Guidance for Industry 

Criteria for Safety and Efficacy 

Evaluation of Oxygen Therapeutics as 

Red Blood Cell Substitutes

DRAFT GUIDANCE 

This guidance is being distributed for comment purposes only. 

Submit comments on this draft guidance by the date provided in the Federal Register notice 

announcing the availability of the draft guidance. Submit comments to the Division of Dockets 

Management (HFA-305), Food and Drug Administration, 5630 Fishers Lane, rm. 1061, 

Rockville, MD 20852. You should identify all comments with the docket number listed in the 

notice of availability that publishes in the Federal Register. 

Additional copies of this draft guidance are available from the Office of Communication, 

Training and Manufacturers Assistance (HFM-40), Suite 200N, 1401 Rockville Pike, Rockville, 

MD 20852-1448, or by calling 1-800-835-4709 or 301-827-1800, or from the Internet at 

http://www.fda.gov/cber/guidelines.htm. 

For questions on the content of this guidance, contact, contact Toby Silverman, M.D. at 301-827-

3524. 

U.S. Department of Health and Human Services 

Food and Drug Administration 

Center for Biologics Evaluation and Research 

October 2004
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Table of Contents 

I. PURPOSE AND RATIONALE FOR RECOMMENDATIONS.................................. 1

II. SCOPE OF RECOMMENDATIONS............................................................................. 1

III. BACKGROUND DISCUSSION...................................................................................... 2

A. General....................................................................................................................... 2

B. Safety Considerations ............................................................................................... 2

C. Efficacy Considerations............................................................................................ 6

1. Local Effects/Regional Perfusion ....................................................................... 7 

2. Perioperative Indications .................................................................................... 8 

3. Trauma ................................................................................................................ 8 

IV. RECOMMENDATIONS.................................................................................................. 9

A. Preclinical Evaluation............................................................................................. 10

1. Characterization of the Product ........................................................................ 10 

2. Animal Safety Testing ...................................................................................... 11 

B. Clinical Evaluation.................................................................................................. 13

1. General.............................................................................................................. 13 

2. Elective Surgery................................................................................................ 15 

3. Trauma .............................................................................................................. 16 
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Guidance for Industry 

Criteria for Safety and Efficacy Evaluation of Oxygen Therapeutics 

as Red Blood Cell Substitutes 

This draft guidance, when finalized, will represent the Food and Drug Administration’s (FDA’s) 

current thinking on this topic. It does not create or confer any rights for or on any person and does 

not operate to bind FDA or the public. You can use an alternative approach if the approach 

satisfies the requirements of the applicable statutes and regulations. If you want to discuss an 

alternative approach, contact the appropriate FDA staff. If you cannot identify the appropriate 

FDA staff, call the appropriate number listed on the title page of this guidance. 

I. PURPOSE AND RATIONALE FOR RECOMMENDATIONS 

The development of oxygen therapeutics for a variety of clinical applications has progressed 

rapidly in recent years. The potential benefits of an oxygen therapeutic include universal 

compatibility, immediate availability, and long-term storage. This guidance document provides 

you, as a sponsor or investigator, with suggested criteria for testing the efficacy and safety of 

oxygen therapeutics as substitutes for red blood cells, and guidance on the design of clinical 

trials to assess risk/benefit ratio of such use. 

The term “blood substitutes” has often been used to describe oxygen therapeutics, although to 

date, these products are not replacements for whole blood, either in terms of its many 

components or the duration of oxygen-carrying capacity. While this guidance document is 

restricted to use of oxygen therapeutics as substitutes for red cells, that is not the only indication 

being evaluated for these investigational new drugs. This guidance should not discourage 

innovation in the development of appropriate endpoints for and the design of clinical trials for 

other uses of oxygen therapeutics. 

FDA’s guidance documents, including this guidance, do not establish legally enforceable 

responsibilities. Instead, guidances describe the FDA’s current thinking on a topic and should be 

viewed only as recommendations, unless specific regulatory or statutory requirements are cited. 

The use of the word should in FDA’s guidances means that something is suggested or 

recommended, but not required. 

II. SCOPE OF RECOMMENDATIONS 

We, the FDA, are providing current recommendations applicable to the development and 

preclinical or clinical evaluation of oxygen therapeutics as blood (red blood cell) substitutes. 

This guidance, when finalized, will supercede the “Points to Consider on the Safety Evaluation 
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of Hemoglobin-Based Oxygen Carriers” dated August 27, 1990, and replaces the draft 

“Guidance for Industry: Efficacy Evaluation of Hemoglobin- and Perfluorocarbon-Based 

Oxygen Carriers” dated September 1997. This guidance is based, in part, on presentations and 

discussions at a workshop entitled “Criteria for Safety and Efficacy Evaluation of Oxygen 

Therapeutics as Red Cell Substitutes” held on September 27-28, 1999. The workshop was 

sponsored by FDA's Center for Biologics Evaluation and Research (CBER), the National Heart, 

Lung, and Blood Institute, the Department of Defense, U.S. Army Medical and Material 

Command, and the Armed Services Blood Program Office. The transcript of this workshop is 

available on the CBER Web Page at www.fda.gov/cber/minutes/workshop-min.htm. We would 

like to acknowledge the participation and contributions of the members of the workshop steering 

committee, workshop panelists, and other speakers whose names are listed in the transcript of the 

workshop proceedings. 

For general guidance, please consult the FDA Web Page, www.fda.gov. This site contains 

information and Internet addresses for other relevant FDA Guidance documents, International 

Committee on Harmonization (ICH) documents, and direct links to the homepage for CBER. 

III. BACKGROUND DISCUSSION 

A. General 

Oxygen therapeutics are derived from hemoglobin- or fluorochemical-based compounds. 

The starting material for hemoglobin-based oxygen carriers may be a stroma-reduced 

hemoglobin, commonly referred to as stroma-free hemoglobin (SFH), 

chromatographically purified hemoglobin obtained from sources including outdated 

human blood or bovine blood, or recombinant hemoglobin. Stable and functional oxygen 

therapeutics are produced from these starting materials by various chemical and/or 

genetic manipulations. The resulting products include intra-tetrameric cross-linked 

hemoglobin, polymers of hemoglobin tetramers (intra- and inter-cross-linked), 

hemoglobin tetramers conjugated to non-protein macromolecules, or genetically 

stabilized tetramers. 

Fluorochemical-based oxygen therapeutics are compounds in which hydrogen atoms on 

cyclic or straight chain hydrocarbons are replaced with fluorine. Some compounds also 

contain other atoms such as bromine. These compounds are emulsified in electrolyte 

solutions containing surface-active agents (surfactants) (perfluorochemical emulsions). 

Fluorochemical-based compounds are capable of dissolving large quantities of oxygen 

(without binding) at high concentrations of inspired oxygen. 

B. Safety Considerations 

A number of new and largely unresolved safety-related problems have arisen during the 

preclinical and clinical development of the current generation of hemoglobin-based 

products and perfluorochemical emulsions. The mechanisms of various observed 
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toxicities have not been elucidated fully. There is evidence to suggest that unmodified 

hemoglobin, which is filtered through the renal glomerulus, is nephrotoxic. This 

evidence suggests that unmodified hemoglobin (i.e., native tetrameric hemoglobin, mw - 

64,000) should not be present in the final product and should be removed to the 

maximum extent possible. However, nephrotoxicity does not explain all the effects 

observed when hemoglobin-based oxygen carriers are infused. For hemoglobin-based 

oxygen carriers, the recommended testing scheme is based on the hypothesis that one 

cause of toxicity involves the interaction of oxygen radicals or iron with cellular 

metabolism. Thus, the modified oxygen carrying capacity of hemoglobin-based oxygen 

carriers could affect safety as a result of excess oxygen supply to cells and tissues, 

deficient supply, or oxidative effects of the hemoglobin moiety itself. Similarly, the 

toxicities observed in animals following administration of perfluorochemical emulsions 

may also be clinically significant. Activation of complement and procoagulant cascades 

may affect the safety of perfluorochemical emulsions and result in dose limiting toxicities 

in clinical practice. For both hemoglobin-based oxygen carriers and perfluorochemical 

emulsions, it is hypothesized that the effects seen are in part the result of activating any 

number of triggered enzyme or cellular systems. 

Current oxygen therapeutic research efforts are aimed at gaining a better understanding of 

reactions in humans and developing safe and effective products. Following is a list of 

toxicities and laboratory findings known or thought to be associated with the use of 

hemoglobin-based oxygen therapeutics. The list is not intended to be all-inclusive, and it 

also includes some hypothetical toxicities that have not been demonstrated 

experimentally or clinically, but which, nevertheless, merit consideration. 

1. Vasoactivity 

Almost all hemoglobin products, regardless of their molecular weight distribution, 

are associated with vasoactivity in humans and in animal models. The etiology 

and clinical consequences of the vasoactive property of hemoglobin are still 

controversial and not completely understood. One hypothesis is that the 

interactions between cell-free hemoglobin and nitric oxide (NO), the 

endothelium-derived relaxing factor, may be a primary event that contributes to a 

vascular inflammatory response progressing to multi-organ failure. Other 

hypotheses suggest that cell-free hemoglobins modulate adrenergic receptor 

sensitivity and stimulate endothelin-1, a peptide with vasoconstrictor activity. 

Even modified hemoglobin products with small effects on blood pressure may 

result in significant vasoconstriction and increased vascular resistance. 

2. Cardiac Toxicity 

Administration of a hemoglobin-based oxygen carrier has been reported to be 

associated with the development of myocardial lesions characterized by mild to 

moderate focal and multifocal degeneration and/or necrosis of cardiac myocytes 

in certain animal models. The lesions have been reported to be seen 24-48 hours 
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after a single infusion of product. Left ventricular myocardium near the base of 

the papillary muscle is most prominently affected, with other affected regions 

including the interventricular septum and the right ventricle. Animal species most 

commonly affected include rhesus monkey and pig. There are significant animal 

species differences in sensitivity to the effect of the oxygen therapeutic; dogs, 

sheep, and rats did not develop the lesion after single infusion of the hemoglobinbased oxygen carrier. 

3. Gastrointestinal Toxicity 

a. Discomfort. 

Symptoms reported in clinical trials include nausea, vomiting, dysphagia, or 

generalized abdominal pain. These symptoms are believed to be related to NO 

scavenging by hemoglobin, causing localized spasm throughout the 

gastrointestinal tract. 

b. Bacterial Translocation. 

Animal experiments indicate changes in the architecture of the intestinal 

microvilli within minutes of infusion of some hemoglobin-based oxygen carriers. 

These experiments suggest the possibility that the incidence or the severity of 

bacterial translocation across the gut epithelium may be increased. 

4. Pro-inflammatory Activity 

Infusion of hemoglobin in small animals stimulates monocyte procoagulant 

activity resulting in disseminated intravascular coagulation. Early in vivo studies 

in rabbits, but not in other animal species, demonstrated pulmonary arteritis and 

thrombotic lesions possibly related to procoagulant activity. In vitro studies on 

the effect of hemoglobin on leukocytes in whole blood demonstrate that in the 

presence of other blood components, leukocytes release pro-inflammatory 

cytokines such as tumor necrosis factor (TNF) and interleukin-8 (IL-8). 

5. Oxidative Stress 

Numerous in vitro and some in vivo reports suggest that hemoglobin solutions 

may induce an oxidative stress. Oxidative stress may, in part, be explained by the 

ability of hemoglobin to serve as a source of toxic oxygen species and/or the 

ability of hemoglobin to remove nitric oxide, an important component of normal 

antioxidant mechanisms. Indirect evidence in humans of such effects is seen with 

reports of increased enzyme activity including creatine phosphokinase (CK), 

lactic dehydrogenase (LDH), and pancreatic enzymes, lipase and amylase (see 

below). 
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6. Pancreatic and Liver Enzyme Elevation 

Reports of elevated pancreatic and liver enzymes in a number of animal models of 

exchange transfusion suggest possible free-radical mediated injury. Increased 

levels of amylase and lipase after hemoglobin infusions in animals and humans 

have been frequently observed. The pattern and magnitude of lactate, CK, and 

LDH elevations seen in animal studies are similar to those reported in humans and 

also suggest possible free-radical mediated injury. 

 

7. Endotoxin Synergy with Hemoglobin 

Endotoxin, a bacterial product, and hemoglobin have been shown to exert 

synergistic toxicity when hemoglobin is given in a clinically relevant dose as a 

resuscitative fluid. Direct biochemical interactions between hemoglobin and 

endotoxin have been shown to reduce clearance of endotoxin from the circulation 

and to enhance lethality in some animal models of sepsis. This is a worrisome 

feature since these products may be given in some instances to patients with 

ongoing infectious processes or to trauma victims with contaminated wounds. 

8. Neurotoxicity 

Although experimental data suggest that acellular hemoglobins scavenge NO and 

may inhibit NO-related neurotoxicity, numerous published reports implicate free 

hemoglobin in the degenerative changes in the brain in experimental models of 

subarachnoid hemorrhage. Direct neurocytotoxicity of modified hemoglobin is 

suggested by pre-clinical studies in which neurons in culture were killed by the 

addition of hemoglobin, leaving glial cells intact. In one clinical report, use of an 

acellular hemoglobin product was an independent predictor of an unfavorable 

outcome at three months following acute ischemic stroke. The mechanism of this 

clinical toxicity has been hypothesized to be, in part, due to the potent 

vasoconstrictor effect of endothelin-1 which was increased in a dose-dependent 

manner by the administration of the acellular hemoglobin product. 

Toxicities known or thought to be associated with one or more of the current 

perfluorochemical emulsions include the following:

1. Thrombocytopenia 

The basis for this side-effect in animals and humans is not fully understood, but 

may be related to metabolism and normal clearance process of these compounds. 

Fluorochemical compounds may lead to some changes in the surface 

characteristics of platelets and subsequent uptake of the altered platelets by the 

liver and the spleen. Platelets may appear “functional” in aggregation and 

bleeding time measurements in vitro, but may potentially have a short half-life in 

circulation even though platelet production is normal. 
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2. Complement Activation and Cytokine Release 

At present, although a hypothetical toxicity, there are no experimental data to 

suggest that complement activation occurs with the use of perfluorochemical 

emulsions. 

3. Reticuloendothelial Blockade 

After intravenous administration, the droplets of perfluorochemical emulsion are 

taken up by the reticulo-endothelial system. It has been hypothesized that 

administration of perfluorochemical emulsions may affect ability to clear 

circulating bacteria. 

4. “Flu-like” Symptoms 

Administration of perfluorochemical emulsions in humans has been associated 

with the development of flu-like symptoms and transient elevations in 

proinflammatory cytokines. The etiology of this phenomenon is not known. 

5. Central Nervous System Effects 

Cerebrovascular accident has occurred in the context of administration of 

perfluorochemical emulsion. The pathophysiologic basis for the association has 

not been elucidated. 

C. Efficacy Considerations 

In addition to the safety considerations described above, oxygen therapeutic products 

present several issues relating to efficacy. Efficacy endpoints may be direct measures of 

clinical benefit (improved survival, alleviation of symptoms) or they may be laboratory 

measurements or physical signs expected to correlate meaningfully with clinical benefit. 

The latter are referred to as surrogate endpoints and, once validated, are especially 

important in the case of oxygen therapeutics since direct demonstration of efficacy is 

likely to be very difficult (as it has been for red blood cells, per se). Validation of a 

surrogate endpoint for a therapy includes generation of clinical data demonstrating that 

effects of the therapy on the surrogate endpoint are reasonably likely to predict clinical 

benefit. See 21 CFR 601.41. Factors of importance when considering acceptability of 

surrogate endpoints include feasibility of using direct clinical measurements, risk/benefit 

assessments, and perhaps most importantly, knowledge and understanding of the disease 

and of the agent. 

There has been extensive clinical experience with red cell transfusion, resulting in a 

practical appreciation of relevant indications, benefits, and risks. There is also an 

extensive collection of data on red blood cells, the anemic state, and their interaction, 
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resulting from years of basic and applied research. Thus, although it is not possible to 

document the clinical benefit of all red cell transfusions with specific endpoints, the 

available knowledge relevant to such transfusions support use of surrogate endpoints 

such as the P50, the oxygen content and the hematocrit as suitable endpoints to 

demonstrate efficacy of red cell transfusions in clinical practice and in some clinical 

trials. Currently, we do not consider these surrogate endpoints to be acceptable as 

measures of the effects of hemoglobin- and perfluorochemical-based red cell substitutes, 

because knowledge of the effects of hemoglobin- and perfluorocarbon-based red cell 

substitutes and of the interaction of these agents with various clinical states is 

rudimentary. Further, no oxygen carrier presently approved by FDA has all the 

properties of the human red cell, nor are any two products identical. We recommend that 

the endpoints used in clinical studies of these agents be selected with these caveats in 

mind. 

Under certain circumstances, oxygen therapeutic agents may qualify for Fast Track 

designation. Criteria and considerations for Fast Track designation may be found in 

“Guidance for Industry: Fast Track Drug Development Programs. Designation, 

Development, and Applicataion Review” dated September 1998. The guidance 

document may be found at http://www.fda.gov/cber/guidelines.htm. 

There are several potential indications for oxygen therapeutics. Below, we discuss the 

following three such uses for these products: 1) local effects/regional perfusion, 2) 

perioperative indications and 3) trauma. As noted earlier, this discussion is not intended 

to represent the only approaches to evaluating clinical use of oxygen therapeutics, nor are 

investigators required to accept these categories. 

1. Local Effects/Regional Perfusion 

This category might best be defined by considering two examples: perfusion 

during coronary angioplasty and enhancement of tumor radiosensitivity. 

Perfusion, via the central lumen of a catheter used for percutaneous transluminal 

coronary angioplasty (PTCA), is an FDA-approved indication for a 

perfluorocarbon preparation (Fluosol). The data that supported this approval 

included clinical studies utilizing surrogate endpoints of left ventricular function 

that had been validated as clinically relevant by recognized cardiologic 

investigations. Future studies for this indication could conceivably utilize similar 

clinical trial design, with specific endpoints appropriately updated. The rationale 

for use of oxygen carriers (systemically administered) in therapy of neoplasms is 

based on the observation that increased tumor tissue oxygen tension will increase 

the sensitivity of tumors to radiation or to chemotherapy more than that of normal 

tissue. Demonstration of increased oxygen tension in the target tumor can 

function as an important supporting argument for efficacy, but would not be likely 

to serve alone as the primary endpoint. Ultimately, we recommend that the 

endpoint used to establish efficacy be similar to that used in evaluation of 

cytotoxic agents for the stage and type of cancer under investigation. 
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2. Perioperative Indications 

Sponsors must monitor their investigations and evaluate pertinent risks to research 

subjects. (21 CFR 312.56.) In the case of investigational oxygen therapeutic 

products for proposed perioperative indications, such risks include the risks to 

subjects receiving the oxygen therapeutic in lieu of allogeneic transfusion (e.g.,

inferior perfusion, undesirable hemodynamic responses, and other adverse drug 

reactions). In situations where red blood cells are used, reduction in the use of 

allogeneic blood is a surrogate endpoint for the avoidance of the risks associated 

with the use of allogeneic blood including, but not limited to, viral transmission, 

incompatibility, etc. In the past, we have accepted reduction in the use of 

allogeneic blood (for instance, in the indications for erythropoietin). However, 

use of both allogeneic transfusion and oxygen therapeutics entail risks. 

Therefore, safety is a critical element in any evaluation of oxygen therapeutic 

products for elective surgical use. In the setting of elective perioperative use, a 

mere delay in the requirement for allogeneic transfusion without reduction in the 

use of allogeneic red blood cells would probably not be considered of benefit to 

patients. The category of peri- and post-operative use of oxygen therapeutics 

includes situations such as hemodilution (with or without autologous predonation) 

and intra- and post-operative replacement. We recommend the investigators be 

aware of the present lack of objective criteria to define a broadly applicable 

transfusion trigger and strive to develop and validate physiologic markers of 

efficacy for individual oxygen carriers. 

A trial to obtain an elective surgical indication alone, without evaluation of the 

product in unstable patients in a trauma setting, is unlikely to assure the safety of 

an oxygen therapeutic in elective surgical patients who become unstable or in 

trauma patients. While an oxygen therapeutic may appear to be safe in patients 

who are euvolemic and anemic, it cannot be assumed that such an oxygen 

therapeutic would be safe in hypovolemic or unstable patients, either surgical or 

trauma. 

3. Trauma 

In trauma, mortality is an unambiguous endpoint that many consider to be the 

most meaningful of the potential indications related to clinical benefit of oxygen 

therapeutics. Although short-term (e.g., 24-48 hours) survival is helpful in 

assessing the physiologic activity of an oxygen therapeutic, long-term survival is 

the primary clinical benefit of interest to the patient and the patient’s family. The 

benefit of short-term survival is limited if it does not lead to long-term survival. 

There is insufficient information at present to correlate short-term survival with 

long-term survival for oxygen therapeutics. 

In designing clinical trials for a potential trauma indication in the hospital setting, 

we recommend that you consider designs where patients are able to provide 
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consent for themselves or where consent can be obtained from a legally 

authorized representative. In the hospital setting, the use of an oxygen therapeutic 

would not be expected to result in a survival advantage over the use of red blood 

cells. Rather, oxygen therapeutics may be interchangeable with red blood cells in 

the short-term. Many, if not all, recipients of the oxygen therapeutic will need 

administration of other transfusion blood components. A clinical trial to 

demonstrate noninferiority, as opposed to superiority, of an oxygen therapeutic to 

blood in terms of mortality might not qualify for exception from informed consent 

under 21 CFR 50.24. 

Historically, many researchers have considered field use as the most likely 

situation in which oxygen therapeutics could improve survival. However, in 

major urban areas with rapid transit to definitive care, there may only be a small 

percentage of patients for whom use of an oxygen therapeutic might provide a 

survival benefit. Some of the most seriously injured patients will die in spite of 

rapid availability of optimum definitive care and other, less seriously injured, 

patients will survive with current resuscitation measures. It is difficult to select 

deliberately and prospectively the small population of trauma patients for whom 

use of an oxygen therapeutic may provide a survival advantage. If a clinical trial 

is not designed to select deliberately and prospectively for this small subset of 

patients who are likely to benefit from treatment, a large number of patients 

would likely need to be tested for a survival advantage. Accordingly, many more 

subjects would receive an oxygen therapeutic agent than are likely to benefit from 

such use; therefore a careful overall safety assessment would be appropriate. In 

addition, for trauma patients who have also sustained head injuries, the 

heterogeneity in the severity of head injury may lead to mortality outcomes 

independent of the effect of blood loss or the use of an oxygen therapeutic agent. 

In rural areas or other situations where there may be prolonged delay to definitive 

care, there may be greater potential for an oxygen therapeutic to provide clinical 

benefit than in urban settings. Such studies are difficult to control and may pose 

complications of trial design and data analysis, due, for example, to practical 

considerations such as differences in the length of time required to transport a 

patient to the hospital. Nevertheless, in the rural setting, a temporary treatment 

that sustains adequate tissue oxygenation and aerobic metabolism prior to control 

of bleeding and/or prior to obtaining cross-matched blood may provide a clinical 

benefit. 

IV. RECOMMENDATIONS 

Given the considerations described above, we provide the following recommendations for 

assessing the safety and efficacy of oxygen therapeutic products: 
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A. Preclinical Evaluation 

We recommend that you consider the following general categories of safety testing when 

developing oxygen therapeutic products: 1) characterization of the product and 2) animal 

safety testing. 

1. Characterization of the Product 

We recommend that you perform a physicochemical characterization of the 

product using available modern technology. This may include, but need not be 

limited to, the following: 

a. Oxygen capacity (P50, Hill coefficient, Bohr, Chloride and CO2

effects). It would be most useful to determine the entire curve of bound 

oxygen as a function of PO2, at least over a physiologically relevant range 

(40-120 mm Hg). 

b. Optical spectrum 

c. High pressure liquid chromatography (HPLC) (size and chargebased), polyacrylamide gel electrophoresis (PAGE), sodium 

dodecylsulfate-PAGE (SDS-PAGE), iso-electric focusing (IEF) 

d. Lipids and lipid phosphate content and identification 

e. Endotoxin level 

f. Free iron content 

g. Methemoglobin, sulfhemoglobin , carbonmonoxy hemoglobin 

levels 

h. Colloid osmotic pressure, viscosity measured at various 

temperatures to mimic mild and moderate hypothermia 

i. pH 

j. Identification and quantitation of electrolytes and trace metals 

We recommend using in vitro biological assays with appropriate test systems that 

may include, but need not be limited to, the following: 

a. tests for generation of oxygen radicals 

b. tests for activation of triggered enzyme/cell systems (e.g., the 

complement/kinin/coagulation cascades, macrophage/neutrophil/platelet 

activation, mediator release [such as histamine, thromboxane metabolites, 

leukotrienes, interleukins]). 

The measurements used in these stages of testing may not involve all of the items 

listed or all stages of product manufacture. However, we recommend that you 

perform a sufficient number of tests on a sufficient number of independent 

batches to assure that the characterization is accurate, and that the properties of 

the product are consistent from batch to batch. 
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We recommend that you make an appropriate subset of these measurements on 

every lot of product and set appropriate specifications (criteria) for acceptability. 

Similarly, we recommend that you choose an appropriate subset of these 

measurements for inclusion in any stability study on the product that should be 

applied initially and after suitable storage intervals and under suitable storage 

conditions including accelerated, high temperature storage conditions. 

For a hemoglobin-based oxygen carrier, we recommend that you develop a 

potency assay that reflects the biological activity sought in clinical studies. For 

use as an oxygen therapeutic in lieu of blood (blood substitute), such a potency 

assay should include a measure of the ability of the hemoglobin product to load, 

carry, and unload oxygen reproducibly. 

2. Animal Safety Testing 

We recommend that you consider the following points when planning and 

executing safety studies of oxygen therapeutics in animals: 

General Recommendations Regarding Toxicology Testing 

a. Perform studies on several animal species and should include late 

effects. A large animal, such as the dog or pig, should be included. 

b. Design toxicology studies to induce toxic effects in the animal at 

some dose level. 

Evaluation of Immune Responses to Oxygen Therapeutic Products 

We recommend that you test for an immune response to the product, e.g., 

development of IgG or IgE response to product administration or appearance of 

delayed hypersensitivity on repeated exposure. 

3. Use of Animal Models in Toxicity Testing 

a. Volume overload and exchange transfusion experiments are of 

limited value in assessing safety. Though such experiments can contribute 

to determination of the dose/toxicity relationship or serve as a model for a 

blood replacement indication, they may not adequately reflect the 

circumstances in other clinical or clinical transfusion situations. 

b. While meaningful information can be obtained from studies in 

normal animals, special animal models will likely be needed to obtain a 

complete safety profile. The animal model, fully instrumented to measure 

cardiac and pulmonary function, should be stressed so as to resemble the 

clinical use of the oxygen therapeutic (e.g., volume depleted for 

resuscitative indication; ischemic model for PTCA; repetitive 
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administration for sickle cell disease; septic shock model for Systemic 

Inflammatory Response Syndrome [SIRS]; cardiopulmonary bypass 

model for cardiac surgery). We recommend that concomitant medications 

and other agents be included, e.g. contrast agents with hemoglobin-based 

oxygen carriers for PTCA indications. Controls should include use of 

approved oxygen carriers and plasma expanders. 

c. You should consider a model designed to produce reperfusion 

injury. Such a model would be a relevant test of clinical situations that 

involved ischemia. 

4. Important Observations in Animal Tests 

a. Evaluation of effects of hemoglobin solutions on microvascular 

circulation and on endothelium. 

b. Serum creatinine or blood urea nitrogen (BUN) levels alone may 

not suffice for evaluation of nephrotoxicity. Rather, evaluation of renal 

function is more appropriately evaluated by utilizing a battery of tests 

including, but not limited to: direct pressure or flow measurements to 

detect changes in the renal arterial bed (e.g., vasoconstriction); a 

combination of clearance tests (inulin or other suitable agent), and 

histological examination to evaluate glomerular function and structure. 

Because of the variable amounts of tubular secretion of creatinine in some 

animal models, the creatinine clearance will not be a satisfactory measure 

of glomerular function. 

c. Measurement (and characterization) of tubular proteinuria or 

enzymuria to determine the status of tubular function or to detect tubular 

damage. 

d. Blood chemistry assays, including: alanine aminotransferase 

(ALT), aspartate amino transferase (AST), CK, Troponin I or Troponin T, 

LDH, creatinine, BUN, and electrolytes. It is recognized that hemoglobin 

will cause interference with colorimetric assays. 

e. Hematological studies, including: hematocrit, hemoglobin, white 

blood count (WBC) with differential, platelet count, prothrombin time 

(PT), activated partial thromboplastin time (PTT), fibrinogen/fibrin split 

products, factor VIII. 

f. Gross and microscopic examination of all vital organs postmortem. 

We recommend that measurements of reliable markers of oxidative damage be 

incorporated in animal models performed as part of the preclinical evaluation of 

hemoglobin-based products. You should evaluate hemoglobin-based oxygen 

carriers in simple biochemical tests of pro-oxidant potential. We are 

recommending this with the recognition of the complex interrelated properties of 

hemoglobin and the various manifestations of injury thought to be mediated by 

iron and heme-mediated free-radical reactions. 
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We suggest that you perform studies in a primate model to evaluate cardiac 

toxicity. Such a model should be sensitive to detecting degenerative changes in 

cardiac myocytes. Histologic evaluation of sections of myocardium should 

include papillary muscle and interventricular septum including the conduction 

system. 

We recommend that you evaluate hemoglobin-based oxygen carriers for time- and 

concentration-dependent effects on neurons in culture. You should perform 

studies in animal models of stroke and/or head injury at a time when the bloodbrain barrier has been demonstrated to be open. 

For stroma-reduced hemoglobin used as source material for further chemical 

modification, you should determine the precise levels and effect on hemoglobin 

potency and stability of residual red cell enzymes that may interfere with oxygencarrying and/or redox activities of the hemoglobin products. 

We recommend that you evaluate and resolve interference of hemoglobin 

solutions with measurements of clinical laboratory parameters for all relevant 

clinical laboratory instrumentation. Colorimetric interference with a number of 

clinical laboratory assessments that are important for individual patient 

management may occur with hemoglobin-based oxygen therapeutics. 

Manufacturers of oxygen therapeutics should anticipate ongoing support of 

clinical laboratories and evaluation of the effects of either hemoglobin-based 

oxygen carriers or perfluorochemical emulsions on new instruments or methods 

of analyte determination. 

Regarding flurochemical products, we recommend that sponsors test for 

fluorochemical-related effects on platelets, in particular the normal survival time 

in circulation and markers of platelet function. 

B. Clinical Evaluation 

1. General 

We recommend that initial studies in humans use a low dose in well-hydrated 

normal volunteers. For the hemoglobin-based oxygen carriers, consideration may 

be given to concomitant phlebotomy, depending on the initial administered 

volume of the hemoglobin-based oxygen carrier. You should monitor subjects 

carefully for at least circulatory and immune function. You should also monitor 

subjects receiving either perfluorochemical emulsions or hemoglobin based 

oxygen carriers for inflammatory mediators, e.g., complement/kinin/coagulation 

cascade, histamine release, thromboxane metabolites, and leukotrienes. If safety 

is clearly established in the initial phase of investigation, the dosage in normal 

volunteers may be carefully increased, with monitoring as indicated above. When 
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safety has been established in normal volunteers, appropriate patients may be 

considered, with the understanding that: 

Patients may be more vulnerable to adverse effects of the product than are normal, 

well-hydrated, healthy subjects 

The specific interactions (if any) between hemoglobin-based oxygen carriers or 

perfluorochemical emulsions and the disease process are poorly understood. 

Clinical situations where the use of blood is indicated differ, making it difficult to 

generalize results obtained from a trial conducted in one setting to other settings. 

Separate safety and efficacy data are therefore generally necessary for each 

clinical setting in which an oxygen therapeutic is needed and for which an 

indication (claim) is sought. The indication for use section of the package insert 

is expected to include information about the clinical setting and the patient 

population. We recommend that you prospectively define the indications sought 

for the use of an oxygen therapeutic before trials are performed, and these claims 

should be amenable to study using outcomes that are direct measures of clinical 

benefit or validated surrogates. 

We recommend a clinical development plan that includes safety and efficacy 

assessments in both trauma and elective surgical settings to gain a full 

understanding of the adverse event profile of an oxygen therapeutic. We believe 

an evaluation in both trauma and elective surgery will provide a full 

understanding of both the benefits and the risks of oxygen therapeutic use in the 

broadest spectrum of situations in which such products may be used. The agency 

believes, however, that clinical development plans for elective surgical 

indications alone may be appropriate in situations where the relative benefits of 

such use outweigh the risks. (See Section III.C.2.) Comments are invited 

regarding other appropriate claims, endpoints, and assessment tools. 

We recommend that clinical studies to evaluate the safety of oxygen therapeutics 

include assessments for all organ systems with particular emphasis on those 

organs and tissues known to be affected by one or more of these products as noted 

above. We recommend that you conduct such studies initially in controlled 

settings such as elective surgery before embarking on studies in unstable surgical 

patients or unstable trauma patients. Because many of the adverse events seen to 

date with oxygen therapeutics are qualitatively similar to adverse events that 

occur in the general surgical population, we recommend that you design clinical 

trials to capture a numerical increase and/or an increase in the intensity of adverse 

events above the underlying background rate/intensity of such events. 

We recommend that you exercise care in designing clinical trials to evaluate 

potential clinical utility in the treatment of patients with head trauma or in clinical 

settings that may be associated with disruption of the blood-brain barrier. We 
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recommend that you make provisions for complete neurologic assessment (before 

and after administration of any oxygen therapeutic) using the same trained clinical 

investigator to conduct the evaluations to minimize inter-observer variability in 

assessment. 

2. Elective Surgery 

We recommend that you evaluate oxygen therapeutics in patients with a wide 

variety of co-existing pathophysiologic processes to assess the safety of the 

products in a wide variety of co-morbid conditions. In a phase III surgery trial, 

we recommend that the population enrolled should reflect the characteristics of 

the population likely to undergo that particular surgery in clinical practice. In 

addition, as these products are likely to be used broadly in general surgery, the 

surgery studied in a phase III trial should be one in which the enrolled study 

population also reflects the characteristics of the general surgical population. 

We recommend that you consider studies in which complete avoidance of 

allogeneic transfusion during and after surgery would be considered one of the 

efficacy endpoints. To this end, we recommend that you conduct 

concentration/dose toxicity trials to determine the maximum tolerated dose of an 

oxygen therapeutic so that future patients will not be administered a toxic dose in 

attempts to avoid allogeneic transfusion. In this regard, you are encouraged to use 

non-linear mixed-effects modeling (NONMEM). If the primary efficacy endpoint 

is avoidance of allogeneic red blood cell transfusion, we recommend that you also 

evaluate total allogeneic exposure (all allogeneic blood components administered) 

as a secondary endpoint. While it may not be possible to substitute completely an 

oxygen therapeutic agent for red blood cells in all patients undergoing elective 

surgery, a suitable trial design should specify and confirm enrollment of patients 

requiring two or more units of red blood cells. 

We recommend that you develop and validate clinical guidelines that are 

routinely available at the bedside for dosing both individual oxygen therapeutics 

and red blood cells in the face of circulating oxygen therapeutic agents. In 

patients treated with an oxygen therapeutic agent, hematocrit will not reflect 

accurately the clinical status of the bleeding patient. Hemoglobin-based oxygen 

therapeutics may be oncotically active, thereby precluding the use of routine 

measures such as total hemoglobin as a reflection of the need for additional 

transfusion/infusion. Perfluorochemical emulsions are functionally distinct from 

red blood cells in the manner in which they load, carry, and unload oxygen and 

will therefore need careful consideration of dosing recommendations and infusion 

criteria. 

Of course, sponsors must assure that clinical trial subjects and investigators are 

fully informed about the risks of an oxygen therapeutic, given that there are also 

uncertain risks associated with the use of red blood cells in clinical practice. See 
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21 CFR 312.53(c)(vi)(d), 312.55(b) Because a clinical trial to assess the relative 

safety of an oxygen therapeutic compared to red blood cells with statistical 

significance could require a very large sample size, at this time we are willing to 

accept a modest level of uncertainty as to whether the oxygen therapeutic is 

actually as safe as red blood cells. 

3. Trauma 

You are encouraged to evaluate and validate surrogate markers that may correlate 

with the mortality endpoint. Surrogate markers/endpoints may be laboratory 

measurements or physical signs that are expected to correlate with clinical benefit. 

At present, there is no general agreement about the usefulness of individual, easily 

measured surrogate markers to predict clinical outcome in trauma. 

We recommend that in a setting where blood is available, a trial to evaluate 

clinical noninferiority of an oxygen therapeutic to blood should provide for 

exclusive use of the oxygen therapeutic in lieu of red blood cells at least until 

bleeding is controlled. While noninferiority to red blood cells in clinical outcome 

(mortality and durable morbidity) may be sufficient for an indication (claim) in 

trauma, such a clinical trial will require a large sample size to assure that 

widespread use of the product is very unlikely to affect adversely mortality, 

durable morbidities, or other safety endpoints. As noted previously under section 

III.C., Efficacy Considerations, since many patients may not be able to provide 

informed consent and their legally authorized representatives may not be 

available, the clinical trial design must take into account the possibility that the 

patient population enrolled in the study might not adequately represent the patient 

population presenting at the hospital in actual clinical practice. 

In a direct study of safety and effectiveness of an oxygen therapeutic in the field 

setting, it would be expected that current approved asanguinous resuscitative 

solutions would be used in the control population and that survival would be the 

primary endpoint. To support a labeling claim in the field setting, survival 

(primary endpoint) in subjects receiving the oxygen therapeutic should be 

superior to survival in subjects receiving control. If the available data support the 

potential of the product for direct benefit to subjects, a field trial may meet the 

criteria for exception from informed consent under 21 CFR 50.24. It should be 

noted that in the urban setting, a large clinical trial would likely be needed to test 

for a superior survival outcome unless a subset of subjects with massive bleeding 

could be identified at baseline. It may be possible to collect information about red 

cell mass at baseline to identify such a subset of subjects. 

Alternatively, an indication (claim) of efficacy where blood is not available or 

cannot be used might also be supported by data from clinical trials in the hospital 

setting where blood is available and used in the control arm of a comparative 

study (see section III.B.3). It may be difficult to extrapolate data generated in the 
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Emergency Room/Operating Room setting where blood is available to the field 

situation where definitive control of bleeding is often not possible. Nevertheless, 

an oxygen therapeutic agent demonstrated to be safe and effective in the treatment 

of trauma or acutely massively bleeding patients in a hospital setting might be 

expected to provide an advantage over current asanguinous resuscitative fluids 

when definitive care is delayed and blood loss is life-threatening.