remaining four comp0unds induced some lethality and were associated with 

lung toxicity. Lung toxicity was typified by increased lung-to-body weight ratios at 

necropsy, and appearance of mottling and/or failure to fully collapse when the thorax was 

opened. The appearance of frothy material at the nares and failure of the 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 of hepatic 

congestion. One of 10 rats exposed to compound 1358 died ~6 min after exposure, and at 

necropsy lungs were moderately to markedly affected. Seven of 10 rats ( 5 of 5 males and 

2 of 5 females) exposed to compound 872 died eith..:r at the end of exposure or after 

exposure, and at necropsy both lungs and livers exhibited abnormal 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 color.