1 

HUMAN HEALTH SAFETY EVALUATION OF 

HALON REPLACEMENT CANDIDATES 

Darol E. Dodd1

, Gary W. Jepson1, and Joseph A. Macko, Jr.2 

1

Air Force Research Laboratory/Operational Toxicology Branch (AFRL/HEST) – 

ManTech Environmental Technology, Inc. 

Wright-Patterson Air Force Base, Ohio 

2

Toxicology Directorate (MCHB-TS-T)/U.S. Army Center for Health Promotion 

& Preventive Medicine 

Aberdeen Proving Ground, Maryland 

Next-Generation Fire Suppression Technology Program (NGP) 

Project 3B/1/89 

Final Report – Section II of II 

This research is part of the Department of Defense’s Next-Generation Fire Suppression 

Technology Program, funded by the DoD Strategic Environmental Research and 

Development Program (SERDP) 
2 

TABLE OF CONTENTS 

Page 

INTRODUCTION..............................................................................................4 

PHASE 1 – TOXICITY SCREENING METHODS.......................................................5 

Chemical/physical properties.......................................................................6 

Existing toxicity literature...........................................................................6 

Preliminary “specific use” scenarios...............................................................7 

Qualitative/quantitative structure activity relationships.........................................7

In vitro screens........................................................................................8 

Acute irritation tests.................................................................................9 

Acute toxicity tests..................................................................................10 

 The “limit test”.............................................................................11 

 The “specific use” scenario test..........................................................12 

Genotoxicity tests (Phase 1).......................................................................13 

DECISION POINT 1..............................................................................14 

Concentration x time (C x T) relationships...........................................18 

Stop or continue testing..................................................................18 

PHASE 2 – TOXICITY TESTING.........................................................................19 

Cardiotoxicity study................................................................................20 

 Linking PBPK modeling to cardiac sensitization potential.........................22 

14-Day dose range-finding study................................................................23 

Genotoxicity tests (Phase 2)......................................................................24 

DECISION POINT 2..............................................................................24 

 Stop or continue testing.................................................................26 

PHASE 3 – TOXICITY TESTING.........................................................................26 

90-Day subchronic study..........................................................................26 

Genotoxicity tests (Phase 3)......................................................................27 

DECISION POINT 3.............................................................................28 

 Stop or continue testing................................................................29 
3 

TABLE OF CONTENTS (continued)

Page 

PHASE 4 – TOXICITY TESTING........................................................................30 

Developmental toxicity study.....................................................................30 

Reproductive toxicity study........................................................................31 

Other studies.........................................................................................31 

SUMMARY OF TOXICITY TESTING PROGRAM...................................................32 

ACKNOWLEDGEMENTS.................................................................................37 

REFERENCES................................................................................................37 

APPENDICES 

Appendix A – Protocol for the Genotoxicity Screen of Volatile Compounds.............41 
4 

HUMAN HEALTH SAFETY EVALUATION OF 

HALON REPLACEMENT CANDIDATES 

INTRODUCTION

Environmental concern over the depletion of stratospheric ozone and global warming has led to 

an international treaty called The Montreal Protocol which calls for the phase out of halons by the 

year 2000. The services within the Department of Defense (DoD) are directed to determine and 

evaluate suitable halon replacement candidates that will optimize performance of missionessential equipment and operations. Part of the evaluation process is to select halon replacement 

candidates that will be in compliance with environment, health, and safety considerations. The 

U.S. Environmental Protection Agency (EPA), under the Clean Air Act, is also directed to 

evaluate and regulate substitutes for ozone-depleting chemicals that are being phased out (Final 

Rule, Federal Register 59: 13044, March 18, 1994). The EPA program that provides these 

determinations is called the Significant New Alternatives Policy (SNAP) program. DoD and 

EPA share a common objective in identifying halon replacement candidates, that begin, to select 

substitutes that offer low (or lower) overall risks to human health and the environment. 

The purpose of this paper is to provide a strategy to human health safety evaluation of halon 

replacement candidates. This is necessary, because there are budget and time constraints 

associated with the identification and development of halon replacements. Decisions have to be 

made to meet project or program goals, yet information is not readily available to assist in making 

decisions on the toxicity evaluation of potential chemical substitutes. This paper will provide a 

stepwise approach in building a chemical toxicity database, specific for halon replacement 

candidates. Information will be provided on the types of toxicity tests or studies to be considered, 

their strengths, weaknesses, costs, and evaluation time. Suggestions will be provided when more 

than one choice arises and a decision has to be made. It is unlikely that a chemical substitute will 

require completion of all parts of the toxicity evaluation process. Once the evaluation process is 

stopped, information will be given on how valuable the database is in terms of uncertainty and 

risk to human health. The strategy described is based on the experience and opinions of the 

authors. Consideration of our comments and recommendations does not assure that no additional 

toxicity testing or research is needed on a halon replacement candidate to provide a complete 

assessment of the overall risks to human health. 
5 

PHASE 1 – TOXICITY SCREENING METHODS

Eight endpoints will be described in the first phase of toxicity evaluation. The endpoints are 

chemical/physical properties, existing toxicity literature, preliminary “use” scenarios, 

qualitative/quantitative structure activity relationships, in vitro screening, acute irritation, acute 

toxicity, and genotoxicity of the halon replacement candidate. Animal or cell studies are not 

required for the first four endpoints, but they are necessary to determine and evaluate a 

candidate’s potential to cause irritation, toxicity, or mutagenicity, unless this information is 

already reported in the literature. Figure 1 is a flow diagram of toxicity testing during Phase 1. 

Details follow on the approach and direction to be taken. 

Figure 1. Flow Diagram - Phase 1 Toxicity Screening Methods

Chemical/physical

properties; existing

toxicology literature;

define specific use

scenario; SARs; in

vitro screens

Acute Irritation

Test

(Liquids/Solids)

Corrosive

Negative

or Mildly

Irritating

Acute 

Toxicity Test

Specific Use

Scenario Test

Decision Point 1 - Stop or Continue to Phase 2 Testing 

Highly

Toxic

Low to

Moderate

Toxicity

Genotoxicity

Test

(Bacteria)

Highly

Mutagenic

Negative

or Mildly

Mutagenic
6 

General chemical/physical properties. Chemical structure is the necessary basis for chemical 

identity. The Chemical Abstracts Service (CAS) registry number is used most frequently to 

associate chemical structure with chemical identity. If a chemical’s structure is unknown, 

analytical methods, such as mass spectroscopy, infrared spectroscopy, nuclear magnetic 

resonance spectroscopy, and gas chromatography may provide valuable information to establish 

chemical structure. Experimental data for physical-chemical properties (i.e., physical state, 

melting point, boiling point, vapor pressure, solubility, octanol-water partition coefficient, 

viscosity, etc.) are necessary for characterization of the chemical. These data may be obtained 

experimentally or by use of computational chemistry tools. In general, the physical-chemical 

properties of a chemical dictate the likely route of human exposure (e.g., inhalation route for 

highly volatile chemicals and gases) and, thus, the likely choice of route of administration of the 

chemical for toxicity testing in laboratory animals. Solubility properties are especially important 

for liquid or solid chemicals that may require a vehicle for dosing either animals or cell/tissue 

preparations. Some assessment of the stability of the chemical under long-term storage 

conditions and as a prepared stock solution is necessary to assure meaningful testing results. 

Nyden and Skaggs (1999) discuss additional physical-chemical properties that are most relevant 

to halon replacement candidates. These include environmental impact properties (e.g., ozone 

depletion potential, global warming potential, and atmospheric lifetime) and materials 

compatibility (e.g., interaction of the halon replacement candidate with metals, gaskets and 

lubricants, and the compatibility of the candidate and its combustion by-products with potentially 

exposed weapons systems). 

Existing toxicity literature. Given the complexity of health endpoints and the expense in 

conducting toxicology testing, a literature search on the halon replacement candidate or 

structurally related analogs to the candidate is extremely useful. Valid analogs should have close 

structural similarity and the same function groups. Structurally related chemicals are likely to 

have similar physical-chemical, environmental, and toxicological properties or follow a 

predictable pattern of effects. Similarities of chemicals may be based on the following list 

adopted from http://www.epa.gov/chemrtk/categuid.htm

• A common functional group (e.g. aldehyde, epoxide, ester, etc.); or 

• The likelihood of common precursors and/or breakdown products, via physical or 

biological processes, which result in structurally similar chemicals; and 

• An incremental and constant change across a group of structurally related chemicals. 
7 

Nyden & Skaggs (1999) present a straightforward, yet thorough approach for conducting a 

literature search to assess toxic properties of candidate fire suppressants. Most database services 

are free of charge, though a cost may be incurred when ordering reports and documents. The 

importance of a thorough literature search cannot be underestimated, since the information 

obtained will provide overall guidance and direction on all subsequent toxicity testing. 

Information gained from a literature search may eliminate the need for select toxicity testing, thus 

reducing the overall cost of testing. 

Preliminary “specific use” scenarios. In general, as halon replacement candidates are being 

developed, they are being investigated as potential total flooding agents or streaming agents. 

Some candidates may also be considered for the replacement of other CFCs that are currently in 

use for other purposes (e.g., refrigerants, solvents, etc.). Knowing the potential end-use of the 

replacement chemical helps define the preliminary exposure scenario. This information, in turn, 

helps guide the type of toxicity testing that would be of greatest benefit for determining health 

consequences. For example, a replacement for a total flooding agent suggests an exposure 

scenario of high concentration (i.e., fire extinguishing levels) for brief periods of time (i.e., few 

minutes) under static air (i.e., defined volume) conditions. A replacement for a streaming agent 

suggests a very different exposure scenario, where specific use conditions include dynamic air 

(i.e., outdoors) movement. In this case, chemical concentrations in the breathing zone of the user 

may be only moderate in value. Examples of how this information applies to toxicity testing 

strategy follows in subsequent sections of this document. Keep in mind that there are several 

more exposure scenarios that have to be considered by regulatory agencies when assessing 

potential human health risk. For example, the accidental release of a chemical during normal 

handling, transportation, or storage procedures is often evaluated for potential harm.

Qualitative/quantitative structure activity relationships. Quantitative structure-activity 

relationships (QSARs), based on scientific judgements by experienced toxicologists, may be used 

as an integral part of health hazard characterization. This approach relies on the toxicologist or 

chemist being able to fit the new chemical into a category of existing chemicals because of 

similarities in molecular structure or chemical functionality. In order for this approach to be of 

value, the existing chemicals category or close structural analog must have its own robust 

toxicology database and the uncertainty of read-across from the close structural analogs or the 

category to the new chemical must be recognized and, if possible, defined. 
8 

QSAR analysis has been formalized and computerized for some health endpoints (e.g., cancer, 

mutagenicity, and teratogenicity) and may be useful with appropriate recognition of the 

limitations of these programs. A review of the various computerized QSAR programs available 

commercially is beyond the scope of this document, but this subject has been reviewed recently 

(Dearden et al. 1997). The U.S. EPA has grouped chemical substances with similar physicalchemical, structural and toxicological properties into working categories. Additional candidate 

categories for the EPA’s new chemical review process are proposed by Toxic Substances Control 

Act (TSCA) New Chemicals Program (NCP) staff based on available data and experience of 

reviewing premanufacturer’s notifications (PMNs) on related substances. These groupings 

enable the user of the NCP Chemical Categories guidance document to benefit from the 

accumulated data and decisional precedents within EPA’s new chemicals review process since 

1987, in order to identify areas of health hazard concern. Currently, there are 51 chemical 

categories listed in the table of contents of the EPA document, the detailed summaries of which 

may be found at URL: http://www.epa.gov/opptintr/newchms/chemcat.htm. 

Close structural analogs may provide data for occupational exposure and/or data for health hazard 

evaluation from occupational experience or epidemiology studies. For close structural analogs 

that have been used for several years, relevant information may be gained from historical 

experience with human exposure from normal handling. For many existing chemicals with many 

years of widespread industrial use, no adverse health effects have been observed. On the other 

hand, in some cases of overexposure or where unexpected toxicity was discovered, adverse 

effects in occupational populations have occurred. When they are available, retrospective (or 

case-control) epidemiological studies for close structural analogs may provide insight as to the 

potential for certain health effects by the new chemical. Recently, Nyden and Skaggs (1999) 

prepared a general listing of chemical classes, functional groups, possible toxic endpoints, and 

references of possible QSARs. 

In vitro screens. In many cases, QSAR can not be performed without a high degree of 

uncertainty, and insufficient human health hazard or animal toxicology data are available on close 

structural analogs of the halon replacement candidate to conduct a preliminary health hazard 

characterization. In instances such as those, in vitro screening tests may be advisable to develop 

the data necessary for a preliminary health hazard characterization. However, caution must be 

exercised when considering this approach, because the role of in vitro toxicity testing in chemical 

hazard characterization has not been formalized by U.S. regulatory agencies. A wide range of in 
9 

vitro screening tests has been developed for toxicology endpoints that are the subject of in vivo 

testing proposed below (e.g., acute irritation tests, acute toxicity tests, 14-day/90-day repeated 

dose toxicity tests, and reproductive/developmental toxicity tests). Cytotoxicity assays are 

promoted as suitable models for screening for human toxicity. A listing of proposed in vitro 

methods that may be used to generate data predictive of more definitive in vivo toxicology results 

and human health hazard potential follow. 

• Skin irritation → In vitro skin corrosivity/irritation testing - CORROSITEXTM or Skin2

(Botham et al. 1994) 

• Eye irritation → In vitro eye corrosivity/irritation testing - EYETEXTM or HET-CAM 

 (Balls et al. 1998) 

• Acute toxicity → Cytotoxicity assays using cultured cells (Seiberts 1996) 

Nyden and Skaggs (1999) prepared a list of in vitro methods for a wide variety of toxicity 

endpoints that may be useful depending on the classes of new chemical replacements. 

Acute irritation tests. This test applies to chemical candidates that are liquids or solids at 

ambient conditions. Irritancy of chemical vapors or gases is assessed in a more general manner 

during acute toxicity testing. The acute irritation test evaluates a chemical for its potential to 

cause irritation in laboratory animals following a single dose. Tests for skin or eye irritation are 

commonly performed in the rabbit, due to the rabbit’s proven responsiveness to irritants and the 

large database of chemical irritant information that exists on this species (almost exclusively the 

New Zealand White). Details of the experimental design that is generally followed in acute eye 

irritation and skin irritation tests are given in EPA’s Office of Prevention, Pesticides and Toxic 

Substances Health Effects Test Guidelines, commonly referred to as the OPPTS 870 Series 

(www.epa.gov/docs/OPPTS_Harmonized/870_Health_Effects_Test_Guidelines/). The specific 

Series numbers are 870.2400 and 870.2500 for acute eye and dermal irritation, respectively. The 

information gained from acute irritation testing is used to determine, in part, the hazard of a 

chemical, i.e., its intrinsic toxicological properties. In general, a toxicologist will place the 

candidate into one of four toxicity categories based on the results of the test. The Department of 

Transportation and other agencies use this approach (hazard classification) to determine 

precautionary labeling of chemicals. 

Before initiating tests for acute irritation, there are some points to consider. Strongly acidic or 

alkaline chemicals (e.g., pH ≤ 2 or ≥ 11.5) need not be tested for eye or skin irritation, since it is 
10 

presumed that these candidates will be irritating to mucosal surfaces. Also, results from well 

validated and accepted in vitro or QSAR tests that predict irritation may preclude the need for 

testing of irritation in animals. However, equivocal results from in vitro or QSAR tests would 

indicate a need to carry out irritation tests in animals. Either the eye or the skin irritation test is 

recommended, though chemicals do not necessarily give similar results in both tests. As part of 

the phase 1 screening method, one test should be sufficient to evaluate a candidate’s irritancy 

potential. The cost to perform a skin or eye irritation test using less than 6 rabbits is 

approximately $1500 to $2000. If a test agent were found to be corrosive to the skin or eyes, then 

further screening of the test agent may not be warranted due to the seriousness of this potential 

health hazard. Expert opinion is needed to decide whether to continue toxicity screening or stop. 

The expected human exposure scenario during or following use of the test agent and the 

consideration of estimated dose, route of entry, and personnel protective measures are factors 

likely to be discussed in making a final decision. 

Acute toxicity tests. The acute toxicity test is considered the cornerstone of the phase 1 

screening method. One reason for this importance is that the EPA’s SNAP program requires 

acute toxicity of a substitute chemical as part of its submission package. The purpose of the test 

is to determine and evaluate mammalian toxicity following a single dose or exposure of the halon 

replacement candidate. Rats are the preferred animal model. Valuable hazard information is 

gained from this test. Similar to the acute irritation test, acute toxicity testing results will place 

the candidate into a toxicity category for the purpose of hazard classification. Keep in mind that 

lethality, as a single end-point of toxicity, is a weak index for hazard assessment. Determination 

of several end-points of toxicity and the evaluation and interpretation of all data collected will 

provide greater confidence in the acute hazard assessment of a chemical. For example, answers 

to the following questions are especially helpful for toxicity evaluation. Did injury or death occur 

rapidly after dosing or were they first observed several days post-treatment? Were clinical signs 

consistent with central nervous system stimulation or depression? Did animals lose body weight 

or was there a depression in weight gain following dosing? Were lesions observed in organs or 

tissues during gross necropsy? Was there recovery of clinical signs and when was it observed? 

A toxicologist compiles this information in order to describe the acute hazard of the candidate 

material in question. Once established, chemicals are frequently compared on the basis of their 

acute hazard. For example, a replacement candidate for Halon 1301 might produce lethality in a 

group of exposed rats at vapor concentrations far below that of Halon 1301. The replacement 

candidate would be considered more potent in producing acute lethality than Halon 1301, but 
11 

other factors, such as fire extinguishing effectiveness need to be considered when determining 

risk management procedures and costs. 

There are several points to consider before initiating an acute toxicity test. One is the route of 

administration. Since inhalation is the most likely route of exposure for halon replacement 

candidates, it is the preferred route. However, inhalation testing is more expensive than other 

routes of chemical testing because it requires more labor effort to carry out the test. Also, more 

test material is needed to conduct the test. Good information is gained from acute oral or dermal 

toxicity tests, but important questions still remain. How much of the test material was absorbed 

following the oral or dermal route? Are the target organs of toxicity identified following the oral 

or dermal route applicable to those that would be identified following inhalation exposure? Since 

it is difficult to answer these questions by current extrapolation methods, more pertinent 

information is gained by performing an acute inhalation test. 

Inhalation exposure of chemicals can be in the form of gas or particulate matter (liquid droplets or 

solid particles). Selection of the most appropriate physical state for inhalation testing is simply a 

matter of considering the “specific use” scenarios that were described previously. One issue that 

needs to be dealt with for aerosol or dust inhalation studies is particle size. The animal inhalation 

exposure needs to contain particles that are respirable. For rats, this is on the order of one micron 

diameter. There are many factors that determine particle size, such as physical properties of the 

chemical candidate, exposure concentration, and particle generators. In some cases, the 

generation of respirable particles is very difficult to attain. Also, there may be discussion that the 

halon candidate would not be used or perhaps effective with such small particle sizes. However, 

the value of inhalation toxicity testing is diminished considerably if the distribution of particle 

sizes generated for toxicity testing of a chemical candidate does not fall within the range of being 

considered respirable for the animal species tested. 

The “Limit Test”. Exposure duration and chemical concentration are the two most important 

points to consider in the experimental design of an acute inhalation toxicity test. The current 

OPPTS Series 870.1300 guidelines recommend 4 hours duration after equilibration of the 

chamber concentration. Selecting a target exposure concentration that produces a defined 

response in animals may be difficult, but in the absence of any toxicity data on the halon 

replacement candidate, EPA allows one to consider using the “limit test” approach. One purpose 

of the limit test is to minimize the number of laboratory animals needed for testing, yet provide an 
12 

estimate of a chemical’s acute hazard. Under DoD Directive #3216.1, stringent polices dictate 

that the most conservative approaches must be taken with regard to the use of laboratory animals 

in research programs. The need to spend time and resources to obtain a once traditional “LC50

value” for a halon replacement candidate is discouraged. In the limit test, five male and five 

female animals (usually rats) are exposed to a chemical concentration of 2 mg/L for 4 hours, or 

where this is not possible due to physical or chemical properties of the test substance, the 

maximum attainable concentration. If no lethality is observed during exposure or for 14 days 

post-exposure, no further testing for acute inhalation toxicity is needed. Chemicals that do not 

cause lethality in a limit test evaluation fall into EPA’s least hazard toxicity category. If lethality 

is observed, additional 4-hour exposures at lower concentrations are strongly recommended, 

unless the test substance is no longer a candidate for consideration due to its acute hazard. 

Depending on the incidence of lethality observed at 2 mg/L, a concentration of 1 mg/L or 0.2 

mg/L is suggested for additional animal exposures. The goal of these exposures would be to 

determine a non-lethal or low (<20% incidence) lethal 4-hour concentration value of the test 

substance. This information is useful for comparing the acute inhalation hazard of chemicals and 

for designing repeated exposure experiments (Phase 2 – Toxicity Testing Methods). The cost to 

perform an acute inhalation toxicity “limit test” is approximately $7,500 to $10,000. Costs can be 

quite variable depending on the analytical method chosen for study and/or the time to develop 

new analytical methods, if necessary. 

The “Specific Use” Scenario Test. In general, the “specific use” scenario for a halon replacement 

candidate is for a few minutes only of exposure duration, but at a concentration much higher than 

the limit test value of 2 mg/L. One might ask what would be the hazard assessment for a 

candidate under these conditions? Exposures that simulate real-world conditions do help 

characterize the overall risks to human health, and risk characterization is a subject of concern for 

regulatory purposes. Thus, this type of inhalation exposure is recommended for halon 

replacement candidates. The goal of this test is to determine the potential for adverse effects 

following a single, high concentration exceedingly short exposure. A logical first choice for test 

atmosphere concentration and exposure duration would be the “worse-case” exposure scenario 

defined previously under the topic of preliminary “specific use” scenarios. The concentration of 

the test substance atmosphere should not be so high that oxygen content of at least 19% could not 

be maintained throughout the exposure duration. Exposure duration of 30 minutes or less is 

difficult to control via whole-body test systems, thus nose-only exposure designs are 

recommended. Once the concentration and duration are selected, the study design is identical to 
13 

that of the “limit test” described previously. Additional animal exposures would need to be 

performed, if toxicity were observed, and more acute inhalation hazard data were desired. At this 

point in the testing scheme, a decision would have to be made to carry out exposures at lower 

concentrations (same duration) or follow the OPPTS guidelines of a 4-hour exposure duration, 

starting with the limit test concentration of 2 mg/L. Making an assumption that one can simply 

apply a concentration x time relationship (Haber’s Law) to acute inhalation toxicity data can be 

erroneous (discussed below). 

As mentioned previously, acute toxicity data may be obtained for some test substances, such as 

liquids and solids stable at ambient conditions, by the oral route. The same general concepts 

described for the inhalation route apply, i.e., selection of limit test and LD50 determination, and 

are discussed in the EPA Health Effects Test Guidelines OPPTS Series 870.1100. The limit test 

value is 2,000 mg/Kg (body weight). Careful consideration should be given to the choice of 

vehicle, if necessary, and the maximum volume of liquid to be administered at one time. 

Recommendations are provided in the EPA Guidelines. 

Similar to the results of skin/eye irritation determination, test substances showing highly toxic 

characteristics (e.g., death, violent convulsions) at small or moderate doses may not warrant 

further consideration for toxicity screening. Again, expert opinion is needed to decide whether to 

continue toxicity screening or stop. The “specific use” scenario and the consideration of 

estimated dose, route of entry, and personnel protective measures are factors likely to be 

discussed in making a final decision. 

Genotoxicity tests (Phase 1). Genotoxicity assays detect and estimate the genetic hazard of 

chemicals. The purpose of the assays is to screen for possible mammalian mutagens and 

carcinogens. There are a wide variety of tests to consider when selecting an assay to determine a 

candidate’s potential to cause genetic damage. For this reason, a battery of three or more assays 

is commonly performed as a first tier approach for assessing genotoxicity. However, to gain as 

much preliminary information as possible with minimal cost, only one assay is recommended as 

part of Phase 1 – Toxicity Screening Methods. The Salmonella typhimurium reverse mutation 

assay, commonly referred as the Ames assay, is probably the most widely used genotoxicity test 

for assessing mutagenicity. Results provide information on whether the test substance causes 

point mutations in the genome of this bacterial organism. Details of the assay are available in 

OPPTS Series 870.5265. Currently, four test strains (TA 1535, TA 1537, TA 98, and TA 100) 
14 

are designated for testing, but a fifth strain (TA 102) is recommended (OPPTS Series 870.5100). 

Each strain is tested in the presence and absence of a metabolic activation system with 

appropriate positive control reference substances to ensure the efficacy of the activation system. 

A preliminary “range-finding” experiment is required to determine the upper limits of a 

candidate’s concentration that will produce cytotoxicity and/or, for relatively insoluble chemicals, 

the limits of solubility. A maximum of 5 mg test substance per plate is acceptable by EPA. 

The Ames assay is fairly straightforward for liquid or solid test materials at ambient conditions. 

The assay is more complicated for gas or vapor halon replacement candidates. This is because of 

the time and skill required exposing bacteria cultures to the test substance. Exposure chambers 

may have to be designed or modified. Vapor generation systems and analytical methods to 

measure the exposure concentration of the test substance have to be developed and implemented. 

A determination of test substance solubility is desirable, particularly when no cytotoxicity is 

observed in the “range-finder” experiment. The cost to perform the Ames assay for liquid or 

solid test materials is about $7,000. For gas or vapor exposures, the cost can be an additional 

$10,000 depending on the development and implementation of analytical methods. However, to 

keep cost at a minimum during Phase 1 testing of volatile halon replacement candidates, a 

protocol for the genotoxicity screen of volatile compounds was prepared (Appendix A). In this 

protocol, two sensitive tester strains of Salmonella (TA100 and TA102) are used to examine 

potential genotoxicity. Tedlar® bags are employed for preparation of vapor test atmospheres and 

exposure of bacteria. 

DECISION POINT 1

The primary objective for establishing decision points in the strategy for safety evaluation of 

potential halon replacement chemicals is to decide if more resources and funds should be 

expended to obtain more data to improve confidence and reduce uncertainty in assessing risk to 

human health. Phase 2 – Toxicity Testing is more time consuming than Phase 1 – Toxicity 

Screening Methods and requires considerable more funds to carry out testing. The information 

gained from Phase 1 – Toxicity Screening Methods includes chemical/physical properties, 

existing toxicity literature, preliminary “use” scenarios, qualitative/quantitative structure activity 

relationships, in vitro screens, acute irritation, acute toxicity, and genotoxicity of the halon 

replacement candidate. The value of this information in terms of human health hazard is 

noteworthy, but relatively small in amount (unless the toxicity literature database is extensive and 

of high quality). In general, hazard classification, precautionary labeling, and handling measures 
15 

(i.e., use of personal protective equipment) can be recommended for a test substance using data 

from Phase 1 – Toxicity Screening Methods. Further, the intrinsic acute hazard of the halon 

replacement candidate can now be compared to chemicals with known properties of irritation, 

acute toxicity, and mutagenicity. For example, the acute toxicity and mutagenicity hazard of 

Halon 1211 (bromochlorodifluoromethane), Halon 1301 (bromotrifluoromethane), CFC 11 

(trichlorofluoromethane), and CFC 12 (dichlorodifluoromethane) are frequently compared to that 

of a replacement candidate (Table 1). 

Table 1. Acute Toxicity of Halon 1211, Halon 1301, CFC 11, and CFC 12 

Halon/CFC Finding 

Halon 1211 Rat LC50 (4-hr) = 31,000 ppm); 

Rat LC50 (15-min) = 200,000 ppm 

Halon 1211 Ames test – negative in 4 strains; positive in 1 strain 

Halon 1301 Rat LClo (4-hr) > 800,000 ppm 

Halon 1301 Ames test – negative in 5 strains 

CFC 11 Rat LC50 (4-hr) = 26,200 ppm); 

Rat LC50 (15-min) = 130-150,000 ppm 

CFC 11 Ames test – negative 

CFC 12 Rat LC50 (4-hr) > 800,000 ppm 

CFC 12 Ames test – negative 

Another useful table is the toxicity categories assigned to products by EPA to assist in the 

selection of personal protective equipment required under the Worker Protection Standard set 

forth in 40 CFR 170.240 (Table 2). Halons 1211 and 1301 and CFCs 11 and 12 would fall into 

Category IV. This provides also a comparison perspective for replacement candidates that were 

tested by oral or dermal routes of administration. 
16 

Table 2. Toxicity Categories* 

Type of Study Category I 

(most hazard) 

Category II Category III Category IV 

(least hazard) 

Acute Oral LD50 Up to and including 

50 mg/Kg 

>50 through 

500 mg/Kg 

>500 through 

5000 mg/Kg 

>5000 mg/Kg 

Acute Dermal 

LD50 

Up to and including 

200 mg/Kg 

>200 through 

2000 mg/Kg 

>2000 through 

5000 mg/Kg 

>5000 mg/Kg 

Acute Inhalation 

LC50 

Up to and including 

0.05 mg/L 

>0.05 through 

0.5 mg/L 

>0.5 through 

2 mg/L 

>2 mg/L 

Eye Irritation 

Corrosive or corneal 

involvement or 

Irritation >21 days 

Corneal involvement 

or irritation clearing 

in 8-21 days 

Corneal involvement 

or irritation clearing 

in ≤7 days 

Minimal effects 

clearing in 

≤24 hours 

Skin Irritation Corrosive Severe irritation 

at 72 hours 

Moderate irritation 

at 72 hours 

Mild or slight 

irritation 

*EPA Health Effects Test Guidelines OPPTS 870.1000 Acute Toxicity Testing – Background 

The Department of Transportation has toxicity categories for assigning hazardous materials to 

hazard zones and packing groups (CFR, Title 49-Transportation, Part 173). Information needed 

for group assignment of gases and dusts/mists includes the material’s 1-hour LC50 value in young 

adult albino rats (Tables 3 and 4, respectively). For volatile liquids, the saturated vapor 

concentration at ambient conditions is needed (Table 5). Packing groups for chemical mixtures 

are based on mole fractions of the individual components (CFR 49, Part 173.133(b)) 
17 

Table 3. Hazard Zone Assignment for Poisonous Gases* 

Hazard Zone Inhalation Toxicity 

A LC50 ≤ 200 ppm 

B LC50 > 200 ppm and ≤ 1000 ppm 

C LC50 > 1000 ppm and ≤ 3000 ppm 

D LC50 > 3000 ppm and ≤ 5000 ppm 

*CFR 49, Part 173.116 

Table 4. Packing Group Assignment for Poisonous Dusts and Mists* 

Packing Group Inhalation Toxicity 

I LC50 ≤ 0.5 mg/L 

II LC50 > 0.5 mg/L and ≤ 2 mg/L 

III LC50 > 2 mg/L and ≤ 10 mg/L 

*CFR 49, Part 173.133 

Table 5. Packing Group Assignment for Poisonous Liquids Based on Inhalation of Vapors* 

Packing Group Vapor Concentration and Toxicity 

I (Hazard Zone A) V** ≥ 500 LC50 and LC50 ≤ 200 ml/m3

*** 

I (Hazard Zone B) V ≥ 10 LC50; LC50 ≤ 1000 ml/m3

; and the criteria for Packing Group I, 

Hazard Zone A are not met 

II V ≥ LC50; LC50 ≤ 3000 ml/m3

; and the criteria for Packing Group I, are 

not met 

III V ≥ 0.2 LC50; LC50 ≤ 5000 ml/m3

; and the criteria for Packing Groups 

I and II, are not met 

*CFR 49, Part 173.133 

**V is the saturated vapor concentration in air of the material in ml/m3

 at 20°C and standard 

atmospheric pressure 

***ml/m3

 = ppm = mg/L x 24,450/MW, where MW = molecular weight of the material and 

24,450 is the molar volume of air in milliliters at 25°C and 760 mm Hg 
18 

Concentration x Time (C x T) Relationships. In 1924, Haber described a relationship between 

concentration, time, and animal death that became known widely as “Haber’s Law” (Haber, 

1924). Haber’s Law states that the product of the concentration (C) and time (T) of exposure 

required to produce a specific physiological effect is equal to a constant (k), C x T = k. Recently, 

attention has been focused on a similar postulate, Cn

 x T = k, where the exponent n is derived 

from probit regression coefficients similar to that described by ten Berge et al., 1986. The Cn

 x T 

= k relationship may be a better predictor for mortality of irritants and systemically acting vapors 

and gases. Clearly, there are limitations to both relationships, but for many volatile chemicals, 

these relationships are applicable especially when lethality is considered the effect and time of 

exposure is acute and within a narrow range, such as several minutes to a few hours. Halons, 

however, may be a class of chemicals that does not follow a C x T = k relationship for the 

endpoint of lethality in laboratory animals. Using the data in Table 1 for Halon 1211 and 

applying Haber’s Law, the product of C x T ranges from 50,000 to 124,000 ppm•hour for the 15-

minute and 4-hour LC50 values, respectively. This difference is approximately 2.5-fold. Halon 

1202 has 15-minute, 2-hour, and 7-hour LC50 (or approximate LC50) values of 110,000, 40,000, 

and 20,000 ppm, respectively, in laboratory rats (DuPont, 1978). The ppm•hour product ranges 

from 27,500 to 140,000, an approximate 5-fold difference. Halon 2402 has 15-minute and 4-hour 

LC50 values of 120,000 and 55,000 ppm, respectively, in laboratory rats (DuPont, 1985). The 

ppm•hour product ranges from 30,000 to 220,000, an approximate 7-fold difference. Thus, 

caution must be exercised in applying C x T relationships for chemicals or chemical classes that 

don’t have databases to support the use of Haber’s Law or the ten Berge et al. postulate. 

Stop or Continue Testing. Expert opinion and advice are recommended to assist in making the 

decision to either drop a candidate from further testing or to proceed to Phase 2 – Toxicity 

Testing. Current scientific literature may have data on toxicity tests or studies that aren’t the 

same as those described in Phase 1 – Toxicity Screening Methods. An expert will have to address 

if acute hazard can be assessed from the existing literature data and compared to other halons. 

For example, the test substance of concern might have a higher 4-hr LC50 value than that of Halon 

1211, but is mutagenic in the Ames test in the presence of a metabolic activation system. Many 

compounds that are positive in the Ames test are mammalian carcinogens; however, there is not a 

perfect correlation between this test and carcinogenicity. Also, how much emphasis should be 

placed on QSAR results? Hazard interpretation of these types of information requires experience 

and knowledge. A test substance candidate that is classified as highly acutely toxic (Category I), 

shows mutagenicity in the Ames test, and lacks additional literature hazard data would likely be 
19 

dropped from further testing consideration. However, important in the decision making process 

is the consideration of preliminary “specific use” scenarios. Since risk is an index of both hazard 

and exposure, a test substance of high or moderate acute toxicity might be appropriate for 

unoccupied spaces or nonresidential uses. These “narrowed use” limitations are part of EPA’s 

assessment process for overall risk in the SNAP Program. 

Halon replacement candidates that show low order of irritancy and acute toxicity, are not 

mutagenic in the Ames assay, and do not indicate signs of delayed or cumulative toxicity based 

on results of QSAR or literature searches, are candidates that will likely proceed to Phase 2 – 

Toxicity Testing. 

PHASE 2 – TOXICITY TESTING 

Three endpoints will be described in the second phase of toxicity evaluation. They are 

cardiotoxicity, repeated dose toxicity, and genotoxicity. Figure 2 is a flow diagram of toxicity 

testing during Phase 2. Details follow on the approach and direction to be taken. 

Figure 2. Flow Diagram - Phase 2 Toxicity Testing

Decision from Phase

1 toxicity screening

to continue testing

Cardiotoxicity

Study of

Potential

Sensitizers

Highly

Cardiotoxic

Negative

or Mildly

Cardiotoxic

14-Day 

Toxicity Test

Genotoxicity

Test

(Mammalian

Cells)

Decision Point 2 - Stop or Continue to Phase 3 Testing 

Highly

Toxic

Low to

Moderate

Toxicity

Highly

Mutagenic

Negative

or Mildly

Mutagenic

Link Data

to PBPK

Modeling
20 

Cardiotoxicity study. One of the most important factors in assessing the safety of a halon 

replacement candidate is cardiac sensitization. Cardiac sensitization refers to heart rate 

arrhythmia experienced after injection of epinephrine and inhalation of the test chemical. The 

phenomenon of sensitizing the heart to epinephrine is associated with a number of unsubstituted 

and halogenated hydrocarbons. There are no regulatory testing guidelines for conducting a 

cardiac sensitization study. However, the experimental design most often followed is that of 

Reinhardt et al., 1971. Dogs are used as test subjects. One of the most important features of the 

experimental design is the establishment of cardiac response of each individual dog to 

epinephrine challenge alone. The typical response consists of a transient increase in heart rate 

followed by a slowing of heart rate and an increase in the height of the T-wave. In some dogs, 

multiple ventricular tachycardia occurs. To establish a predictable and minimal (i.e., baseline) 

cardiac response, epinephrine is administered intravenously at doses ranging from 1 to 12 μg/kg 

(body weight) at a rate of 0.1 ml/kg. It is not uncommon to find that the baseline response to 

epinephrine challenge will range from 1 to 12 μg/kg in a small (<10) group of naïve dogs, and 

that a few dogs from the group will be eliminated from the study due to unpredictable or greater 

than minimal response to epinephrine. This feature of the experimental design, the concern of the 

use of canine as an animal model that results frequently in mortality (due to ventricular 

fibrillation), and the issue of toxicological significance attributed to single animal reponses, have 

led to considerable debate among scientists and regulators, including the search for alternative 

methods in assessing cardiotoxicity of replacements to chlorofluorocarbons (Snyder et al., 1997). 

The cardiac sensitization study results in determining two critical endpoints for a test chemical, 

the lowest-observable-adverse-effect-level (LOAEL) and the no-observable-adverse-effect-level 

(NOAEL). However, these determinations are not calculated or extrapolated from the study data, 

but are determined simply by the selection of target exposure concentrations defined by the 

experimental design. The determination of the LOAEL and NOAEL become the key factor in 

assessing the safety of a halon replacement agent. However, physiologically based 

pharmacokinetic (PBPK) models have been developed to better define the relationship between 

acute exposure and cardiosensitization effect (see Linking PBPK Modeling). 

From a regulatory perspective (e.g., EPA), obtaining information on the cardiac sensitization 

potential of a halon replacement candidate is essential, assuming that the replacement candidate 

has chemical structure and properties analogous to halogenated hydrocarbons. Under EPA’s 

SNAP, cardiac sensitization LOAELs and NOAELs help define a replacement chemical’s use 
21 

restrictions. For example, when using halon substitutes as total flooding agents the following 

conditions, which were adopted from an OSHA safety and health standard (29 CFR 1910 Subpart 

L), generally apply: 

• Where egress from a normally occupied area cannot be accomplished within one 

minute, the employer shall not use the agent in concentrations exceeding its NOAEL. 

• Where egress from a normally occupied area takes longer than 30 seconds but less 

than one minute, the employer shall not use the agent in a concentration greater than 

its LOAEL. 

• Agent concentrations greater than the LOAEL are only permitted in areas not 

normally occupied by employees provided that any employee in the area can escape 

within 30 seconds. The employer shall assure that no unprotected employees enter 

the area during agent discharge. 

Halon substitutes being used as streaming agents are restricted to nonresidential uses. EPA’s 

SNAP conditions on agent use are different for substitutes of technologies unrelated to 

halogenated hydrocarbons or the potential to produce cardiac sensitization, such as inert gases, 

water mist, or powdered aerosols. 

The cost of testing one compound for cardiac sensitization is between $40-65K, and the cost of 

running two to three test materials is about $70-80K due to the fact that set-up costs would be 

done only once and study animals may be used repeatedly among the testing of different agents. 

The time to complete a cardiotoxic study is approximately two months. Only a few laboratories 

perform this testing—those with both inhalation and canine capabilities. One of the reasons the 

number of laboratories is limited is that physiological equipment and personnel to perform and 

interpret electrocardiograms are necessary. 

As mentioned previously with test substances that were either corrosive to the skin and eyes or 

highly toxic following single small doses, test materials that are potent cardiac sensitizers may 

not warrant further consideration for toxicity evaluation. Expert opinion is needed to decide 

whether to continue toxicity testing or stop. The “specific use” scenario and the consideration of 

estimated dose, route of entry, and personnel protective measures are factors likely to be 

discussed in making a final decision. An additional consideration, if applied, is the results and 

interpretation from physiologically based pharmacokinetic (PBPK) modeling of the test substance 

and its cardiac sensitization response. 
22 

Linking PBPK Modeling to Cardiac Sensitization Potential. Note: excerpts are taken from 

Vinegar and Jepson (1996). As mentioned previously, human exposure to halon replacement 

chemicals may be regulated on the basis of cardiac sensitization potential. After a range of 

chemical concentrations has been evaluated using the cardiac sensitization protocol, the doseresponse data can be used to establish the NOAEL and LOAEL. Utilization of the animal testing 

data for assessing potential risks to humans requires development of appropriate risk assessment 

methods. These methods must address the unusual exposure circumstances involved in the use of 

chemicals as fire suppressants. In particular, potential exposures would be for relatively brief, but 

varying, periods of time at concentrations high enough to effectively extinguish fires. Egress 

times need to be established for people occupying a facility at the time of chemical agent 

discharge. Currently, the LOAEL determined in dogs has been applied directly by the EPA in 

evaluating acceptable use and allowable exposure limits for humans. Establishing egress times 

from the 5 min exposure LOAEL requires careful consideration of the relationship between 

exposure concentration, duration, and the temporal aspects of the biological response. Under 

steady state conditions, the concentration in blood and tissues would be constant regardless of the 

exposure duration. With pre-steady state conditions, concentrations vary with time. Using the 

quantitative relationship between inhalation exposure at the LOAEL and the resulting 

concentration of chemical in blood is a way to make scientifically based decisions about the 

egress times. Cardiac sensitization is considered to be a function of the concentration of parent 

chemical in heart tissue which under perfusion limited conditions is proportional to parent 

chemical in blood. Therefore, the approach described herein involves estimating a target level 

defined as the chemical concentration in human blood that is achieved after 5 min of inhalation 

exposure at the chemical LOAEL as determined in dogs. 

A PBPK model provides the quantitative link between exposure concentration and blood levels of 

chemical achieved following inhalation. The PBPK model is a mathematical description of the 

uptake, distribution, metabolism, and elimination of a chemical in the species of interest. The 

physiological compartments that compose the model are based on appropriate physiological and 

anatomical properties for the species of interest as well as the chemical specific properties of the 

test compound. The use of PBPK models for kinetic description of chemical interaction with 

biological systems has been well represented in the scientific literature and is widely accepted by 

the scientific community as a tool for risk assessment. Full details of the development and 

application of the human PBPK model in support of evaluating halon replacement chemicals are 
23 

described in Section I of this report. A list of data requirements for use of the PBPK model for 

short term inhalation of fire suppressant chemicals is included in Section I. 

14-Day dose range-finding study. This is the first study in the proposed series of toxicity 

testing to assess and evaluate the potential toxic characteristics of a replacement chemical 

following repeated doses (or exposures). Though important data can be obtained from a 14-day 

study, it is often performed as a “dose range-finder” for the 90-day study (see Phase 3 – Toxicity 

Testing). Thus, the 14-day study is streamlined to use fewer animals, take less time to complete, 

and cost about 20% that of a 90-day study. Since the 14-day study is considered primarily to be 

a dose range-finder for longer term studies, the experimental design can be flexible. The 

following list provides some guidelines in experimental design that maximize the obtainment of 

information and minimize costs. 

• Use adult rats as the species for testing; 5 males and 5 females per dose group 

• Use 3 or 4 dose groups; 2 or 3 treated and one untreated (control) 

• For inhalation studies, expose animals 6 hr/day, 5 days/wk for 2 weeks; for other 

routes of administration, dose animals for 13 continuous days 

• Endpoints should include daily clinical observations, body weight measurements 

every 2-3 days, routine hematology and blood biochemistry determinations (at 

conclusion of the study), gross necropsy, and select organ weight measurements (e.g., 

liver, lungs, kidneys, adrenals, testes) 

• Preserve select organs and tissues in neutral buffered formalin for possible 

histopathological examination 

Following these guidelines will result in providing useful toxicological information at a cost of 

less than $50,000 and a period of time less than 45 days. The selection of doses (or exposure 

concentrations for inhalation studies) is important and requires careful consideration. The 

purpose of the highest dose is to produce some toxicity, even mortality. The intermediate and 

lowest doses may also produce toxicity, though usually less severe. It is not necessary to 

determine a NOAEL, because this determination is reserved for the longer term, more detailed 

(with endpoints) 90-day study. Two suggestions follow on selecting a high dose for a 14-day 

study. For chemicals that appear to be mildly toxic, i.e., categories III and IV (Table 2), select 

EPA’s “limit test” dose for 90-day studies, unless one suspects that human exposure might be 

higher. The limit test value is 1 mg/L via the inhalation route, and 1,000 mg/kg body weight for 
24 

oral administration. Consideration should be given also to minimize cost and select only two 

treated groups for the 14-day study. For chemicals that appear to be more potent, i.e., categories I 

and II (Table 2), start with an exposure concentration (or dose) that is 50-75% of the acute LC50

or LD50 value. Three treated groups are recommended with a concentration range of 

approximately one order of magnitude between the highest and lowest dose groups. 

Genotoxicity tests (Phase 2). A bacterial cell system was recommended for genotoxicity 

screening in Phase 1 – Toxicity Screening Methods. However, additional tests need to be carried 

out to provide greater confidence in predicting potential genotoxicity health hazard. In this phase 

of testing, assessment of genotoxicity involves a mammalian cell system. There are several 

assays to consider, but the two most common are the in vitro gene mutation assays in Chinese 

hamster ovary (CHO) cells and mouse lymphoma (L5178Y) cells. EPA guidelines for these tests 

are available (OPPTS 870.5300). Each test costs $17-22K for liquid or solid test materials and 

takes 2 to 3 months to complete. Again, if the halon replacement chemical can be tested as a 

vapor only, the cost and time to complete each assay will increase. Two references for testing 

volatile substances in CHO cells are Krahn et al. (1982) and Zamora et al. (1983). A reference 

for testing vapor of halon replacement candidates in L5178Y cells is Dodd et al. (1997). Only 

one assay needs to be selected for chemical testing, because both assays give similar type of 

genotoxicity information. The choice of an assay depends primarily on the historical background 

and familiarity of the laboratory performing the test. Similar to the Ames assay described 

previously, a preliminary “range-finding” experiment is performed to determine the upper limits 

of a candidate’s concentration that will produce cytotoxicity. The use of an exogenous source of 

metabolic activation to mimic mammalian in vivo conditions is part of the study design. 

DECISION POINT 2

Information gained from Phase 2 – Toxicity Testing includes a test candidate’s potential for 

cardiac sensitization, short-term repeated exposure toxicity, and genotoxicity in a mammalian cell 

system. By now, a much more realistic profile of the toxicity of the halon replacement should be 

available. The value of this information in terms of human health hazard is noteworthy, 

especially with regard to cardiac sensitization. As discussed previously, the intrinsic hazard of 

the halon replacement candidate can be compared with other chemicals, such as Halon 1211, 

Halon 1301, CFC 11, and CFC 12 (Tables 6, 7, and 8). 
25 

Table 6. Cardiac Sensitization NOAELs and LOAELs (Canine) 

Halon/CFC NOAEL (5-min) LOAEL (5-min) Comments 

Halon 1211 5,000 ppm 10,000 ppm EC50 = 19,000 ppm 

Halon 1301 50,000 ppm 75,000 ppm EC50 = 200,000 ppm 

CFC-11 3,200 ppm 3,500 ppm EC50 = 9,000 ppm 

CFC-12 40,000 ppm 50,000 ppm EC50 = 77,000 ppm 

 N.A. = not available 

 EC50 = A calculated concentration that represents an effect in 50% of the animals in the study 

 group 

Table 7. Short-Term Repeated Exposure Toxicity Data (Rat) 

Halon/CFC Exposure Duration NOAEL or LOAEL 

Halon 1211 6 hr/day x 15 days 10,000 ppm LOAEL 

3,300 ppm NOAEL 

Halon 1301 2 hr/day x 15 days 500,000 ppm NOAEL 

CFC-11 1-hr (twice)/day x 15 days 50,000 ppm LOAEL 

25,000 ppm NOAEL 

CFC-12 3.5 hr/day x 20 days 100,000 ppm NOAEL 

Table 8. In Vitro Mammalian Cells Genotoxicity Data 

Halon/CFC InVitro 

Mammalian cells 

Halon 1211 Negative 

Halon 1301 N.A. 

CFC-11 Negative 

CFC-12 Negative 

 N.A. = not available 
26 

Stop or Continue Testing. Expert opinion and advice are recommended to assist in making the 

decision to either drop a candidate from further testing or to proceed to Phase 3 – Toxicity 

Testing. Since the results of cardiac sensitization testing is very important to the EPA with 

respect to the candidate’s use in occupied areas, this information will be the most critical to 

evaluate. Results of PBPK modeling will help clarify the association between safe concentration 

and egress time. EPA will most likely designate a potent cardiac sensitizer for use in unoccupied 

areas only. Thus, potent cardiac sensitizers may continue to have potential use; however, the 

concern for issues of safe transportation, storage, and handling of a halon replacement candidate 

that is restricted for use in unoccupied areas remains to be addressed. In this phase of evaluation, 

results of repeated exposure toxicity studies begin to play a more important role in predicting 

health hazard. In the 14-day dose range-finding study, indicators of toxicity include unusual or 

persistent clinical signs (e.g., decreased activity), decreases in body weight, abnormalities in 

blood hematology or serum chemistries, alterations in organ weights, and the presence of gross 

organ/tissue lesions. Depending on the nature and severity of the effect and the exposure 

concentration required to elicit the effect, a decision to stop or continue testing can be made. 

Similar to the previous discussion on interpretation of genotoxicity results from the Ames test, a 

positive finding of mutagenicity in an in vitro mammalian cell system does not indicate absolute 

certainty that a chemical will cause cancer in humans. For greater confidence in predicting 

genotoxicity consequences in humans, more specific testing is required. 

Halon replacement candidates that are not potent cardiac sensitizers, show low order of toxicity in 

the repeated dose range-finding study, and are not mutagenic in mammalian cell systems, are 

candidates that will likely proceed to Phase 3 – Toxicity Testing. 

PHASE 3 – TOXICITY TESTING 

Only those candidates that are seriously being considered for halon replacement should proceed 

with Phase 3 - Toxicity Testing, due to the high cost in performing these studies. Two tests will 

be presented; the 90-day subchronic study and an in vivo genotoxicity study that will complete 

the “battery of tests” for genotoxicity potential. 

90-Day subchronic study. EPA considers a single, well-conducted, subchronic mammalian 

bioassay by the appropriate route as a minimum database for estimating a reference dose (RfD) or 

reference concentration (RfC). Rats are the most common mammalian species chosen for 
27 

investigation. The RfD and RfC are defined by EPA as an estimate (with uncertainty ranging 

approximately an order of magnitude) of a daily exposure to the human population (including 

sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects during a 

lifetime. An important component of the RfD is the NOAEL of the toxicology study that 

evaluates the most important biological endpoints of the chemical in question. In many cases, the 

results of the 90-day study provide the NOAEL used for setting RfDs and RfCs, because target 

organs and systemic effects are identified and evaluated in the study. 

The biological endpoints of a 90-day study include daily clinical observations; measurements of 

body weight, food consumption, and organ weights; hematological, serum chemistry, and urine 

determinations; gross necropsy; and histopathology of numerous tissues and organs. Depending 

on the toxicity profile to-date of the halon replacement to be evaluated, assessments of 

neurotoxicity, reproductive toxicity, and immunotoxicity can become part of the 90-day study 

design. EPA guidelines are available for the basic 90-day study designs (OPPTS 870.3465 for 

inhalation route of administration and OPPTS 870.3100 for the oral route in rodents). The cost of 

basic 90-day study designs ranges from $130,000 (oral) to approximately $200,000 (inhalation), 

but add-ons for specific assessments can increase the cost another $40,000 to $80,000. 

Selection of doses or exposure concentrations for the 90-day should be fairly straightforward, 

because the results of the 14-day dose range-finder study (phase 2) are the primary determinants. 

It is important that the highest dose selected for the 90-day study cause some adverse effects, and 

that the lowest dose selected produce no adverse effects, i.e., a NOAEL. A dose range of an 

order of magnitude (e.g., 10,000 ppm to 1,000 ppm) is not unusual for 90-day studies. 

Genotoxicity tests (Phase 3). As discussed previously, the prediction of a chemical to produce 

cancer in humans from the results of short-term testing in cell culture systems is more reliable 

when information is available from several genotoxicity test systems, i.e., a “battery of tests.” 

The conductance of an in vivo mammalian cell genotoxicity test will complete the “battery of 

tests” that is often used to determine genotoxicity potential. An in vivo mammalian cell test is 

especially relevant to assessing mutagenic hazard in that it allows consideration of factors of in 

vivo metabolism, pharmacokinetics, and DNA-repair processes although these may vary among 

species, among tissues, and among genetic endpoints. The in vivo mammalian cell system used 

frequently in a genotoxicity test battery is the mouse micronucleus assay. The mouse 

micronucleus test detects the damage of chromosome or mitotic apparatus caused by a chemical 
28 

in immature (polychromatic erythrocyte; PCE) cells of treated animals. Micronuclei are believed 

to be formed from chromosomes or chromosome fragments left behind during cell division. 

After cell division, these fragments may not be included in the nuclei of the daughter cells and 

form single or multiple micronuclei in the cytoplasm. So the micronucleus test can serve as a 

rapid screen for clastogenic agents and test materials that interfere with normal mitotic cell 

division, effecting spindle fiber function or formation. The assay is based on the increase in the 

frequency of micronucleated PCEs in bone marrow of the treated animals. 

General guidelines to be followed in the mouse micronucleus assay are described in EPA’s 

Health Effects Test Guidelines OPPTS 870.5395. More specifically, the assay is conducted using 

animals of both sexes and 3 to 4 concentrations of the test agent or negative control (e.g., air-only 

exposed or saline dosed). Treatment of animals is either by inhalation (volatile test chemicals) or 

by gavage for three consecutive days. A positive control agent, cyclophosphamide, dissolved in 

physiological saline, is given by intraperitoneal injection as a single dose. Twenty-four hours 

following the last exposure/dose, mice are sacrificed, and bone marrow cells are collected and 

processed. The frequency of micronucleated cells is observed by counting 2000 PCEs per 

animal. Animal mortality and the ratio of PCE to mature erythrocytes (normochromatic 

erythrocyte; NCE) are used as indicators of test agent toxicity. The difference in the 

micronucleated PCE frequency and the ratio of PCE /NCE between treated and control animals 

are statistically evaluated by Chi-square analysis, and the dose dependent response is examined 

by linear regression. A test agent is considered to have elicited a positive response in the mouse 

micronucleus test if there is a dose-related increase in micronuclei and if one or more of the doses 

induces a statistically significant (p < 0.05) increase in micronuclei induction. The assay 

typically costs $20,000 to $35,000, depending on the route of administration of the halon 

replacement to be tested. 

DECISION POINT 3

Information gained from Phase 3 – Toxicity Testing includes a chemical candidate’s potential for 

intermediate-term (90-day) repeated exposure toxicity and genotoxicity in an in vivo mammalian 

cell system. Much more confidence of the toxicity profile of the halon replacement is now 

available for predicting human health hazard. As discussed previously, the intrinsic hazard of the 

halon replacement candidate can be compared with other chemicals, such as Halon 1211, Halon 

1301, CFC 11, and CFC 12 (Tables 9 and 10). 
29 

Table 9. 90-Day Repeated Exposure Toxicity Data (Rat) 

Halon/CFC NOAEL or LOAEL Target Organ of 

Concern for Toxicity 

Halon 1211 N.A. - 

Halon 1301 23,000 ppm NOAEL* Respiratory tract* 

CFC-11 1,000 ppm (24 hr/day) 

NOAEL 

Cardiotoxicity; 

CNS (depression) 

CFC-12 10,000 ppm NOAEL CNS (depression) 

 N.A. = not available 

 CNS = central nervous system 

 *18-week study

Table 10. In Vivo Mouse Micronucleus Assay Data 

Halon/CFC InVivo Mouse 

Micronucleus Result 

Halon 1211 50,000 ppm NOAEL 

(6 hr/day x 1 day) 

Halon 1301 N.A. 

CFC-11 N.A. 

CFC-12 N.A. 

 N.A. = not available 

Stop or Continue Testing. Expert opinion and advice are recommended to assist in making the 

decision to either drop a candidate from further testing or to proceed to Phase 4 – Toxicity 

Testing. By now, a considerable amount of toxicity information is available on a halon 

replacement candidate and estimates of RfCs or RfDs can be made. Again, the RfC and RfD are 

defined by EPA as an estimate (with uncertainty ranging approximately an order of magnitude) of 

a daily exposure to the human population (including sensitive subgroups) that is likely to be 

without an appreciable risk of deleterious effects during a lifetime. Histopathology data from the 

90-day study and results form the genotoxicity test battery provide moderate to strong confidence 
30 

in predicting carcinogenic potential of a chemical. However, important biological endpoints such 

as developmental toxicity and reproductive toxicity remain to be addressed more thoroughly. 

The decision to begin Phase 4 – Toxicity Testing implies that a chemical candidate is being 

seriously considered as a halon replacement. Occupational exposure is likely due to industrial 

manufacturing, processing, distribution, and storage of the new chemical. The general public 

may be at risk as well. Thus, it becomes imperative to determine and evaluate the potential of a 

new chemical that is being considered for industrial production to cause more specific endpoints 

of toxicological concern. Phase 4 – Toxicity Testing addresses some of these concerns. 

PHASE 4 – TOXICITY TESTING 

As discussed previously, only those candidates that are seriously being considered for 

manufacture and use should proceed with Phase 4 – Toxicity Testing, due to the high cost in 

performing these studies, especially by the inhalation route of exposure. Two tests will be 

presented in detail (developmental and reproductive toxicity studies), but other tests will be listed 

for completeness. 

Developmental toxicity study. Developmental toxicity is any adverse effect observed in the 

fetus/neonate induced during the period from conception through puberty. The major types of 

developmental toxicity are embryo-lethality, structural abnormalities, altered growth and 

functional deficiencies. Chemical effects on the developing fetus may be mediated through 

toxicity in the parents. These effects are not generally considered to be developmental toxicity. 

However, effects in the fetus that result from the direct interaction of the chemical with 

developmental processes are attributed to developmental toxicity and generally raise the level of 

concern about chemical hazard. 

A developmental toxicity study in rodents typically costs $100,000, requires approximately eight 

months from initiation to final report, and utilizes between 100 and 150 animals. The purpose 

and principle of the test method are described in EPA’s Health Effects Test Guidelines OPPTS 

870.3700. The test substance is administered to pregnant animals at least from implantation to 

one day prior to the expected day of parturition. For inhalation developmental toxicity studies in 

rodents, the exposure regimen is usually 6 hr/day for gestation days 6 through 15. Shortly before 

the expected date of delivery, the pregnant females are terminated, the uterine contents are 

examined, and the fetuses are processed for visceral and skeletal evaluations. Because of the 
31 

concern for chemicals that may produce malformations in humans but are not detected in rodent 

animal models (e.g., thalidomide), a non-rodent animal model using rabbits is often times 

considered. The testing of rabbits for developmental toxicity is a separate study from that of 

rodents and would be an additional cost. 

Reproductive toxicity study. Reproductive toxicity covers all phases of the reproductive cycle, 

and includes impairment of male or female reproductive function or capacity and the induction of 

nonheritable adverse effects on offspring (including death, growth retardation, structural 

abnormalities and functional effects). A two-generation reproduction study in rats typically costs 

$450,000, requires approximately twelve months from initiation to final report, and utilizes about 

150 rats initially. The test substance is administered to parental animals prior to and during their 

mating, during the resultant pregnancies, and through the weaning of their F1 offspring. The 

substance is then administered to selected F1 offspring during their growth into adulthood, 

mating, and production of an F2 generation, until the F2 generation is weaned. 

Data from hundreds of pups are generated from the two generations of reproduction. The purpose 

and principle of the test method are described in EPA’s Health Effects Test Guidelines OPPTS 

870.3800. Briefly, the two-generation reproductive toxicity study is designed to provide general 

information concerning the effects of a test substance on the integrity and performance of the 

male and female reproductive systems, including gonadal function, the estrous cycle, mating 

behavior, conception, gestation, parturition, lactation, and weaning, and on the growth and 

development of the offspring. The study may also provide information about the effects of the 

test substance on neonatal morbidity, mortality, target organs in the offspring, and preliminary 

data on prenatal and postnatal developmental toxicity and serve as a guide for subsequent tests. 

Additionally, since the study design includes in utero as well as postnatal exposure, this study 

provides the opportunity to examine the susceptibility of the immature/neonatal animal. For 

further information on developmental effects and functional deficiencies, additional study 

segments can be incorporated into the protocol, utilizing the guidelines for developmental toxicity 

(described previously) or developmental neurotoxicity. 

Other studies. The most definitive test for carcinogenic potential using laboratory animal 

models is a chronic (lifetime) exposure of the chemical at a maximum tolerated dose. For 

rodents, this is generally referred to as a two-year bioassay. For most chemicals, the subchronic 

(e.g., 90-day) toxicity study and the in vitro/in vivo genetic toxicity test battery are sufficient to 

assess the potential for long-term toxicity and carcinogenicity. Carcinogenicity studies are not 
32 

generally conducted for nonregulated chemicals for which the exposures are primarily 

occupational. An inhalation carcinogenicity study in rodents typically costs $1-1.5 million, 

requires approximately 36 months from initiation to final report, and utilizes a minimum of 480 

rats or mice. The purpose of this study type is described in EPA’s Health Effects Test Guidelines 

OPPTS 870.4300. If a two-year bioassay is conducted, it is important to keep in mind the 

extreme conditions (i.e. lifetime exposure at the maximum tolerated dose) used to elicit a 

tumorigenic response when interpreting the results. 

Neurotoxicity studies examine the potential functional and morphological effects of chemical 

exposure to the nervous system that may occur in young adults or, in the case of developmental 

neurotoxicity, the offspring from maternal exposure during pregnancy and lactation. Some 

neurotoxicity tests are designed to assess specific neurotoxic endpoints, such as 

acetylcholinesterase or neurotoxic esterase activities (EPA’s Health Effects Test Guidelines 

OPPTS 870.6100). More recently, there are specific tests for assessing a chemical’s potential to 

produce immunotoxicity and endocrine disruption. The neurotoxicity, developmental 

neurotoxicity, immunotoxicity, and endocrine disruptor tests may be considered if there is reason 

to suspect alterations in the nervous, immune, or endocrine systems. 

Metabolism and toxicokinetic studies are useful for determining the uptake of a chemical by the 

body from various exposure routes, distribution of the chemical in the organs and tissues of the 

body, and elimination of the chemical and its metabolites from the body. Also, metabolism and 

toxicokinetic studies are useful for setting dose/exposure levels for subchronic (e.g., 90-day) and 

chronic (e.g., carcinogenicity) studies and for extrapolating toxicology data from animals to 

humans. A toxicokinetic study typically costs $50,000 to $200,000, requires approximately four 

to twelve months from initiation to final report, and utilizes a minimum of four to greater than 

100 animals, depending on study design. Analytical methods are an important component of 

toxicokinetic studies. Although this report does not recommend specific levels of concern for 

results from toxicokinetic studies, issues such as a long half-life for a chemical or metabolism to a 

reactive chemical intermediate (e.g. epoxides, free radical, etc.) tend to raise the level of concern 

for a chemical. 

SUMMARY OF TOXICITY TESTING PROGRAM

A strategy to human health safety evaluation of halon replacement candidates is provided. A 

step-wise approach in building a chemical toxicity database, specific for halon replacement 
33 

candidates, allows decisions to be made with budget and time constraints in mind. Four phases of 

toxicity testing are described. The confidence in predicting human health hazard increases as one 

proceeds from one phase of testing to the next, but the cost of each phase increases as well. 

Information is provided at the end of each phase that allows one to evaluate the overall benefit 

and cost of the tests performed before deciding to continue or stop toxicity testing of the 

replacement candidate. Figures 1 and 2 are flow diagrams that outline the strategy to be taken for 

toxicity testing Phases 1 and 2. Table 11 summarizes the cost and time associated with each of 

the phases of testing that are described in detail in this report. Figure 3 summarizes the 

relationship between duration, cost, gain in confidence, and likely regulatory decision for each of 

the four phases of toxicity testing. 
34 

Table 11. Summary of Toxicity Testing of Halon Replacement Candidates 

Phase Test or Method Estimated 

Cost ($)* 

Time for Completion

(start to final report) 

1 Chemical/physical properties; 

literature search; define 

specific use scenario; SARs 

10,000** 7 days** 

1 In vitro screening 1,500 – 3,500 2 weeks 

1 Acute irritation 1,500 – 2,000 3 weeks 

1 Acute toxicity and “specific 

use” scenario test 

7,500 – 15,000 4 weeks 

1 Genotoxicity (in vitro 

bacterial cells) 

7,000 – 17,000 6 – 8 weeks 

2 Cardiac sensitization 40,000 – 65,000 6 – 8 weeks 

2 PBPK modeling link to 

cardiac sensitization 

10,000 – 25,000 4 – 12 weeks 

2 14-Day dose range-finder 50,000 6 weeks 

2 Genotoxicity (in vitro 

mammalian cells) 

17,000 – 22,000 

(higher for inhalation) 

2 – 3 months 

3 90-Day subchronic study 130,000 – 200,000 6 – 8 months 

3 Genotoxicity (in vivo

mammalian) 

20,000 – 35,000 2 – 3 months 

4 Developmental toxicity 100,000 8 months 

4 Reproductive toxicity 450,000 12 months 

4 Carcinogenic 2-year bioassay 1,000,000 – 1,500,000 36 months 

4 Metabolism/toxicokinetics 50,000 – 200,000 4 – 12 months 

*1999 Estimates 

**Nyden and Skaggs, 1999 
35 

Phase 4 Testing

Time: 36 months

Cost: $1.6-2.25M

Phase 3 Testing

Time: 8-11 months

Cost: $150-235K

Phase 2 Testing

Time: 12-20 weeks

Cost: $117-162K

Phase 1 Testing

Time: 6-8 weeks

Cost: $25-50K

Gain in DataConfidence RegulatoryAcceptance

Figure 3. Relationship of Phase Testing with Regulatory Decisio

37 

ACKNOWLEDGEMENTS 

Dr. Sheela Sharma, ManTech Environmental Technology, Inc., for preparation of the protocol for 

genotoxicity screening of volatile compounds. Dr. Andrey Nikiforov, Toxicology/Regulatory 

Services, for comments and citations on data development strategy. 

REFERENCES AND SELECTED READINGS 

Balls, M., Berg, N., Bruner, L.H., Curren, R.D., de Silva, O., Earl, L.K., Esdaile, D.J., Fentem, 

J.H., Liebsch, M., Ohno, Y., Prinsen, M.K., Spielmann, H., and Worth A.P. (1998). Eye 

Irritation Testing: The Way Forward. The Report and Recommendations of ECVAM Workshop 

34. 

Botham, P.A., Chamberlain, M., Barratt, M.D., Curren, R.D., Esdaile, D.J., Gardner, J.R., 

Gordon, V.C., Hildebrand, B., Lewis, R.W., Liebsch, M., Logemann, P., Osborne, R., Ponec, M., 

Regnier, J.F., Steiling, W., Walker, A.P., and Balls M. (1994). A Pre-validation Study on In 

vitro Skin Corrosivity Testing. The Report and Recommendations of ECVAM Workshop 6. 

Dearden, J.C., Barratt, M.D., Benigni, R., Bristol, D.W., Combes, R.D., Cronin, M.T.D., Judson, 

P.N., Payne, M.P., Richard, A.M., Tichy, M., Worth, A.p., and Yourick, J.J. (1997). The 

Development and Validation of Expert Systems for Predicting Toxicity. The Report and 

Recommendations of ECVAM Workshop 24. Atla. 25:223-252. 

Dodd, D.E., Ledbetter, A.D., and Mitchell, A.D. (1997). Genotoxicity testing of the halon 

replacement candidates trifluoroiodomethane (CF3I) and 1,1,1,2,3,3,3-heptafluoropropane (HFC227ea) using the Salmonella typhimurium and L5178Y mouse lymphoma mutation asssays and 

the mouse micronucleus test. Inhalation Toxicol 9: 111-131. 

DuPont Haskell Laboratory Review of Halon 2402, Unpublished, Updated July 9, 1985, 11 

pages. 

EPA Health Effects Guidelines. 
38 

Haber, F. (1924). [Five lectures for the years 1920-1923.] Springer-Verlag, Berlin (in German). 

Krahn, D.F., Barsky, F.C., and McCooey, K.T. (1982). CHO/HGPRT mutation assay: 

Evaluation of gases and volatile liquids. (Eds.) Tice, R.R., Costa, D.L., and Schaich, K.M. 

Genotoxic Effects of Airborne Agents. New York, Plenum, pp. 91-103 

Nikiforov, A. (1999). Final Report: Data Develoment Strategy for Occupational Health Hazard 

Evaluation of New Chemicals, prepared for ManTech Environmental Technology, Inc., October 

19, 49 pages. 

Nyden, M.R., and Skaggs, S.R. (1999). Screening Methods for Agent Compatibility with People, 

Materials, and the Environment, NISTIR 6323, April, 1999, 67 pages. 

Reinhardt, C.F., Azar, A., Maxfield, M.E., Smith, P.E., and Mullin, L.S. (1971). Cardiac 

arrhythmias and aerosol sniffing. Arch Environ Health 22: 265-279. 

Seibert, H., Balls, M., Fentem, J.H., Bianchi, V., Clothier, R.H., Dierickx, P.J., Ekwall, B., Garle, 

M.J., Gomez-Lechón, M.J., Gribaldo, L., Gülden, M., Liebsch, M., Rasmussen, E., Roguet, R., 

Shrivastava, R., and Walum, E. (1996). Acute Toxicity Testing In vitro and the Classification 

and Labeling of Chemicals. The Report and Recommendations of ECVAM Workshop 16. Atla. 

24: 499-510. 

Snyder, R., Bakshi, K.S., and Wagner, B.M. (1997). Abstracts of the workshop on toxicity of 

alternatives to chlorofluorocarbons. Inhal Toxicol 9: 775-810. 

ten Berge, W.F., Zwart, A., and Appelman, L.M. (1986). Concentration-time mortality response 

relationship of irritant and systemically acting vapours and gases. J Hazard Mat 13: 301-309. 

Vinegar, A., and Jepson, G.W. (1996). Cardiac sensitization thresholds of halon replacement 

chemicals predicted in humans by physiologically-based pharmacokinetic modeling. Risk 

Analysis 16: 571-579 
39 

Zamora, P.O. et al. (1983). Evaluation of an exposure system using cells grown on collagen gels 

for detecting highly volatile mutagens in the CHO/HGPRT mutation assay. Environ Mutagen 5:

795-801. 

CFC-11

ACGIH Documentation of TLV-TWA, Revised 1992 

DuPont Haskell Laboratory Review, Unpublished, Updated October 24, 1988, 36 pages 

EPA Environmental Hazard Assessment of One and Two Carbon Fluorocarbons, Final Report, 

EPA-560/2-75-003, 1975 

CFC-12

ACGIH Documentation of TLV-TWA, Revised 1991 

DuPont Haskell Laboratory Review, Unpublished, Updated February 19, 1992, 32 pages 

EPA Environmental Hazard Assessment of One and Two Carbon Fluorocarbons, Final Report, 

EPA-560/2-75-003, 1975 

Halon 1211

Clark, D.G., and Tinston, D.J. (1982). Acute inhalation toxicity of some halogenated and nonhalogenated hydrocarbons. Human Toxicol 1: 239-247. 

DuPont Haskell Laboratory Review, Unpublished, Updated May 30, 1993, 25 pages 

EPA Environmental Hazard Assessment of One and Two Carbon Fluorocarbons, Final Report, 

EPA-560/2-75-003, 1975 

Orlowski, J. (1981). Determination of Halon 1211 toxicity. Part I. Acute inhalation toxicity. 

Bromatol Chem Toksykol 14: 53-55. 
40 

Styles, J.A., Richardson, C.R., Callander, R.D., Cross, M.F., Bennett, I.P., and Longstaff, E. 

(1985). Activity of bromochlorodifluoromethane (BCF) in three mutation tests. Mutat Res 142:

187-192. 

Van Stee, E.W., Aerospace Medical Research Laboratory Technical Report, AMRL-TR-74-143, 

1974 

Halon 1301

ACGIH Documentation of TLV-TWA, Revised 1992 

DuPont Haskell Laboratory Review, Unpublished, Updated May 25, 1990, 30 pages 

EPA Environmental Hazard Assessment of One and Two Carbon Fluorocarbons, Final Report, 

EPA-560/2-75-003, 1975 

National Research Council (1984). Emergency and Continuous Exposure Limits for Selected 

Airborne Contaminants, Volume 3 – Bromotrifluoromethane. Committee on Toxicology. 

Washington, DC: National Academy Press. 22 pages. 

Van Stee, E.W., Aerospace Medical Research Laboratory Technical Report, AMRL-TR-74-143, 

1974 

Waritz, R.S. (1968). Acute inhalation toxicity of bromotrifluoromethane (“Freon 13B1”). 

Haskell Lab. Report No. 46-68. Medical Research Project No. 876. 
41 

Appendix A 

Protocol for the 

Genotoxicity Screen of Volatile Compounds 

HUMAN HEALTH SAFETY EVALUATION OF 

HALON REPLACEMENT CANDIDATES 

Final Report – Section II of II 

Next-Generation Fire Suppression Technology Program (NGP) 

Project 3B/1/89 
42 

Protocol for the Genotoxicity Screen of Volatile Compounds

I. Overall Objectives: 

Research will be conducted to determine the potential genotoxicity associated with the exposure 

to (test material), a chemical that is being considered as a fire suppressant for potential military 

application. 

A short-term genotoxic assay, Salmonella/Mammalian microsome reverse mutation assay, 

per EPA (OPPTS) Health Effect Testing Guidelines (870.5100, 1998), will be used to examine 

the genotoxicity of this volatile chemical. 

II. Salmonella/Mammalian Microsome Reverse Mutation Assay 

II-A. Purpose 

The Salmonella/Mammalian microsome reverse mutation system is a microbial assay which 

measures the reversion from his- (histidine dependent) to his+ (histidine independent) induced by 

chemicals which cause base changes or frameshift mutations in the genome of this organism. 

II-B. Background 

A reverse mutation assay using Salmonella typhimurium detects mutations in a gene of a histidine 

requiring strains to produce a histidine independent strain of this organism. A reverse mutation 

can be achieved by base pair changes, which may occur at the site of the original mutation or at a 

second site in the chromosome; or by frameshift mutations resulted from the addition or deletion 

of single or multiple base pairs in the DNA molecule. 

In the standard plate incorporation Salmonella/mammalian microsome reverse mutation assay, 

the test agent, bacteria and the metabolic activation mixture (+S9) or buffer (-S9) will be mixed 

with 450

 C overlay agar, spread on a plate of bottom agar and incubated for 48 hours at 370 C. 

However, volatile chemicals may evaporate before there is adequate time for bacteria to be 

exposed to the chemical. Therefore, in this assay, a vaporization technique will be used for test 

materials which are volatile at ambient temperatures. Briefly, the bacteria which are in the top 

agar with and without a metabolic activation system and plated onto histidine deficient minimum 

agar medium will be exposed to the vapor phase of a test agent in a Tedlar bag which will 

increase the contact time between the volatiles and the bacterial test system. After a suitable 

period of exposure, plates will be removed from the bag to avoid extra toxicity and 

contamination, and incubate for an additional 24 to 48 hours. Revertant colonies will be counted 

and compared with the number of spontaneous revertants in an untreated (air) and / or vehicle 

control culture. The mutagenicity of the test agents will be evident from the increased number of 

revertants. 
43 

II-C. Test Methods 

1. Tester strains: 

Two most sensitive tester strains for volatile chemicals with boiling points below 63 o

C, will be 

used in this assay, which included TA100 for the detection of GC base pair mutagens, and TA102 

for the detection of AT base pair mutagens (Hughes et al, 1987). The tester strains will be 

obtained from Dr. Bruce N. Ames in University of California, CA. 

2. Confirmation of the genotypes of the tester strains: 

Following genotypes will be confirmed in each tester strain based on the methods described by 

Maron and Ames (1983) prior to the mutagenesis study: 

a. Requirement of histidine for growth (His-

) 

b. Sensitivity to Crystal violet (rfa mutation) 

c. Resistance to ampicillin or ampicillin +tetracycline for TA102 only (R 

factor) 

d. Sensitivity to U.V. light (uvrB factor). 

e. Spontaneous revertants 

3. Bacteria growth: 

Fresh culture of the tester strains will be used for each assay. The bacteria are cultured in nutrient 

broth at 37o

C in a environmental shaker incubator for 10-12 hours (no more than 16 hours) to 

reach the late exponential or early stationary phase of growth (108

-109 cells per ml). 

4. Metabolic activation: 

The test compound will be examined both in the presence and absence of an appropriate 

metabolic activation system. The most commonly used activation system in this assay is S9 

mixture, a cofactor supplemented postmitochondrial fraction prepared from the liver of rats 

treated with enzyme inducers such as Aroclor-1254. 

5. Test agent preparation: 

The vapor phase test atmospheres of volatile chemicals will be prepared fresh in Tedlar® bags as 

they are needed. A measured volume of sterile breathing quality air is injected into an evacuated 

Tedlar® bag. The volume of air injected should not exceed 75% of the volume of the bag. A 

measured volume of the volatile liquid chemical will then be injected into the inflated bag. The 

bag will then be warmed (Pegram 1997) to ensure complete vaporization of the liquid chemical. 

Mixing of the chemical vapor and air will be accomplished by alternately pressing and releasing 

the bag several times. After preparation of the test atmosphere the bag should be stored in the 

dark or under yellow lights (Pegram 1997) and allowed to return to room temperature. As the bag 

returns to room temperature the chemical vapor may condense on the inside surface of the 

Tedlar® bag. This indicates that a saturated vapor is present in the bag and is a condition that 

should be avoided because of concentration stability problems. The chemical atmosphere 

contained in this bag will be the standard used to prepare other test atmosphere concentrations. 
44 

Each test atmosphere concentration will require a separate Tedlar® bag. For each desired vapor 

concentration, a measured volume of the test atmosphere prepared above is injected into the bag. 

Then, a measured of volume of sterile breathing quality air is injected. The contents of the bag is 

thoroughly mixed by alternately pressing and releasing the bag several times. The vapor 

concentration of each bag (determined by vapor phase dilution) will then be in a fixed ratio to the 

concentration in the standard bag which can be measured or calculated. A separate Tedlar bag 

will be used for the negative (air) control. For the dose selection a prescreening test using four 

test vapor concentrations will be conducted using both strains. Toxicity will be evident by a 

reduction in the spontaneous revertants per plate, and /or a clearing of the background lawn. Five 

test atmosphere concentrations ranging from low to high with adequate intervals will be selected 

and tested in the mutagenesis assay. 

6. Controls: 

In each assay, following concurrent controls will be set up: 

a. Negative (air) controls 

Cultures with and without S9 mixture will be exposed to sterile air as described in the chemical 

preparation except no chemical will be injected into Tedlar bag. They are used for the 

measurement of spontaneous revertants, which will serve as the background level of reverse 

mutation. Appropriate solvent controls will also be included in each assay by using regular plate 

incorporation method. 

 b. Positive control 

 Positive controls with known mutagens shall ensure the responsiveness of the tester strains as 

well as the efficacy of the activation system. Anthramine will be used as positive control with 

activation system (+S9) for both tester strains. In the non activation system (-S9), the positive 

controls will be sodium azide for TA 100 and Mitomycin C for TA102 . The above positive 

control agents will be dissolved in DMSO and tested using the regular plate incorporation 

method. 

7. Mutagenesis assay 

A Tedlar ®bag vaporization technique will be used in this assay. All dose groups (with and 

without S9 mixture) will be set up in triplicates. 0.1 ml of the culture is added to 2 ml of top agar 

which is melt and held at 45o

C heating block along with 0.5 ml of S9 mixture or incubation buffer 

The contents are mixed and poured onto the surface of a minimum glucose agar plate. After the 

top agar solidifies, the plates with the lid on the bottom are placed in a Tedlar® bag. The bag is 

then sealed and evacuated. The contents of the Tedlar bag (prepared in Step 5) containing the 

desired chemical vapor concentration are transferred into the evacuated bag (do not fill the bag 

with the plates to more than 50% of it’s capacity). The bag with the plates and chemical vapor 

will then be incubated at 37o

C for 24 hours. After 24 hours of exposure, plates will be removed 

from the bag and incubated for an additional 24 hours for TA100 and 48 hours for TA102. 

Negative (air) control will be included in each experiment. Regular plate incorporation method 

will be used for solvent and positive control. The number of revertants per dish will be counted 

manually or by an automatic colony counter. 
45 

II-D. Data collection and reporting 

The number of revertants per dish will be determined and the results are stored and processed by 

a computer using Excel spreadsheets. Following specific information will be reported: (1) Tester 

strains used (results of genotypic confirmation), (2) Metabolic activation system used (source, 

amount, cofactors, method for preparation), (3) Test agent preparation, dose levels and the 

rationale for their selection, (4) Positive and negative controls, (5) Individual plate counts, means, 

and standard deviation, and (6) Dose response relationship if applicable. 

II-E. Result analysis and interpretation 

1. Criteria for acceptability: 

The data generated will be considered acceptable if: 

a. The spontaneous revertant frequency is in the normal range as reported 

in the literature or within the laboratory's historical range. 

b. A sufficient number of nontoxic concentrations have been tested. 

c. The strain-specific positive mutagens significantly increase the revertant 

in the corresponding strains. 

2. Criteria for interpretation: 

a. Positive result: 

A compound will be considered positive in this assay if a dose-dependent increase in the number 

of revertants is observed in three concentrations, or the highest increase equal to two times the 

spontaneous control value (Brusick and Hayes, 1994). Sometimes the precise fold increase will 

not be necessary if a clear dose-dependent pattern is noted over several concentrations. A positive 

result in Salmonella/microsome mutagenesis indicates that under the experimental conditions, the 

test compound induces point mutation by base changes or frameshift in the genome of this 

organism. 

b. Negative result: 

A test agent will be considered negative in this assay if the criteria for positive response are not 

met, and the tester strains are sensitive to the positive mutagens. 

A negative result indicates that under the experimental conditions, the test compound is not 

mutagenic in Salmonella typhimuriumm 
46 

III. Identification, Handling and Storage and of the Test Agent 

The test agent, eg.2-bromo, 3-3-3 trifluro propene will be provided by the sponsor in liquid form. 

All chemical and physical identifications will be verified and information will be provided by the 

sponsor. The compound is presumed to be soluble in DMSO and stable at ambient temperature. 

It will be kept in original container and stored at appropriate temperature, avoiding direct sunlight 

and sudden temperature rise. Safety glasses, rubber gloves, and protective clothing will be used 

for handling. 

IV. Good Laboratory Practice and Quality Assurance 

All assays will be conducted in accordance with the provisions of the United States 

Environmental Protection Agency/Toxic Substances Control Acts (EPA/TSCA) Good Laboratory 

Practice (GLP) Standards as defined in the Federal Register (40 CFR, Part 792, 1998) and the 

TSCA Test Guidelines (40 CFR 798.5265,1998). All the procedures are performed in accordance 

with the Standard Operating Procedures (SOPs) of ManTech Environmental for the 

Salmonella/microsome mutagenesis assay. 

The Quality Assurance Auditor of ManTech Environmental Inc. will document inspections on all 

procedures used in this study. After the initiation of the study, modifications of the protocol will 

be in the form of Protocol Amendments, which will state the specific modifications and the 

reasons for the modifications. 

V. Schedule 

In the starting phase (about 4 weeks), all the test agents, media, equipments and tester bacteria 

will be ordered. The genotypes of the tester strains will be confirmed. The prescreening studies 

for dose selection will also be conducted in this period. The mutagenesis assay will be completed 

within 4 weeks including an independent confirmatory experiment. Another two weeks will be 

contributed to data analysis and report preparation. 

VI. Reports and Deliverables

The technical part of the study is to be completed after 2 months of receiving the test material. A 

draft final report will be submitted after 2 weeks of completion of the study and the final report 

will be submitted one week after receiving review comments on draft final report from the 

sponsor. The final report constitutes the study’s deliverable and ManTech considers acceptance 

of the deliverable to occur when it is received by U.S. Air force. 
47 

VII. References:

A. Araki, T. Noguchi, F. Kato and T.Mateushima (1994) Improved method for mutagenecity 

testing of gaseous compounds by using a gas sampling bag. Mutation Research 307: 335-344. 

Brusick D. (1994) In: Principles and Methods of Toxicology, Second Edition, A.W. Hayes Ed., 

Raven Press, NY, p545. 

D.M. Maron and B.N. Ames (1983) Revised methods for the Salmonella mutagenicity test. 

Mutation Res., 113, 173-215. 

E. Zeiger, B. Anderson, S. Haworth, T. Lawlor and K. Mortelmans (1992) Salmonella 

Mutagenecity Test: V. Results from the Testing of 311 chemicals. Environmental and Molecular 

Mutagenesis, Vol 19, 21: 2-141 

R. A. Pegram, M. E. Anderson, S> H. Warren, T.m M. Ross and L. D. Claxton (1997) 

Glutathione S- Transferase - mediated mutagenecity of Trihalomethans in salmonella 

typhimurium: Contrasting results with bromodichloromethane and chloroform. Toxicology and 

Applied Pharmacology 144: 183-188. 

T.J Hughes, D. M. Simmons, L.G.Monteith, and L.D.Claxton (1987) Vaporization technique to 

measure mutagenic activity of volatile organic chemicals in the Ames/Salmonella assay. 

Environmental Mutagenesis 9:421-441.