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Toxicity Summary of 2‐Bromo‐3,3,3‐Trifluoropropene

Prepared By: Stephanie Skaggs, Universal Technical Services Inc., and Bradford Colton, American 

Pacific Corp. 

Date: November 26, 2013 

As of November 2013, the following toxicological, aquatic and environmental studies were 

conducted on 2-bromo-3,3,3-trifluoropropene (BTP) since 1999 and reports have been issued: 

 Acute (30-min) inhalation toxicity study in rats 

 Acute (4-hr) inhalation toxicity study in rats 

 Acute (10-min) cardiac sensitization study in dogs 

 Skin irritation study in rabbits 

 Eye irritation study in rabbits 

 In Vitro genotoxicity studies: 

o Salmonella typhimurium and Escherichia coli mutagenicity 

o Chromosomal aberration in human lymphocytes 

o Mouse lymphoma L5178Y cells 

 Repeated exposure 14-day inhalation toxicity studies in rats 

 90-day subchronic inhalation toxicity study in rats 

 Reproductive toxicity screening test in rats 

 Physiologically based pharmacokinetic (PBPK) modeling 

 Activated sludge respiration inhibition (ASRIT) 

 Assessment of ready biodegradability 

 Abiotic Degradation 

All animal studies were conducted using acceptable practices of good animal husbandry and 

principles of Good Laboratory Practice (GLP) were followed. More complete details of materials, 

methods, and results are available from the references cited at the end of this paper. 

Acute Toxicity

Male and female rats were exposed to BTP for 30 minutes at a concentration of 44,000 ppm, and 

then observed for a subsequent 2 hours post exposure.1

 The animals were then sacrificed and the 

major organs were examined. The rats experienced relaxed breathing and apparent sedation during 

the exposure both of which returned to normal within 10-minutes during the post-exposure recovery 

period. There was no mortality during the testing and all organs appeared normal after sacrificing the 

animals. 

Additional acute toxicity testing was performed in rats exposed to BTP for four hours to a target 

concentration of 25,000 ppm and 5000 ppm.2

 Observations were performed during the exposure 

periods as well as following the exposure for up to 14 days. Macroscopic examination of selected 

organs was done after death or sacrifice and the lungs tissues were examined microscopically. 

Exposure to 26,580 ppm results in mortality of all the rats either during the exposure or as a result of 

euthanasia for humane reasons. None of the animals died during or after the exposure to 5,173 ppm. 

The 4-hour LC50 (95% confidence limits) for both sexes was calculated to be 11,726 ppm. The rats 

experienced labored breathing during the exposure with clinical signs of nasal discharge, excessive 
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salivation, and moist rales. Gasping and decreased motor activity was noted at the higher 

concentration. There was no apparent effect on the organs weighed. In the higher concentration 

group, all rats had bronchiolar lesions consisting of desquamated epithelium, inflammation with 

intra-luminal debris ranging from slight to moderate in severity. Alveolar macrophages were also 

present in the higher dose group. 

Cardiotoxicity

The cardiac sensitization (CS) potential of BTP was investigated by Huntingdon Life Sciences Ltd. 

in 2002.3 The CS study was conducted according to the experimental procedure described by 

Reinhardt, et al.4

 Beagle dogs were exposed to nominal concentrations of 5,000, 10,000 or 15,000 

ppm BTP for the 10-min exposures. Individual responses to epinephrine alone were selected for each 

dog prior to the testing regimen for the purpose of establishing an epinephrine dose at which there 

was a clear but minimal effect on the electrocardiogram (ECG), ideally with a few ectopic beats. The 

range of intravenous doses used was 1 to 12 μg epinephrine per kg body weight. There were no 

indications of cardiac sensitization to epinephrine at target BTP concentration of 5000 ppm. At 

10,000 ppm BTP, one out of six dogs gave a positive cardiac sensitization response. At 15,000 ppm, 

one dog responded positively with an episode of multi-ventricular ectopic activity approximately 

thirteen seconds after the second epinephrine challenge. Clinical signs included agitation, deep slow 

breathing and muscle tremors. These results indicate that the cardiac sensitization LOAEL for BTP is 

10,000 ppm and the NOAEL is 5,000 ppm. 

Skin Irritation		

Three rabbits received a single four-hour, occlusive, dermal administration of BTP and were 

observed for four days.5

 Approximately 0.5 ml of BTP was applied under a 2-ply 25 mm x 25 mm 

porous gauze pad secured with ‘blenderm’ surgical tape to intact skin sites on three animals. An 

additional skin site was similarly treated with the exception of test substance and acted as its own 

animal’s control. Each treatment site was covered with a square piece of aluminum foil, all four sides 

of which were secured to the skin by impermeable surgical tape (Blenderm). No dermal reaction was 

observed in any animal throughout the duration of the study. There was no sign of toxicity or ill 

health in any rabbit during the observation period. According to the criteria of the Globally 

Harmonised System and European Commission regulation 1272/2008, BTP does not require any 

classification and labeling related to skin irritation. 

Eye Irritation		

A study was performed to test the eye irritation potential of BTP in rabbits.6

 The study design was 

based on the OECD Guideline for Testing Chemicals No. 405. Three rabbits were each administered 

a single ocular dose of a volume of 0.1 mL of BTP and observed for four days after instillation. No 

sign of reaction to treatment was apparent in any animal at any time during the observation period. 

Instillation of BTP gave rise to practically no or a slight pain response. According to the criteria 

developed by Kay and Calandra,7

 BTP was classified as “non-irritating” to the eye and did not 

require labeling in accordance with the Globally Harmonised System and European Commission 

regulation 1272/2008. 
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Genotoxicity Results

BTP was tested for mutagenic potential in an in vitro mammalian cell mutation assay.8

 This test 

system is based on detection of a forward mutation in the mouse lymphoma L5178Y cells, from the 

heterozygous condition at the thymidine kinase locus (TK+/-) to the thymidine kinase deficient 

genotype (TK-/-). Following 3-hour treatment in the absence and presence of S9 mix, there were no 

increases in the mean mutant frequencies of any of the test concentrations assessed that exceeded the 

sum of the mean concurrent vehicle control mutant frequency and the Global Evaluation Factor 

(GEF), within acceptable levels of toxicity. It was concluded that BTP did not demonstrate 

mutagenic potential in this in vitro cell mutation assay, under the experimental conditions described. 

In genotoxicity test using four strains of Salmonella typhimurium (TA97a, TA98, TA100, and TA 

1535) and one strain of Escherichia coli (WP2 uvrA) in the presence and absence of an exogenous 

metabolic activator, BTP showed no evidence of mutagenic activity at concentrations ranging from 5 

to 5000 μg/plate.9

 No substantial increases in revertant colony numbers of any of the bacterial 

strains were observed following BTP treatment at any dose level, in the absence or presence of 

S-9 (prepared rat liver fraction of stimulated mixed function oxidase system). Concurrent positive 

control compounds demonstrated the sensitivity of the assay and the metabolizing activity of the S-9 

preparations. 

BTP was evaluated for in vitro clastogenic (chromosome-damaging) potential in human peripheral 

blood lymphocytes in the absence and presence of an exogenous metabolic activation system.10 An 

initial cytotoxicity/chromosome aberration assay was conducted to establish the dose range for 

testing. Dose levels of 0.088 to 0.625 μL/mL were selected for mitotic index analysis. No 

statistically significant increases were observed in the proportion of aberrant cells treated with BTP 

compared to the solvent control cultures in the absence or presence of S-9. Positive control 

compounds caused large statistically significant increases in the proportion of aberrant cells, 

demonstrating the efficacy of the S-9 fraction and the sensitivity of the test system. 

Repeated Exposure Toxicity Tests

14‐day Inhalation Toxicology Tests

In a range-finder study, groups of three male and three female rats were exposed to BTP at target 

exposure levels of 0, 5,000, 10,000 or 20,000 ppm for 6 hours/day, 5 days/week for a total of 2 

weeks.11 During the study, clinical condition, detailed physical and arena observations, sensory 

reactivity, grip strength, motor activity, bodyweight, food consumption, organ weight, 

macropathology and histopathology investigations were undertaken. None of the test animals died 

during or after the exposure to any BTP concentration tested. Clinical signs were those consistent 

with exposure to a respiratory irritant and mild sedating compound. Bodyweight losses were evident 

in male rats and to a lesser extent in females exposed to the highest concentration, but only in the 

males at the lower two BTP concentrations. There were no test chemical related effects on organ 

weights. At necropsy, a reduction in the size of the thymus and depressions or roughening of the 

stomach was evident in animals exposed to 20,000 ppm. Histopathological changes related to BTP 

treatment were seen in the nasal turbinates, larynx and trachea. Overall, the most marked changes 
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were in a dose-response of the nasal cavity, with less severe changes in the larynx and trachea. A 

NOAEL was not determined due to irritant effects in the nasal turbinates at all concentrations tests. 

A second 14-day study was also conducted at lower concentrations to determine a NOAEL.12 Target 

concentrations of 500, 1200 and 3000 ppm were selected for whole-body exposure of three male and 

three female rats per concentration for 6 hours a day, 5 days a week, for 2 weeks. Transient 

treatment-related clinical signs were evident during and after exposure. Impaired bodyweight 

gain was evident, over the 2 week treatment period, for treated males in a dose-related manner. 

Histopathological changes related to treatment were seen mainly in the nasal turbinates and, to a 

much lesser extent, in the larynx, trachea and lungs of rats exposed to 1171 or 2980 ppm. The no 

observed adverse effect level (NOAEL) was 533 ppm, as this level was not associated with any 

histopathological changes or adverse in-life findings. 

90‐day Inhalation Toxicology Tests

A 90-day repeat exposure study was undertaken in CD rats for 13 weeks followed by a 4-week 

recovery period.13 Six groups of ten male and ten female rats were exposed by whole-body 

inhalation to nominal concentrations of 0, 200, 500, and 3,000 ppm and 0 or 3000 ppm with a 4 week 

recovery period after the 13 week exposure. There were no deaths directly related to the exposure to 

BTP at any dose level studied. Treatment-related, but transient, clinical signs, related to inhalation of 

an irritant material or possible CNS effects, were evident during exposure at all concentrations. 

Reductions in grip strength and motor activity were evident for animals exposed to the higher two 

concentration were seen and only partially reversible for animals previously exposed to 3000 ppm 

after a 4 week recovery period. Lower bodyweight gain was evident, over the 13 week treatment 

period, for treated males and females exposed to 500 ppm or higher, in a dosage related manner. 

Total food consumption was similarly lower for treated males and females exposed to 3000 ppm. 

Effects on bodyweight and food consumption showed evidence of recovery for animals previously 

exposed to 3000 ppm after a 4 week recovery period. Histopathological changes were seen in the 

liver (centrilobular hepatocyte hypertrophy), pancreas (acinar cell degranulation), heart (myocardial 

inflammatory cell infiltration/ fibrosis), spleen (capsular inflammation and/or thickening and 

adhesion), thymus (involution/atrophy), nasal turbinates (findings related to minor local irritants), 

larynx (ventral squamous metaplasia) and teeth (pulp cavity necrosis). The majority of these findings 

were seen with animals at 500 and 3000 ppm. Chronic inflammation in the heart and acinar cell 

degranulation in the pancreas were evident for animals exposed to 200 ppm, but these changes were 

not considered adverse. Following the 4 week recovery period, there was complete recovery of test 

material related histopathological changes seen in the liver, pancreas, heart, thymus, larynx and teeth 

and partial recovery for the findings see in the spleen (capsular inflammation and/or thickening) and 

nasal turbinates (atrophy/disorganization/vacuolation of the olfactory epithelium and nasolacrimal 

duct inflammation). Because of the reversibility of effects and the non-adverse nature, the NOAEL 

was determined to be 200 ppm.

Reproductive Toxicity

A whole-body inhalation tests was performed in CRL:CD (SD) rats.14 Three groups of ten male and 

ten female adult rats received nominal concentrations of 200, 500, or 3000 ppm for 6 hours per day, 7 

days per week. A control group receiving only air underwent the same study procedures. Exposures 

occurred for 15 days prior to pairing, through pairing, and then up until Day 10 after the birth of the 

offspring. The offspring did not receive direct exposure, only in utero and possibly through 
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lactation. Clinical conditions, body weight, food consumption, macroscopic findings, microscopic 

pathology, and fertility parameters were examined in all groups. Dose-related clinical signs during 

exposure were underactivity, unresponsiveness, piloerection and partially closed eyelids; these 

clinical signs were reversible. Treated animals had lower bodyweight gain and lower food intake 

compared to controls. There was a dose-related lengthening of the estrous cycles and gestation 

periods. At the highest dose, more females showed irregular cycles or being acyclic and there was 

reduced fertility, smaller size of the corpora lutea, lower post-implantation survival rate, live birth 

index, and live litter size, with only one female littering and the offspring subsequently dying. In the 

500 ppm treated group, there was a lower post implantation survival, live birth index, and live litter 

size, with only half of the litters surviving to Day 10 of lactation. At the lowest dose, there was a 

slightly lower implantation rate where two out of 10 litters experienced lower litter size and viability, 

but the remaining litters were within normal ranges. There were no treatment-related effects on the 

offspring that survived to Day 10 of lactation, nor were there any gross physical abnormalities in any 

of offspring. Dose-dependent effects on sperm analysis included reduced sperm velocity (but not 

motility), reduced sperm count (highest dose only), and irregular sperm morphology (did not affect 

sperm vigor). Macroscopic examination of male reproductive organs indicated reduced prostate, 

epididymides, seminal vesicle and pituitary weights. Because there were effects seen even at the 200 

ppm level, a clear NOAEL was not determined. A follow-up reproductive study is being undertaken 

at lower concentrations to determine a NOAEL. 

Physiologically Based Pharmacokinetic (PBPK) Modeling

Physiologically based pharmacokinetic (PBPK) modeling was performed using an Environmental 

Protection Agency (EPA)-approved model that was developed for halon alternatives used in fire 

suppression.15 Arterial blood concentrations were measured in a cardiac sensitization-like test 

protocol, except the canines were not delivered the epinephrine challenge.16 Arterial blood samples 

were taken at timed intervals during and after canines were administered 1%vol BTP (cardiac 

sensitization LOAEL) by inhalation to determine the concentration-time profile of BTP in the blood 

that is then used in the PBPK model. The acceptable arterial blood concentration for BTP at the 

LOAEL was determined to be 30.6 mg/L, based on the lowest 5-minute arterial concentration. The 

human and rat partition coefficients for BTP used as inputs to the PBPK model were measured by 

Battelle, Pacific Northwest Division.17 The 5-minute PBPK allowable concentration is defined as 

the highest constant concentration level that a human may be exposed to for 5 minutes without 

exceeding the acceptable arterial blood concentration. The 5-minute PBPK allowable concentration 

was determined to be 0.95%vol. The FAA provides safe use guidance for exposure from the 

discharge of agent from handheld extinguishers onboard aircraft that is based on PBPK modeling. 

Data tables were created matching the format used by the FAA for guidance.15

Aquatic and Environmental Studies

In addition to the studies listed below, the Acute Toxicity to Rainbow Trout, Acute Toxicity to 

Daphnia Magna, and Algal Growth Inhibition Assay studies have been completed and test reports 

are not yet complete. These reports will be submitted to the EPA when complete. 
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Activated sludge respiration inhibition (ASRIT)		

A preliminary investigation was conducted to assess if test concentrations of BTP could be 

adequately maintained under the conditions of the activated sludge respiration inhibition test.18

Results of the analytical measurements show that at the start of the test, recoveries of the test material 

were between 42% and 71% of nominal. At the end of the incubation phase measured test levels had 

decreased to <1% of nominal. The loss of the test material was so significant, that if the test 

substance was used in a definitive test, concentrations would not have been maintained during the 

exposure phase. Due to the volatile nature of the test compound and the losses observed during the 

preliminary investigations it would not be expected that sewage micro-organisms would be impacted 

by this compound. 

Assessment of Ready Biodegradability

Biodegradability was assessed in the Sealed-Vessel CO2 Evolution test.19 Test, control and reference 

mixtures were incubated for 28 days on a reciprocating shaker at a temperature of 22 2C. After 28 

days, the mean carbon dioxide (CO2) production by the controls was 13.4% of the applied organic 

carbon. Production of CO2 by mixtures containing the test substance was equivalent to, at most, 1.1% 

of the theoretical maximum by Day 21. Substances are considered to be readily biodegradable if CO2 

production is equal to or greater than 60% of the theoretical value within ten days of the level 

achieving 10%. Therefore, BTP was considered not to be readily biodegradable under the conditions 

of the test. 

Abiotic Degradation

A study was performed to determine the rate of hydrolysis of BTP as a function of pH. The 

preliminary study showed that at each of pH 4, 7, and 9 at 50 0.5oC, less than 10% hydrolysis had 

occurred after 5 days, equivalent to a half-life of greater than 1 year under environmental conditions 

(25°C). BTP was determined to be hydrolytically stable under acidic, neutral and basic conditions.20

 

1

 Finch, G. L., et al. Acute Inhalation Toxicity of Candidate Halon Replacement Compounds in Rats, Lovelace 

Respiratory Research Institute, June 15, 1999. 

2

 Hoffman, G. M., 2-Bromo-3,3,3-Trifluoropropene, E-1-Bromo-3,3,3-Trifluoropropene and 1-Bromo-2-

Trifluoromethyl-3,3,3-Trifluoropropene: An Acute (4-Hour) Inhalation Toxicity Study in the Rat Via Nose-Only 

Exposure, Huntingdon Life Sciences, 02-5449, January 14, 2004. 3

 Horrell, P.J., et al., Agent 873: An Inhalation Study to Investigate the Cardiac Sensitization Potential in the Beagle 

Dog, Huntingdon Life Sciences, AAS 001/014630, September 12, 2002. 4

 Reinhardt, et al., Cardiac Arrhythmias and Aerosol Sniffing, Arch. Env. Hlth., 22: 265-279, 1971. 5

 Rees, P. B., 2-Bromo-3,3,3-trifluoropropene: Skin Irritation to the Rabbit, Huntingdon Life Sciences, WAG0010, 

September 27, 2012.

6

 Rees, P. B., 2-Bromo-3,3,3-trifluoropropene: Eye Irritation to the Rabbit, Huntingdon Life Sciences, WAG0011, 

October 1, 2012.

7

 Kay J.H., Calandra J.C. Interpretation of eye irritation tests. J. Soc. Cosmet. Chem. 13: 281–289, 1962. 
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 8

 Woods, I., 2-bromo-3,3,3-trifluoropropene: In Vitro Mutation Test Using Mouse Lymphoma L5178Y Cells, 

Huntingdon Life Sciences, WAG0017, July 24, 2012. 

9 Gladnick, N. L., AAWG #873, AAWG #903, and AAWG #1116: Bacterial Reverse Mutation Test in Salmonella 

typhimurium and Escherichia coli, DuPont Pharmaceuticals Company, Laboratory Project ID: DuPont-3908, 

February 29, 2000.

10 Curry, P. T., AAWG #873: In Vitro Chromosome Aberration Test in Human Peripheral Blood Lymphocytes, 

DuPont Pharmaceuticals Company, Laboratory Project ID: DuPont-4436, November 20, 2000. 11 Horrell, P.J., et al., 2-Bromo-3,3,3-trifluoropropene: Dose Range Finding Study by Inhalation Administration to 

Rats for 2 Weeks, Huntingdon Life Sciences, WAG0013 Report, August 21, 2012. 12 Horrell, P.J., et al., 2-Bromo-3,3,3-trifluoropropene: Dose Range Finding Study by Inhalation Administration to 

Rats for 2 Weeks, Huntingdon Life Sciences, WAG0024 Report, August 31, 2012. 13 Horrell, P.J., et al., 2-Bromo-3,3,3-trifluoropropene: Toxicity Study by Inhalation Administration to CD Rats for 

13 weeks Followed by a 4-Week Recovery Period, Huntingdon Life Sciences, WAG0014, October, 2013. 14 Brooker, A.J., et al. 2-Bromo-3,3,3-trifluoropropene: Reproductive/Developmental Toxicity Screening Study in 

the CD Rat by Inhalation Administration, Huntingdon Life Sciences, WAG0015 Report, July 2013. 15 Colton, B. and Poet, T. Setting Acute Exposure Limits For The Halotron® Brx (2‐Bromo‐3,3,3‐Trifluoropropene) 

Clean Agent Onboard Aircraft Using Physiologically Based Pharmacokinetic Modeling, American Pacific 

Corporation and Battelle, Pacific Northwest Division, October 25, 2013. 

16 Horrell, P.J., et al., 2-Bromo-3,3,3-trifluoropropene: Measurement of Arterial Blood Levels Following Inhalation 

Administration to Beagle Dogs, Huntingdon Life Sciences, WAG0016 Report, September, 2013. 17 Poet, T. Determination of Tissue to Air Partition Coefficients for 2-Bromo-3,3,3-Trifluorpropene (Halotron BrX),

Battelle, Pacific Northwest Division, Project Number 64197, April 2013. 

18 Davies, J., et al., 2-bromo-3,3,3-trifluoropropene:Activated Sludge Respiration Inhibition Test, Huntingdon Life 

Sciences, WAG0020 Report, October 21, 2013. 

19 Dickinson, R.A., 2-bromo-3,3,3-trifluoropropene: Assessment of Ready Biodegradability - Sealed-Vessel Carbon 

Dioxide Evolution Test, Huntingdon Life Sciences, WAG0021 Report, April 30, 2013. 20 Sydney, P., 2-Bromo-3,3,3-Trifluoropropene Abiotic Degradation: Hydrolysis as a Function of pH (Preliminary 

Test), Huntingdon Life Sciences, WAG0007 Report, July 26, 2012.