Osteoporosis:  
Nonclinical Evaluation 
of Drugs Intended for 
Treatment 
Guidance for Industry 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
August 2019 
Pharmacology/Toxicology 
   
Osteoporosis:  
Nonclinical Evaluation 
of Drugs Intended for 
Treatment 
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 
 
 
 
 
 
 
 
 
 
 
 
U.S. Department of Health and Human Services 
Food and Drug Administration 
Center for Drug Evaluation and Research (CDER) 
 
August 2019 
Pharmacology/Toxicology 
   
TABLE OF CONTENTS 
 
I.  INTRODUCTION............................................................................................................. 1 
II.  BACKGROUND ............................................................................................................... 2 
III.  NONCLINICAL STUDIES.............................................................................................. 2 
A.  Toxicology Studies ......................................................................................................................... 2 
B.  Bone Quality Studies...................................................................................................................... 3 
1. General ............................................................................................................................................ 3 
a.  Animal models .......................................................................................................................... 3 
b.  Animal species .......................................................................................................................... 3 
c.  Osteoporosis indications ........................................................................................................... 3 
2. Study Design .................................................................................................................................... 3 
a.  Dose selection ........................................................................................................................... 3 
b.  Dosing regimen and administration route ................................................................................. 4 
c.  Treatment initiation and duration .............................................................................................. 4 
d.  Data analysis ............................................................................................................................. 4 
3. Evaluations ...................................................................................................................................... 4 
a.  Bone turnover ............................................................................................................................ 4 
b.  Bone mass and density .............................................................................................................. 5 
c.  Bone structure and architecture ................................................................................................. 5 
d.  Bone strength ............................................................................................................................ 5 
e.  Additional evaluations .............................................................................................................. 6 
C.  Biologics .......................................................................................................................................... 6 
D.  Anabolic Agents ............................................................................................................................. 7 
IV.  REGULATORY ASPECTS ............................................................................................. 7 
REFERENCES .............................................................................................................................. 8 
 
  Contains Nonbinding Recommendations 
1  Osteoporosis:  Nonclinical Evaluation of  
2  Drugs Intended for Treatment 
3  Guidance for Industry1 
4   
5   
6   
7   
8  This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on 
9  this topic.  It does not establish any rights for any person and is not binding on FDA or the public.  You 
10  can use an alternative approach if it satisfies the requirements of the applicable statutes and regulations.  
11  To discuss an alternative approach, contact the FDA office responsible for this guidance as listed on the 
12  title page. 
13   
14   
15   
16   
17  I.  INTRODUCTION 
18   
19  The purpose of this guidance is to provide recommendations to industry for designing 
20  nonclinical bone quality studies to support the approval of drugs and biologics intended for the 
21  treatment of osteoporosis.2 
22   
23  We recommend sponsors review the following guidances for industry before initiating clinical 
24  trials of drugs intended to treat osteoporosis:3 
25   
26    General Considerations for the Clinical Evaluation of Drugs (January 1997) 
27   
28    Providing Clinical Evidence of Effectiveness for Human Drug and Biological Products 
29  (May 1998) 
30   
31    Study of Drugs Likely to be Used in the Elderly (November 1989) 
32   
33  In general, FDAs guidance documents do not establish legally enforceable responsibilities.  
34  Instead, guidances describe the Agencys current thinking on a topic and should be viewed only 
35  as recommendations, unless specific regulatory or statutory requirements are cited.  The use of 
36  the word should in Agency guidances means that something is suggested or recommended, but 
37  not required. 
                                                 
1 This guidance has been prepared by the Division of Bone, Reproductive, and Urologic Products in the Center for 
Drug Evaluation and Research at the Food and Drug Administration.   
 
2 For the purposes of this guidance, drugs refers to drug and biological products regulated in CDER. 
 
3 We update guidances periodically.  To make sure you have the most recent version of a guidance, check the FDA 
Drugs guidance web page at https://www.fda.gov/drugs/guidance-compliance-regulatory-information/guidances-
drugs. 
  1  Contains Nonbinding Recommendations 
38  II.  BACKGROUND 
39   
40  In addition to the pharmacology and toxicology studies required for all new drugs,4,5  long-term 
41  nonclinical studies to evaluate bone tissue (bone quality studies) should be conducted for drugs 
42  intended to treat osteoporosis.  These studies are warranted because of concerns about long-term 
43  adverse effects of pharmaceutical agents on bone quality (Harris et al. 1993; Kleerekoper and 
44  Vieth 1996; Van der Meulen and Boskey 2012) and because there are no validated and reliable 
45  methods for the noninvasive assessment of bone quality in humans.  Bone quality refers to those 
46  structural and material properties of bone that determine its biomechanical behavior in ways that 
47  are not accounted for by bone quantity or mass (Hernandez and Keaveny 2006).  An adverse 
48  effect on bone quality can be identified by an unfavorable change in the correlation between 
49  bone mass (i.e., bone mineral density (BMD) or bone mineral content (BMC)) and bone strength.  
50  The nonclinical bone quality studies are intended to evaluate this correlation and can provide 
51  support for the validity of BMD as a surrogate marker for fracture risk in clinical studies.  
52  However, increases in BMD and reductions in the incidences of bone fractures must still be 
53  established in clinical trials.6 
54   
55   
56  III.  NONCLINICAL STUDIES  
57   
58  A.  Toxicology Studies 
59   
60  Pharmacology and toxicology studies are needed to support clinical development of new drugs 
61  and biologics for osteoporosis indications.7  In addition to conducting these standard 
62  pharmacology and toxicology studies, the sponsor should conduct nonclinical bone quality 
63  studies for drugs intended to treat osteoporosis. 
64   
                                                 
4 21 CFR 312.23(a)(8) 
 
5 See the ICH guidances for industry S7A Safety Pharmacology Studies for Human Pharmaceuticals (July 2001), 
M3(R2) Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for 
Pharmaceuticals (January 2010), and S6(R1) Preclinical Safety Evaluation of Biotechnology-Derived 
Pharmaceuticals (May 2012). 
 
6 21 CFR 314.50(d)(2) and 21 CFR 314.50(d)(5) 
 
7 See the ICH guidances for industry S7A Safety Pharmacology Studies for Human Pharmaceuticals, M3(R2) 
Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for 
Pharmaceuticals, and S6(R1) Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals. 
  2  Contains Nonbinding Recommendations 
65  B.  Bone Quality Studies 
66   
67  1.  General 
68   
69  a.  Animal models8 
70   
71  Various animal models are available for the study of osteoporosis (Turner 2001; Jerome and 
72  Peterson 2001).  For the bone quality studies, the sponsor should select osteoporosis models that 
73  are relevant to the specific clinical indication for which the drug is being developed.  For 
74  postmenopausal osteoporosis, bone quality studies should be conducted in ovariectomized 
75  animals.  For other forms of osteoporosis, appropriate animal models (such as the mature 
76  orchidectomized rodent for male osteoporosis and the glucocorticoid-treated rabbit for 
77  glucocorticoid-induced osteoporosis) and transgenic animal models may provide relevant 
78  information (see section III. C., Biologics). 
79   
80  b.  Animal species 
81   
82  Generally, the sponsor should conduct bone quality studies in two animal species.  For 
83  postmenopausal osteoporosis, one of the studies should be conducted in the ovariectomized rat.  
84  Generally, a study in a larger ovariectomized nonrodent species with more extensive cortical 
85  remodeling (e.g., nonhuman primate, sheep, pig, or dog) should also be conducted.  For other 
86  osteoporosis indications, one bone quality study should be conducted in rodents and another in 
87  an appropriate animal model for the intended indication.  Biologics may be exempted from the 
88  two-species recommendation (see section III. C., Biologics). 
89   
90  c.  Osteoporosis indications 
91   
92  When a drug has been approved for a specific osteoporosis indication supported by bone quality 
93  studies in indication-specific animal models, the need for additional nonclinical studies to 
94  support another osteoporosis indication depends on the concern about the skeletal safety of the 
95  drug in the other form of osteoporosis.  The recommended evaluation may be limited to a 
96  relatively short-term study in a relevant animal model that can serve as a bridge to the original 
97  bone quality studies.  Additional animal studies to support osteoporosis indications involving 
98  combination drug treatments may also be needed depending on the level of scientific concern. 
99   
100  2.  Study Design 
101   
102  a.  Dose selection 
103   
104  The sponsor should generally conduct the bone quality studies with at minimum two doses, 
105  including a dose that induces an optimal pharmacological effect on bone mass and a high dose 
106  that is an adequate multiple of the optimally effective dose.  An optional low dose can be useful 
                                                 
8 We support the principles of the 3Rs (reduce/refine/replace) for animal use in testing when feasible.  The FDA 
encourages sponsors to consult with review divisions when considering a nonanimal testing method believed to be 
suitable, adequate, validated, and feasible.  The FDA will consider if the alternative method could be assessed for 
equivalency to an animal test method. 
  3  Contains Nonbinding Recommendations 
107  for determining dose-effect relationships.  Dose selection should be determined in dose range-
108  finding studies.  The optimal dose should be based on BMD and biochemical markers of bone 
109  turnover and the high dose should be selected to identify adverse bone effects.  The dose 
110  selection may be influenced by nonskeletal toxicities. 
111   
112  b.  Dosing regimen and administration route 
113   
114  The dosing regimen and route of administration should reflect the intended clinical use.  Dosing 
115  intervals should be selected on the basis of the pharmacokinetic profile of the investigational 
116  drug and the respective bone remodeling cycle durations in animals and humans.  For follow-up 
117  indications with different clinical dosing regimens or routes of administration, the need for 
118  additional nonclinical studies should be based on scientific rationale. 
119   
120  c.  Treatment initiation and duration 
121   
122  The sponsor should determine treatment initiation time (e.g., after ovariectomy) based on the 
123  intended clinical use of the drug.  The animals age during treatment should be adequate to 
124  ensure the evaluation of the drugs effects on already-formed bone rather than bone growth (i.e., 
125  animals should be skeletally mature).  The sponsor should determine treatment duration based on 
126  the intended clinical treatment duration and the bone remodeling cycle duration in the species 
127  studied.  For example, assuming the duration of the bone turnover cycle is 120 days to 200 days 
128  in humans (Eriksen 2010), 40 days in rats (Baron et al. 1984), and 75 days in monkeys (Schock 
129  et al. 1972), a study duration of 7 to 12 months in the rat and 14 to 23 months in the monkey 
130  would be adequate to support at least 3 years of human exposure.  Study duration also can be 
131  affected by other species-specific considerations, such as the age of the animals at treatment 
132  initiation.  The sponsor should consider the use of relevant nonrodent models other than 
133  monkeys.  The sponsor should discuss the timing and design of the long-term bone quality 
134  studies with the division, as early in development as possible (see section IV, Regulatory 
135  Aspects). 
136   
137  d.  Data analysis 
138   
139  Studies should be sufficiently powered to demonstrate statistically significant effects on BMD 
140  and biomechanical strength parameters at the optimal dose.  Group sizes for rats of 20 to 25 per 
141  group and group sizes for larger animals of 10 to15 per group are generally adequate. 
142   
143  3.  Evaluations 
144   
145  a.  Bone turnover 
146   
147  The sponsor should measure biochemical markers of bone resorption and formation in the bone 
148  quality studies to provide information on bone turnover.  Bone resorption markers include serum 
149  or urine cross-linked telopeptides of type I collagen, such as collagen type I cross-linked N-
150  telopeptide (NTx) or collagen type I cross-linked C-telopeptide (CTx), and urinary pyridinium 
151  cross-links of collagen, such as pyridinoline (PYD) or deoxypyridinoline (DPD).  Bone 
152  formation markers include serum osteocalcin (OC), procollagen type I C-terminal propeptide 
  4  Contains Nonbinding Recommendations 
153  (PICP), procollagen type I N-terminal propeptide (PINP), and bone-specific alkaline phosphatase 
154  (BSAP).  Data on bone turnover should be collected at interim time points (e.g., at 3, 6, 12, and 
155  18 months) and at the end of the study.  Bone turnover markers do not by themselves provide 
156  information on bone quality but may help to explain or interpret changes in other bone 
157  parameters. 
158   
159  b.  Bone mass and density 
160   
161  The sponsor should use established noninvasive techniques for the assessment of BMD and 
162  BMC, such as dual energy X-ray absorptiometry and peripheral quantitative computed 
163  tomography (pQCT), in the bone quality studies.  Both axial (spine) and appendicular (long 
164  bone) skeletal sites should be examined.  The pQCT data should be collected for both cancellous 
165  and cortical bone.  Geometrical bone properties also should be estimated using densitometric 
166  techniques.  Ex vivo measurements can be carried out at end of study, but in vivo densitometric 
167  measurements in anesthetized animals can be performed at interim time points. 
168   
169  c.  Bone structure and architecture 
170   
171  The sponsor should perform a qualitative histological evaluation of the microscopic bone 
172  structure, with optional histological staining, to identify bone cell and matrix components.  In 
173  addition, the sponsor should employ static and dynamic histomorphometry of cortical and 
174  cancellous bone at axial and appendicular skeletal sites to obtain quantitative information on 
175  bone architecture and remodeling dynamics (Parfitt et al. 1987; Dempster et al. 2013).  Other 
176  imaging or spectroscopic techniques (microcomputed tomography, high-resolution pQCT, 
177  magnetic resonance imaging, Raman or infrared spectroscopy, polarized light microscopy, small- 
178  and wide-angle X-ray scattering, or advanced forms of computed tomography) can be used to 
179  provide additional information on bone structure at different hierarchical levels.  Evaluations 
180  should be carried out at the end of the study, but data also can be collected at interim time points. 
181   
182  d.  Bone strength 
183   
184  The sponsor should perform biomechanical testing of both axial and appendicular sites in the 
185  bone quality studies.  Tests can include compression tests of vertebrae or vertebral bodies, 
186  bending tests of long bones, and femoral neck loading tests.  Both extrinsic (e.g., ultimate force, 
187  stiffness, work-to-failure) and intrinsic (e.g., ultimate strength, yield strength, elastic modulus) 
188  mechanical parameters should be determined (Turner and Burr 1993).  Characterization of pre-
189  yield as well as post-yield bone mechanical properties is recommended.  The sponsor should 
190  justify the choice of the biomechanical parameter or parameters used both to describe the bones 
191  mechanical properties and to demonstrate an effect of the therapeutic drug.  Geometric and 
192  densitometric parameters of the mechanically tested bone types should also be evaluated. 
193   
194  An analysis of the correlation between densitometric parameters (BMC, BMD) and mechanical 
195  parameters (e.g., ultimate force, stiffness, work-to-failure, ultimate strength, yield strength, or 
196  toughness) is essential and should be carried out to provide information about the value of BMD 
197  as a strength predictive parameter for the investigational drug.  BMD can be correlated to mass-
198  normalized strength parameters, but BMC should be associated with whole bone (extrinsic) 
  5  Contains Nonbinding Recommendations 
199  mechanical properties.  Importantly, the sponsor should determine potential differences in the 
200  relationship between bone mass and strength parameters between control and treatment groups 
201  by adequate statistical analysis.  Finite element analysis based on computed tomography images 
202  can be carried out, but finite element analysis is currently not considered to be a substitute 
203  measure of bone strength.  Biomechanical assessments should be carried out in animals 
204  sacrificed at the end of the study but also can be performed in animals sacrificed at interim time 
205  points. 
206   
207  e.  Additional evaluations 
208   
209  Bone quality evaluation is a continually evolving field that seeks to characterize bone-tissue 
210  properties and their relationship to the bones mechanical behavior using the latest scientific 
211  advances.  As described above, bone quality is not captured by the measurement of one 
212  particular bone parameter but is, in part, reflected by the relationship between specific bone 
213  mechanical and densitometric parameters.  The sponsor can include measurement of additional 
214  bone mechanical properties (e.g., fatigue life, fracture toughness, hardness) in the animal studies. 
215   
216  In addition to the evaluation of skeletal effects, bone quality studies may be suitable for 
217  toxicological assessments based on drug- and indication-specific safety concerns and as 
218  appropriate for the animal model utilized. This could include measurement of standard 
219  toxicological parameters as well as histologic evaluation of target organs of toxicity.  The 
220  sponsor should evaluate pharmacokinetic parameters (C , area under the curve (AUC)) in the 
max
221  bone quality studies to determine human exposure multiples. 
222   
223  Skeletal endpoints in long-term toxicology studies may also serve to provide additional 
224  nonclinical support for the bone safety and efficacy of therapeutic drugs. 
225   
226  C.  Biologics 
227   
228  Biologics (e.g., recombinant proteins and monoclonal antibodies) are typically selected on the 
229  basis of their high specificity for their human target receptor or antigen.  This target may be 
230  absent in common animal test species, or the nonhuman target (the ortholog) may not optimally 
231  interact with the biologic.  Because of these potential limitations, it may be appropriate to 
232  conduct bone quality studies as well as toxicology studies in a single pharmacologically 
233  responsive animal species.  The sponsor should characterize the immunogenicity of the biologic 
234  and the effect of the immune response on systemic exposure, pharmacodynamic response, and 
235  toxicity of the drug.  In cases where no relevant test species exists, the sponsor should consider 
236  the use of alternative models, such as the use of an analogous drug (surrogate) against the 
237  orthologous target or the use of a transgenic model in which the animal is made to express the 
238  human target.  For biologics to be used for the treatment of osteoporosis, the sponsor should also 
239  consult the ICH guidance for industry S6(R1) Preclinical Safety Evaluation of Biotechnology-
240  Derived Pharmaceuticals. 
241   
  6  Contains Nonbinding Recommendations 
242  D.  Anabolic Agents 
243   
244  A toxicological issue for the development of bone anabolic agents for the treatment of 
245  osteoporosis is the potential for carcinogenicity due to osteoblast stimulation.  In previous 
246  nonclinical studies, rats and mice dosed with parathyroid hormone (PTH) or parathyroid 
247  hormone-related peptide (PTHrP) drugs for 4 to 24 months developed bone tumors, including 
248  osteosarcomas, at low multiples of human exposure (AUC).  Because carcinogenicity studies 
249  with anabolic agents may entail unique design features, the sponsor should submit study 
250  protocols for review by both the Division of Bone, Reproductive, and Urologic Products and the 
251  Executive Carcinogenicity Assessment Committee (ECAC) before study initiation. 
252   
253   
254  IV.  REGULATORY ASPECTS 
255   
256  The sponsor is encouraged to consult with the division regarding the conduct and design of the 
257  bone quality studies as early in development as possible.  Study protocols with a detailed 
258  description of testing procedures should be submitted for review by the division.  Data from 
259  dose-range finding studies of relatively short-term duration (e.g., 3 months in rodents, 6 months 
260  in large animals) may be needed to evaluate drug-specific bone safety concerns, support the 
261  initiation of phase 2 or phase 3 clinical trials, and inform the design of the long-term studies.  
262  The sponsor should submit study reports of the bone quality studies by the end of phase 3 or at 
263  the time of submission of the new drug application or biologics license application.  For some 
264  drugs, the sponsor may consider modifications of the study program, including extent of studies, 
265  study timing, and study design, based on toxicological or clinical safety concerns or the available 
266  relevant animal models. 
  7  Contains Nonbinding Recommendations 
267  REFERENCES 
268   
269  Baron, R, Tross, R, and Vignery, A, 1984, Evidence of Sequential Remodeling in Rat Trabecular 
270  Bone:  Morphology, Dynamic Histomorphometry, and Changes During Skeletal Maturation, 
271  Anat Rec, 208(1):137145. 
272   
273  Dempster, DW, Compston, JE, Drezner, MK, Glorieux, FH, Kanis, JA, Malluche, H, Meunier, 
274  PJ, Ott, SM, Recker, RR, and Parfitt, AM, 2013, Standardized Nomenclature, Symbols, and 
275  Units for Bone Histomorphometry:  A 2012 Update of the Report of the ASBMR 
276  Histomorphometry Nomenclature Committee, J Bone Miner Res, 28(1):217. 
277   
278  Eriksen, EF, 2010, Cellular Mechanisms of Bone Remodeling, Rev Endocr Metab Disord, 
279  11:219227. 
280   
281  Harris, ST, Watts, NB, Jackson, RD, Genant, HK, Wasnich, RD, Ross, P, Miller, PD, Licata, 
282  AA, and Chesnut III, CH, 1993, Four-Year Study of Intermittent Cyclic Etidronate Treatment of 
283  Postmenopausal Osteoporosis:  Three Years of Blinded Therapy Followed by One Year of Open 
284  Therapy, Am. J Med, 95(6):557567. 
285   
286  Hernandez, CJ and Keaveny, TM, 2006, A Biomechanical Perspective on Bone Quality, Bone, 
287  39(6):11731181. 
288   
289  Jerome, CP and Peterson, PE, 2001, Nonhuman Primate Models in Skeletal Research, Bone, 
290  29(1):16. 
291   
292  Kleerekoper, M and Vieth, R, 1996, Fluoride and the Skeleton, Crit Rev Clin Lab Sci, 
293  33(2):139161. 
294   
295  Parfitt, AM, Drezner, MK, Glorieux, FH, Kanis, JA, Malluche, H, Meunier, PJ, Ott, SM, and 
296  Recker, RR, 1987, Bone Histomorphometry:  Standardization of Nomenclature, Symbols, and 
297  Units: Report of the ASBMR Histomorphometry Nomenclature Committee, J Bone Miner Res, 
298  2(6):595610. 
299   
300  Schock, CC, Noyes, FR, and Villanueva, AR, 1972, Measurement of Haversian Bone 
301  Remodeling by Means of Tetracycline Labeling in Rib of Rhesus Monkeys, Henry Ford Hosp 
302  Med J, 20(3):131144. 
303   
304  Turner, AS, 2001, Animal Models of Osteoporosis  Necessity and Limitations, Eur Cell Mater, 
305  1:6681. 
306   
307  Turner, CH and Burr, DB, 1993, Basic Biomechanical Measurements of Bone:  A Tutorial, Bone 
308  14(4):595608. 
309   
310  Van der Meulen, MC and Boskey, AL, 2012, Atypical Subtrochanteric Femoral Shaft Fractures:  
311  Role for Mechanics and Bone Quality, Arthritis Res Ther, 14:220227. 
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