Setting Endotoxin Limits 
During Development of 
Investigational Oncology Drugs 
and Biological Products 
Guidance for Industry 
 
 
DRAFT GUIDANCE
 
 
  This guidance document is being distributed for comment purposes only. 
 
Comments and suggestions regarding this draft document should be submitted within 60 days of 
publication in the Federal Register of the notice announcing the availability of the draft 
guidance.  Submit electronic comments to https://www.regulations.gov.  Submit written 
comments to the Dockets Management Staff (HFA-305), Food and Drug Administration, 5630 
Fishers Lane, Rm. 1061, Rockville, MD  20852.  All comments should be identified with the 
docket number listed in the notice of availability that publishes in the Federal Register. 
 
For questions regarding this draft document, contact (CDER) Patricia Keegan at 301-796-1387, 
or (CBER) Office of Communication, Outreach and Development at 800-835-4709 or 240-402-
8010. 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Oncology Center of Excellence (OCE) 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
 
 
July 2020 
Clinical/Medical 
 
 
Endotoxin Draft Guidance   
Setting Endotoxin Limits 
During Development of 
Investigational Oncology Drugs 
and Biological Products 
Guidance for Industry 
 
 
Additional copies are available from: 
 
Office of Communications, Division of Drug Information  
Center for Drug Evaluation and Research 
Food and Drug Administration 
10001 New Hampshire Ave., Hillandale Bldg., 4th Floor  
Silver Spring, MD 20993-0002  
Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353 
Email: druginfo@fda.hhs.gov 
https://www.fda.gov/drugs/guidance-compliance-regulatory-information/guidances-drugs 
and/or 
Office of Communication, Outreach, and Development  
Center for Biologics Evaluation and Research 
 Food and Drug Administration 
10903 New Hampshire Ave., Bldg. 71, Rm. 3128 
Silver Spring, MD 20993-0002 
Phone: 800-835-4709 or 240-402-8010 
Email: ocod@fda.hhs.gov 
https://www.fda.gov/vaccines-blood-biologics/guidance-compliance-regulatory-information-biologics/biologics-guidances 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Oncology Center of Excellence (OCE) 
Center for Drug Evaluation and Research (CDER) 
Center for Biologics Evaluation and Research (CBER) 
 
 
July 2020 
Clinical/Medical 
 
 
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
TABLE OF CONTENTS 
 
I.  INTRODUCTION............................................................................................................. 1 
II.  BACKGROUND ............................................................................................................... 2 
III.  ENDOTOXIN CONTROL AND LIMITS...................................................................... 2 
IV.  SETTING ENDOTOXIN LIMITS DURING DRUG DEVELOPMENT ................... 4 
A.  Early Clinical Development .......................................................................................................... 4 
1. Parenteral (Excluding Intrathecal or Intraocular) Route of Administration .................................. 4 
2. Intrathecal Route of Administration ................................................................................................ 5 
B.  Late Stage Clinical Development .................................................................................................. 5 
REFERENCES .............................................................................................................................. 6 
 
  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
1  Setting Endotoxin Limits During Development of Investigational 
2  Oncology Drugs and Biological Products 
3  Guidance for Industry1 
4   
5   
6   
7  This draft guidance, when finalized, will represent the current thinking of the Food and Drug 
8  Administration (FDA or Agency) on this topic.  It does not establish any rights for any person and is not 
9  binding on FDA or the public.  You can use an alternative approach if it satisfies the requirements of the 
10  applicable statutes and regulations.  To discuss an alternative approach, contact the FDA staff responsible 
11  for this guidance as listed on the title page. 
12   
13   
14   
15  I.  INTRODUCTION 
16   
17  New approvals in oncology often build on prior success by adding new drugs to current regimens 
18  or by combining products in a novel treatment regimen, creating new multidrug regimens2 that 
19  may have greater efficacy.3 
20   
21  This guidance describes FDAs recommendations to investigational new drug sponsors for 
22  setting endotoxin limits during the development of investigational drugs intended for use in 
23  combination with other approved drugs or for the codevelopment of two or more investigational 
24  drugs. The scope of this guidance is limited to anticancer drugs, including combination products 
25  under 21 CFR Part 3,4 as described further in this guidance and administered parenterally (except 
26  for intraocular administration) to treat serious and life-threatening cancers based on histology or 
27  stage of disease. This guidance does not apply to the development of drugs for adjuvant or 
28  neoadjuvant treatment or for cancer subtypes that can be cured or where prolonged survival can 
29  be achieved with available therapy. 
30   
31  In general, FDAs guidance documents do not establish legally enforceable responsibilities.  
32  Instead, guidances describe the Agencys current thinking on a topic and should be viewed only 
33  as recommendations, unless specific regulatory or statutory requirements are cited. The use of 
                                                 
1 This guidance has been prepared by the Oncology Center of Excellence in cooperation with the Center for Drug 
Evaluation and Research and the Center for Biologics Evaluation and Research at the Food and Drug 
Administration. 
 
2 For the purpose of this guidance, regimen refers to two or more therapeutic products that are (or will be) marketed 
separately but are being tested for use in combination based upon one or more adequate and well-controlled trials. 
 
3 See the guidance for industry Codevelopment of Two or More New Investigational Drugs for Use in Combination 
(June 2013).  We update guidances periodically.  To make sure you have the most recent version of a guidance, 
check the FDA guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents. 
 
4 Additional considerations regarding endotoxin exposure may apply for delivery devices and combination products 
with device constituent parts under 21 CFR 812. 
  1  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
34  the word should in Agency guidances means that something is suggested or recommended but 
35  not required. 
36   
37   
38  II.  BACKGROUND 
39   
40  Investigational drugs for treating patients with incurable cancers who have short life expectancy 
41  are often studied in combination with approved drugs in an attempt to identify multidrug or 
42  multimodality regimens that may prolong survival. Administration of one or more drugs within 
43  the same 60-minute time frame as the investigational drug may pose challenges regarding the  
44  endotoxin limits for that investigational drug at a time when the manufacturing process for that 
45  investigational drug has not been optimized. This guidance addresses endotoxin limits for 
46  investigational drugs for the treatment of advanced cancer, when evaluated in early (pilot) 
47  clinical trials as part of a multidrug regimen. 
48   
49  FDA recognizes that based on the potential benefits of investigational drugs administered to 
50  patients with life-threatening incurable cancers, such patients possible exposure to increased 
51  levels of endotoxin exceeding the recommendations in USP General Chapter <85> Bacterial 
52  Endotoxins Test may be considered an acceptable risk under appropriate circumstances. 
53   
54  This guidance discusses FDAs thinking regarding setting endotoxin limits for investigational 
55  drugs during early product development for incurable cancers. The recommendations are 
56  consistent with the abbreviated product testing for feasibility clinical trials of monoclonal 
57  antibodies in patients with serious or immediately life-threatening conditions for which no 
58  effective alternative treatment exists.5 In this guidance, FDA recommends a risk-based approach, 
59  weighing the potential risks of possible exposure to increased levels of endotoxin across all 
60  components of a multidrug regimen against the potential benefits to patients with serious and 
61  life-threatening cancers. 
62   
63   
64  III.  ENDOTOXIN CONTROL AND LIMITS 
65   
66  Bacterial endotoxins are lipopolysaccharides found in the outer membrane of gram-negative 
67  bacteria. Endotoxins are released upon cell death and lysis and have the potential to contaminate 
68  drug and biological products. When administered in high amounts, endotoxins can cause 
69  pyrogenic reactions, severe inflammatory responses, septic shock, and death. Therefore, it is 
70  important to prevent the introduction of bacterial endotoxins into drug and biological products 
71  and their components (as defined in 21 CFR 201.3) during the product and component 
72  manufacturing processes, and to conduct appropriate testing for the presence of endotoxins on 
73  each batch of clinical and commercial product. Following are several guidances that refer to the 
74  need for pyrogen and endotoxin control and testing during sterile drug and biological product 
75  development: 
76   
                                                 
5 See Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use 
(February 1997). 
  2  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
77    Guidance for industry Content and Format of Investigational New Drug Applications 
78  (INDs) for Phase 1 Studies of Drugs, Including Well-Characterized, Therapeutic, 
79  Biotechnology-derived Products (November 1995) 
80   
81    Guidance for industry INDs for Phase 2 and Phase 3 StudiesChemistry, Manufacturing, 
82  and Controls Information (May 2003) 
83   
84    Guidance for reviewers and sponsors Content and Review of Chemistry, Manufacturing, 
85  and Control (CMC) Information for Human Somatic Cell Therapy Investigational New 
86  Drug Applications (INDs) (April 2008) 
87   
88     Guidance for industry Chemistry, Manufacturing, and Control (CMC) Information for 
89  Human Gene Therapy Investigational New Drug Applications (INDs) (January 2020) 
90   
91    Guidance for industry CGMP for Phase 1 Investigational Drugs (July 2008) 
92   
93   
94  Controls on pyrogen and endotoxins are part of the chemistry, manufacturing, and control 
95  information to be included in an investigational new drug, a new drug, or a biologics license 
96  application for most drug products (21 CFR 312.23(a)(7)(iv)(b), 21 CFR 314.50(d)(1)(ii)(a), and 
97  21 CFR 601.2(a) and (c), respectively). Similarly, current good manufacturing practice 
98  regulations also require control of pyrogen for containers and closures (see 21 CFR 211.94(c) 
99  and (d) and 21 CFR 600.11(h) and for most drugs (see 21 CFR 211.165(a), 21 CFR 211.167(a), 
100  and 21 CFR 610.13(b)). As described in the guidance for industry Pyrogen and Endotoxins 
101  Testing: Questions and Answers (June 2012), the requirement in 21 CFR 610.13 for rabbit 
102  pyrogen testing for certain biological products may be waived if a method equivalent to the 
103  pyrogen test is demonstrated in accordance with 21 CFR 610.9.6 
104   
105  Controls on pyrogen and endotoxins are part of the chemistry, manufacturing, and control 
106  information to be included in an investigational new drug, a new drug, or a biologics license 
107  application (see 21 CFR 312.23(a)(7)(iv)(b), 21 CFR 314.50(d)(1)(ii)(a), and 21 CFR 601.2(a) 
108  and (c), respectively). Similarly, current good manufacturing practice regulations also require 
109  control of pyrogen (see 21 CFR 211.94(c)(d), 21 CFR 211.165(a), 21 CFR 211.167(a), 21 CFR 
110  600.11(h), 21 CFR 610.9 (equivalent methods and processes), and 21 CFR 610.13(b)). 
111   
112  The compendial recommendations for threshold pyrogenic doses of endotoxins for injectable 
113  products are based upon the results of human and animal studies demonstrating the deleterious 
114  effects of bacterial endotoxins administration. United States Pharmacopeia (USP) General 
115  Chapter <151> Pyrogen Test provides a test method and acceptance criteria for the absence of 
116  pyrogens by measuring febrile reactions to injectable products. The endotoxin limits for 
117  parenteral drugs recommended in USP General Chapter <85> Bacterial Endotoxins Test are 
118  defined by the formula K/M, where K is the threshold pyrogenic dose of endotoxins per kg of 
                                                 
6 FDA supports the principles of the 3Rs, to reduce, refine, and replace animal use in testing when feasible. We 
encourage sponsors to consult with us if they wish to use a nonanimal testing method they believe is suitable, 
adequate, validated, and feasible. We will consider if the alternative method could be assessed for equivalency to an 
animal test method. 
  3  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
119  body weight or m2 of body surface area per hour and M is the maximum dose to be administered 
120  to the patient within a 60-minute period. K is equal to 5 EU (endotoxin units) per kg of body 
121  weight or 100 EU per m2 of body surface area per hour for nonintrathecal injections. K is equal 
122  to 0.2 EU per kg of body weight per hour for intrathecal injections. These compendial 
123  recommendations represent the upper limit for endotoxin content for injectable products so 
124  stricter limits may be warranted. 
125   
126  These values represent the maximum recommended exposure to endotoxins, based on the 
127  absence of clinically important increases in body temperature at these exposures. FDA expects 
128  that sponsors of investigational new drug applications will justify the proposed endotoxin 
129  acceptance criteria for each investigational therapeutic biologic, drug, or combination therapy 
130  based on manufacturing experience and established control strategies, such as careful selection 
131  of ancillary materials, aseptic processing, and the use of closed systems. 
132   
133   
134  IV.  SETTING ENDOTOXIN LIMITS DURING DRUG DEVELOPMENT 
135   
136  In keeping with the principles of facilitating drug development for serious and life-threatening 
137  diseases, this guidance outlines FDAs current thinking on a risk-based approach to setting 
138  acceptance criteria for endotoxins during the clinical development of drugs intended to treat 
139  serious and life-threatening cancers. Additionally, sponsors should refer to recommendations 
140  regarding testing discussed in the guidance for industry Pyrogen and Endotoxins Testing: 
141  Questions and Answers. 
142   
143  A.  Early Clinical Development 
144   
145  During early clinical trials (e.g., dose-finding or activity-estimating trials) conducted in patients 
146  with a serious and life-threatening cancer based on stage of disease and expected prognosis for 
147  that cancer subtype: 
148   
149  1.  Parenteral (Excluding Intrathecal or Intraocular) Route of Administration 
150   
151    For investigational products that are small molecules or certain therapeutic biological 
152  products,7 the endotoxin limits of an investigational drug or the combined endotoxin 
153  limits of multiple investigational drugs administered concomitantly by a parenteral route 
154  should not exceed the limit specified in USP General Chapter <85>, that is, 5 USP-EU 
155  per kg body weight per hour or 100 EU per m2 body surface area (BSA) per hour. The 
156  sponsor of an investigational new drug need not consider the potential endotoxin 
157  contribution from approved and/or licensed components of a combination regimen when 
158  calculating the acceptable limits for endotoxin exposure from the investigational drug. 
159   
160    For all other investigational products, including cellular therapy and gene therapy 
161  products, in order to assess causality of observed adverse events, the combined endotoxin 
162  exposure from all agents (investigational drug and other approved and/or licensed 
                                                 
7 See Transfer of Therapeutic Products to the Center for Drug Evaluation and Research (CDER) at 
https://www.fda.gov/AboutFDA/CentersOffices/OfficeofMedicalProductsandTobacco/CBER/ucm133463.htm. 
  4  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
163  concomitantly administered drugs) administered parenterally should not exceed the limits 
164  specified in USP General Chapter <85>; this will allow better identification of adverse 
165  reactions of investigational products that may overlap with the onset of and mimic the 
166  signs and symptoms of endotoxin exposure. 
167   
168  2.  Intrathecal Route of Administration 
169   
170  When investigational products will be administered intrathecally with other agents, the combined 
171  endotoxin exposure from all drugs (investigational drug and other concomitantly administered 
172  investigational, approved, or licensed drugs) should not exceed that specified in USP General 
173  Chapter <85>, that is, 0.2 USP-EU per kg body weight per hour. 
174   
175    In the rare case that the combined endotoxin exposure exceeds the limits described above, 
176  sponsors should justify that such limits cannot be achieved based on specific aspects of 
177  product manufacturing and provide a rationale to support a conclusion that the risks to 
178  human subjects are reasonable considering the preliminary evidence of clinical activity of 
179  the investigational product, the seriousness of the disease, and the availability of 
180  satisfactory alternative therapies. 
181   
182   
183  B.  Late Stage Clinical Development 
184   
185  During clinical trials intended to support the approval of an investigational drug to be 
186  administered concomitantly with other drugs: 
187   
188    Sponsors should tighten the specifications for endotoxin limits to ensure that by the time 
189  they submit a marketing application for that drug, the endotoxin limits will not exceed 
190  that specified in USP General Chapter <85> for a parenterally administered drug 
191  considering the combined endotoxin exposure of the investigational drug and all 
192  concomitantly administered drugs cited in the INDICATIONS AND USAGE section of 
193  product labeling. 
194   
195     
  5  Contains Nonbinding Recommendations 
Draft  Not for Implementation 
196  REFERENCES 
197   
198  Burrell R, Human Responses to Bacterial Endotoxin, 1994, Circ Shock, 43:137153. 
199   
200  Hochstein HD, EA Fitzgerald, FG McMahon, and R Vargas, 1994, Properties of US Standard 
201  Endotoxin (EC-5) in Human Male Volunteers. J Endotoxin Res, 1(1):5256. 
202   
203  Reeve AJ, S Patel, A Fox, K Walker, and L Urban, 2000,  Intrathecally Administered Endotoxin 
204  or Cytokines Produce Allodynia, Hyperalgesia and Changes in Spinal Cord Neuronal Responses 
205  to Nociceptive Stimuli in the Rat, Eur J Pain, 4(3):247257. 
206   
207  Simmons RL, TB Ducker, and AM Martin Jr., 1969, Comparative Pathology after Intrathecal 
208  Endotoxin in the Rabbit, Dog, and Monkey. Experentia, 25(6):622623.   
209   
210  Suffredini AF, RE Fromm, MM Parker, M Brenner, JA Kovacs, RA Wesley, and JE Parrillo, 
211  1989, The Cardiovascular Response of Normal Humans to the Administration of Endotoxin, N 
212  Engl J Med, 321(5):280287. 
213   
214  Suffredini AF and RJ Noveck, Human Endotoxin Administration as an Experimental Model in 
215  Drug Development, 2014, Clin Pharmacol Ther, 96(4):418422. 
  6