NTP TECHNICAL REPORT

ON THE

TOXICOLOGY AND CARCINOGENESIS

STUDIES OF 

CHROMIUM PICOLINATE MONOHYDRATE

(CAS NO. 27882-76-4)

IN F344/N RATS AND B6C3F1 MICE

(FEED STUDIES)

Scheduled Peer Review Date: February 27-28, 2008

NOTICE

This DRAFT Technical Report is distributed solely for the purpose of predissemination peer review under the applicable information

quality guidelines. It has not been formally disseminated by the NTP. It does not represent and should not be construed to represent

NTP determination or policy.

NTP TR 556

NIH Publication No. 08-5897

National Toxicology Program

National Institutes of Health

Public Health Service

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
FOREWORD

The National Toxicology Program (NTP) is an interagency program within the Public Health Service (PHS) of the

Department of Health and Human Services (HHS) and is headquartered at the National Institute of Environmental

Health Sciences of the National Institutes of Health (NIEHS/NIH). Three agencies contribute resources to the 

program: NIEHS/NIH, the National Institute for Occupational Safety and Health of the Centers for Disease

Control and Prevention (NIOSH/CDC), and the National Center for Toxicological Research of the Food and Drug

Administration (NCTR/FDA). Established in 1978, the NTP is charged with coordinating toxicological testing

activities, strengthening the science base in toxicology, developing and validating improved testing methods, and

providing information about potentially toxic substances to health regulatory and research agencies, scientific and

medical communities, and the public. 

The Technical Report series began in 1976 with carcinogenesis studies conducted by the National Cancer Institute.

In 1981, this bioassay program was transferred to the NTP. The studies described in the Technical Report series

are designed and conducted to characterize and evaluate the toxicologic potential, including carcinogenic activity,

of selected substances in laboratory animals (usually two species, rats and mice). Substances selected for NTP 

toxicity and carcinogenicity studies are chosen primarily on the basis of human exposure, level of production, and

chemical structure. The interpretive conclusions presented in NTP Technical Reports are based only on the results

of these NTP studies. Extrapolation of these results to other species, including characterization of hazards and

risks to humans, requires analyses beyond the intent of these reports. Selection per se is not an indicator of a 

substance’s carcinogenic potential.

The NTP conducts its studies in compliance with its laboratory health and safety guidelines and FDA Good

Laboratory Practice Regulations and must meet or exceed all applicable federal, state, and local health and safety

regulations. Animal care and use are in accordance with the Public Health Service Policy on Humane Care and

Use of Animals. Studies are subjected to retrospective quality assurance audits before being presented for public

review.

NTP Technical Reports are indexed in the NIH/NLM PubMed database and are available free of charge 

electronically on the NTP website (http://ntp.niehs.nih.gov) or in hardcopy upon request from the NTP Central

Data Management group at cdm@niehs.nih.gov or (919) 541-3419.
NTP TECHNICAL REPORT

ON THE

TOXICOLOGY AND CARCINOGENESIS

STUDIES OF 

CHROMIUM PICOLINATE MONOHYDRATE

(CAS NO. 27882-76-4)

IN F344/N RATS AND B6C3F1 MICE

(FEED STUDIES)

Scheduled Peer Review Date: February 27-28, 2008

NOTICE

This DRAFT Technical Report is distributed solely for the purpose of predissemination peer review under the applicable information

quality guidelines. It has not been formally disseminated by the NTP. It does not represent and should not be construed to represent

NTP determination or policy.

NTP TR 556

NIH Publication No. 08-5897

National Toxicology Program

National Institutes of Health

Public Health Service

U.S. DEPARTMENT OF HEALTH AND HUMAN SERVICES
2

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft

National Toxicology Program

Evaluated and interpreted results and reported findings

M.D. Stout, Ph.D., Study Scientist

A. Nyska, Ph.D., Study Pathologist

J.B. Bishop, Ph.D.

D.W. Bristol, Ph.D.

J.R. Bucher, Ph.D.

R.S. Chhabra, Ph.D.

B.J. Collins, M.S.

P.M. Foster, Ph.D.

R.A. Herbert, D.V.M., Ph.D.

M.J. Hooth, Ph.D.

A.P. King-Herbert, D.V.M.

G.E. Kissling, Ph.D.

D.E. Malarkey, D.V.M., Ph.D.

J.H. Roycroft, Ph.D.

J.M. Sanders, Ph.D.

C.S. Smith, Ph.D.

G.S. Travlos, D.V.M.

N.J. Walker, Ph.D.

K.L. Witt, M.S.

Southern Research Institute

Conducted studies and evaluated pathology findings

C.D. Hébert, Ph.D., Principal Investigator

J.E. Heath, D.V.M.

J.F. Mann, D.V.M.

Experimental Pathology Laboratories, Inc.

Provided pathology review

M.H. Hamlin, II, D.V.M., Principal Investigator

E.T. Adams, D.V.M., Ph.D.

N. Allison, D.V.M., Ph.D.

A. Brix, D.V.M., Ph.D.

G.C. Hard, D.V.M., Ph.D., D.Sc.

J.C. Peckham, D.V.M., M.S., Ph.D.

TherImmune Research Corporation

Provided SMVCE analysis

G.W. Wolfe, Ph.D., Principal Investigator

H.S. Seung, M.S.

CONTRIBUTORS

Dynamac Corporation

Prepared quality assessment audits

S. Brecher, Ph.D., Principal Investigator

S. Iyer, B.S.

V. Tharakan, D.V.M.

Constella Group, Inc.

Provided statistical analyses

P.W. Crockett, Ph.D., Principal Investigator

L.J. Betz, M.S.

K.P. McGowan, M.B.A.

NTP Pathology Working Group

Evaluated slides and prepared pathology report on rats

(July 13, 2006)

A.W. Suttie, B.V.Sc., Ph.D., Coordinator

ILS, Inc.

E.T. Adams, D.V.M., Ph.D.

Experimental Pathology Laboratories, Inc.

A. Brix, D.V.M., Ph.D.

Experimental Pathology Laboratories, Inc.

S.A. Elmore, D.V.M., Ph.D.

National Toxicology Program

G.P. Flake, M.D.

National Toxicology Program

G.C. Hard, D.V.M., Ph.D., D.Sc.

Experimental Pathology Laboratories, Inc.

R.A. Herbert, D.V.M., Ph.D.

National Toxicology Program

J.B. Nold, D.V.M.

GlaxoSmithKline

A. Nyska, D.V.M.

ILS, Inc.

J.C. Peckham, D.V.M., M.S., Ph.D.

Experimental Pathology Laboratories, Inc.

B.P. Singh, D.V.M., M.S., Observer

National Toxicology Program
NOT FOR DISTRIBUTION OR ATTRIBUTION

Chromium Picolinate Monohydrate, NTP TR 556 3

Evaluated slides and prepared pathology report on mice

(May 25, 2006)

A.W. Suttie, B.V.Sc., Ph.D., Coordinator

ILS, Inc.

E.T. Adams, D.V.M., Ph.D.

Experimental Pathology Laboratories, Inc.

N. Allison, D.V.M., Ph.D.

Experimental Pathology Laboratories, Inc.

S.A. Elmore, D.V.M., Ph.D.

National Toxicology Program

R.A. Herbert, D.V.M., Ph.D.

National Toxicology Program

J.B. Nold, D.V.M.

GlaxoSmithKline

A. Nyska, D.V.M.

ILS, Inc.

J.C. Peckham, D.V.M., M.S., Ph.D.

Experimental Pathology Laboratories, Inc.

A. Remick, D.V.M., Observer

National Toxicology Program

N. Wakamatsu, D.V.M., Ph.D., Observer

National Toxicology Program

Board Draft 

Biotechnical Services, Inc.

Prepared Technical Report

S.R. Gunnels, M.A., Principal Investigator

P.A. Gideon, B.A.

B.F. Hall, M.S.

L.M. Harper, B.S.

D.C. Serbus, Ph.D.
4

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY . . . . . . . . . . . . . . . . 12

TECHNICAL REPORTS REVIEW SUBCOMMITTEE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS . . . . . . . . . . . . 14

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

DISCUSSION AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

APPENDIX A Summary of Lesions in Male Rats in the 2-Year Feed Study

of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A-1

APPENDIX B Summary of Lesions in Female Rats in the 2-Year Feed Study

of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B-1

APPENDIX C Summary of Lesions in Male Mice in the 2-Year Feed Study

of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C-1

APPENDIX D Summary of Lesions in Female Mice in the 2-Year Feed Study

of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-1

APPENDIX E Genetic Toxicology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-1

APPENDIX F Clinical Pathology Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-1

APPENDIX G Organ Weights and Organ-Weight-to-Body-Weight Ratios . . . . . . . . . . . . . . . . . . . . . . . G-1

APPENDIX H Reproductive Tissue Evaluations and Estrous Cycle Characterization . . . . . . . . . . . . . . H-1

APPENDIX I Chemical Characterization and Dose Formulation Studies . . . . . . . . . . . . . . . . . . . . . . . . I-1

APPENDIX J Feed and Compound Consumption in the 2-Year Feed Studies 

of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J-1

CONTENTS
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Chromium Picolinate Monohydrate, NTP TR 556 5

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APPENDIX K Ingredients, Nutrient Composition, and Contaminant Levels 

in NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K-1

APPENDIX L Sentinel Animal Program . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L-1

APPENDIX M Chromium Tissue Distribution Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M-1

APPENDIX N Absorption, Distribution, Metabolism, and Excretion Studies . . . . . . . . . . . . . . . . . . . . . N-1
6 Chromium Picolinate Monohydrate, NTP TR 556 

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NOT FOR DISTRIBUTION OR ATTRIBUTION

7

Board Draft 

Chromium picolinate monohydrate is the commercially available form of chromium picolinate. Chromium

picolinate is one of a number of compounds that contain chromium in the trivalent state (Cr III), which is the

predominant form of chromium in nature. Humans ingest Cr III in food and dietary supplements. The major uses

of Cr III in the chemical and manufacturing industries include production of chromium pigments and leather

tanning. Chromium picolinate was nominated by the National Cancer Institute and a private individual for testing

based on the potential for widespread consumer exposure from use as a dietary supplement. Male and female

F344/N rats and B6C3F1 mice were exposed to chromium picolinate monohydrate (95% to 96% pure) in feed for

3 months or 2 years. Genetic toxicology studies with chromium picolinate monohydrate were conducted in

Salmonella typhimurium and mouse peripheral blood erythrocytes. Genetic toxicology studies with chromium

picolinate were conducted in S. typhimurium and rat bone marrow cells.

ABSTRACT

CHROMIUM PICOLINATE MONOHYDRATE

CAS No. 27882-76-4

Chemical Formula: C18H12CrN3O6@H2O Molecular Weight: 436

Synonyms: Chromium 2-pyridine-carboxylate; chromium tripicolinate; chromium tris (picolinato)-; 

chromium, tris (2-pyridinecarboxylato-N(1), O(2))-(9CI); picolinate acid, chromium salt

Trade name: Chromax®

N

O

N

N

O

O

Cr3+

O

O

O

H2O @
8 Chromium Picolinate Monohydrate, NTP TR 556 

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3-MONTH STUDY IN RATS

Groups of 10 male and 10 female rats were fed diets containing 0, 80, 240, 2,000, 10,000, or 50,000 ppm

chromium picolinate monohydrate (equivalent to average daily doses of approximately 7, 20, 160, 800, and

4,240 mg chromium picolinate monohydrate/kg body weight to males and 6, 20, 160, 780, and 4,250 mg/kg to

females) for 14 weeks. All rats survived to the end of the study. Mean body weights and feed consumption of all

exposed groups of males and females were similar to those of the control groups throughout the study. No

exposure-related lesions occurred in males or females.

3-MONTH STUDY IN MICE

Groups of 10 male and 10 female mice were fed diets containing 0, 80, 240, 2,000, 10,000, or 50,000 ppm

chromium picolinate monohydrate (equivalent to average daily doses of approximately 17, 50, 450, 2,300, and

11,900 mg chromium picolinate monohydrate/kg body weight to males and 14, 40, 370, 1,775, and 9,140 mg/kg to

females) for 14 weeks. All mice survived to the end of the study. Mean body weights and feed consumption of all

exposed groups were similar to those of the control groups throughout the study. No exposure-related lesions

occurred in male or female mice.

2-YEAR STUDY IN RATS

Groups of 50 male and 50 female rats were fed diets containing 0, 2,000, 10,000, or 50,000 ppm chromium

picolinate monohydrate (equivalent to average daily doses of approximately 90, 460, and 2,400 mg/kg to males and

100, 510, and 2,630 mg/kg to females) for 105 weeks. Survival of all exposed groups of males and females was

similar to that of the control groups. Mean body weights and feed consumption of exposed groups of males and

females were generally similar to those of the controls throughout the study. The incidence of preputial gland

adenoma was significantly increased in males exposed to 10,000 ppm and exceeded the historical control ranges. 
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Chromium Picolinate Monohydrate, NTP TR 556 9

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2-YEAR STUDY IN MICE

Groups of 50 male and 50 female mice were fed diets containing 0, 2,000, 10,000, or 50,000 ppm chromium

picolinate monohydrate (equivalent to average daily doses of approximately 250, 1,200, and 6,565 mg/kg to males

and 240, 1,200, and 6,100 mg/kg to females) for 105 weeks. Survival of all exposed groups of males and females

was similar to that of the control groups. Mean body weights of exposed groups of males were generally similar to

those of the controls throughout the study; mean body weights of 50,000 ppm females was 10% less than the

control group at 1 year. Feed consumption by exposed groups of males and females was similar to that by the

controls throughout the study. No neoplasms or nonneoplastic lesions were attributed to exposure to chromium

picolinate monohydrate.

GENETIC TOXICOLOGY

In the standard screening assays conducted by the NTP, chromium picolinate monohydrate showed no clear

evidence of genotoxicity. It was not mutagenic in Salmonella typhimurium strains TA98 or TA100 or

Escherichia coli strain WP2 uvrA/pKM101 when tested with or without exogenous metabolic activation (S9). No

increase in the frequency of micronucleated normochromatic erythrocytes was observed in male B6C3F1 mice

administered chromium picolinate monohydrate in feed for 3 months. A small increase in micronucleated

normochromatic erythrocytes was seen in female mice at the highest exposure concentration tested, and the results

in female mice were considered equivocal.

Additional genotoxicity testing was conducted with chromium picolinate (not the monohydrate form of the

compound), and results were also negative. No induction of gene mutations was observed in two independent

studies conducted in several strains of S. typhimurium with and without S9. No induction of micronucleated

polychromatic erythrocytes was observed in bone marrow of male F344/N rats treated with chromium picolinate

by oral gavage three times at 24-hour intervals.
10

CONCLUSIONS

Under the conditions of these 2-year feed studies there was equivocal evidence of carcinogenic activity* of

chromium picolinate monohydrate in male F344/N rats based on an increase in the incidence of preputial gland

adenoma. There was no evidence of carcinogenic activity of chromium picolinate monohydrate in female F344/N

rats or in male or female B6C3F1 mice.

__________

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 12.

Chromium Picolinate Monohydrate, NTP TR 556 

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Chromium Picolinate Monohydrate, NTP TR 556 11

Board Draft 

Summary of the 2-Year Carcinogenesis and Genetic Toxicology Studies 

of Chromium Picolinate Monohydrate and Genetic Toxicology Studies of Chromium Picolinate

Male Female Male Female

F344/N Rats F344/N Rats B6C3F1 Mice B6C3F1 Mice

Concentrations 

in feed

Body weights

Survival rates

Nonneoplastic effects

Neoplastic effects

Equivocal findings

Level of evidence of

carcinogenic activity

0, 2,000, 10,000,

50,000 ppm

Exposed groups similar

to control group

37/50, 36/50, 35/50,

28/50

None

None

Preputial gland:

adenoma (1/50, 1/50,

7/50, 4/50)

Equivocal evidence

0, 2,000, 10,000,

50,000 ppm

Exposed groups similar

to control group

36/50, 35/50, 36/50,

40/50

None

None

None

No evidence

0, 2,000, 10,000,

50,000 ppm

Exposed groups similar

to control group

46/50, 43/50, 38/50,

45/50

None

None

None

No evidence

0, 2,000, 10,000,

50,000 ppm

50,000 ppm group was

10% less than the control

group at 1 year

45/50, 44/49, 44/50,

39/50

None

None

None

No evidence

Genetic toxicology 

for chromium picolinate monohydrate

Salmonella typhimurium gene mutations:

Micronucleated erythrocytes

Mouse peripheral blood in vivo:

Genetic toxicology 

for chromium picolinate

Salmonella typhimurium gene mutations:

Micronucleated erythrocytes

male rat bone marrow in vivo:

Negative in strains TA98 and TA100 with and without S9; negative in Escherichia

coli strain WP2 uvrA/pKM101 with and without S9

Negative in males; equivocal in females

Negative in strains TA97, TA98, TA100, TA102, TA104, and TA1535 with and

without S9

Negative
12 Chromium Picolinate Monohydrate, NTP TR 556 

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EXPLANATION OF LEVELS OF EVIDENCE OF CARCINOGENIC ACTIVITY

The National Toxicology Program describes the results of individual experiments on a chemical agent and notes the strength of the evidence for

conclusions regarding each study. Negative results, in which the study animals do not have a greater incidence of neoplasia than control

animals, do not necessarily mean that a chemical is not a carcinogen, inasmuch as the experiments are conducted under a limited set of

conditions. Positive results demonstrate that a chemical is carcinogenic for laboratory animals under the conditions of the study and indicate

that exposure to the chemical has the potential for hazard to humans. Other organizations, such as the International Agency for Research on

Cancer, assign a strength of evidence for conclusions based on an examination of all available evidence, including animal studies such as those

conducted by the NTP, epidemiologic studies, and estimates of exposure. Thus, the actual determination of risk to humans from chemicals

found to be carcinogenic in laboratory animals requires a wider analysis that extends beyond the purview of these studies.

Five categories of evidence of carcinogenic activity are used in the Technical Report series to summarize the strength of the evidence observed

in each experiment: two categories for positive results (clear evidence and some evidence); one category for uncertain findings (equivocal

evidence); one category for no observable effects (no evidence); and one category for experiments that cannot be evaluated because of major

flaws (inadequate study). These categories of interpretative conclusions were first adopted in June 1983 and then revised in March 1986 for

use in the Technical Report series to incorporate more specifically the concept of actual weight of evidence of carcinogenic activity. For each

separate experiment (male rats, female rats, male mice, female mice), one of the following five categories is selected to describe the findings.

These categories refer to the strength of the experimental evidence and not to potency or mechanism.

• Clear evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a dose-related 

(i) increase of malignant neoplasms, (ii) increase of a combination of malignant and benign neoplasms, or (iii) marked increase of 

benign neoplasms if there is an indication from this or other studies of the ability of such tumors to progress to malignancy.

• Some evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a chemical-related increased 

incidence of neoplasms (malignant, benign, or combined) in which the strength of the response is less than that required for clear 

evidence.

• Equivocal evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing a marginal increase of 

neoplasms that may be chemical related.

• No evidence of carcinogenic activity is demonstrated by studies that are interpreted as showing no chemical-related increases in 

malignant or benign neoplasms.

• Inadequate study of carcinogenic activity is demonstrated by studies that, because of major qualitative or quantitative limitations, 

cannot be interpreted as valid for showing either the presence or absence of carcinogenic activity.

For studies showing multiple chemical-related neoplastic effects that if considered individually would be assigned to different levels of evidence

categories, the following convention has been adopted to convey completely the study results. In a study with clear evidence of carcinogenic

activity at some tissue sites, other responses that alone might be deemed some evidence are indicated as “were also related” to chemical

exposure. In studies with clear or some evidence of carcinogenic activity, other responses that alone might be termed equivocal evidence are

indicated as “may have been” related to chemical exposure.

When a conclusion statement for a particular experiment is selected, consideration must be given to key factors that would extend the actual

boundary of an individual category of evidence. Such consideration should allow for incorporation of scientific experience and current

understanding of long-term carcinogenesis studies in laboratory animals, especially for those evaluations that may be on the borderline between

two adjacent levels. These considerations should include:

• adequacy of the experimental design and conduct;

• occurrence of common versus uncommon neoplasia;

• progression (or lack thereof) from benign to malignant neoplasia as well as from preneoplastic to neoplastic lesions;

• some benign neoplasms have the capacity to regress but others (of the same morphologic type) progress. At present, it is impossible to 

identify the difference. Therefore, where progression is known to be a possibility, the most prudent course is to assume that benign 

neoplasms of those types have the potential to become malignant;

• combining benign and malignant tumor incidence known or thought to represent stages of progression in the same organ or tissue;

• latency in tumor induction;

• multiplicity in site-specific neoplasia;

• metastases;

• supporting information from proliferative lesions (hyperplasia) in the same site of neoplasia or in other experiments (same lesion in 

another sex or species);

• presence or absence of dose relationships;

• statistical significance of the observed tumor increase;

• concurrent control tumor incidence as well as the historical control rate and variability for a specific neoplasm;

• survival-adjusted analyses and false positive or false negative concerns;

• structure-activity correlations; and

• in some cases, genetic toxicology. 
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NATIONAL TOXICOLOGY PROGRAM BOARD OF SCIENTIFIC COUNSELORS

TECHNICAL REPORTS REVIEW SUBCOMMITTEE

The members of the Technical Reports Review Subcommittee who evaluated the draft NTP Technical Report on chromium picolinate

monohydrate on February 27-28, 2008, are listed below. Subcommittee members serve as independent scientists, not as representatives of any

institution, company, or governmental agency. In this capacity, subcommittee members have five major responsibilities in reviewing the NTP

studies:

• to ascertain that all relevant literature data have been adequately cited and interpreted,

• to determine if the design and conditions of the NTP studies were appropriate,

• to ensure that the Technical Report presents the experimental results and conclusions fully and clearly,

• to judge the significance of the experimental results by scientific criteria, and

• to assess the evaluation of the evidence of carcinogenic activity and other observed toxic responses.

Nancy Kerkvliet, Ph.D., Chairperson

Department of Environmental and Molecular Toxicology

Oregon State University

Corvallis, OR

Christopher Bradfield, Ph.D.

McArdle Laboratory for Cancer Research

University of Wisconsin

Madison, WI

Tracie E. Bunton, D.V.M., Ph.D.

Toxicology Consultant

Eicarte LLC

Mechanicsburg, PA

Russell C. Cattley, V.M.D., Ph.D.

Amgen

Thousand Oaks, CA

Kenny S. Crump, Ph.D.

ENVIRON International Corporation

Monroe, LA

Jon Mirsalis, Ph.D.

SRI International

Menlo Park, CA

Raymond F. Novak, Ph.D.

Institute of Environmental Health Sciences

Wayne State University

Detroit, MI

Michael V. Pino, D.V.M., Ph.D.

Drug Safety Evaluation

Sanofi-aventis

Bridgewater, NJ

Keith Soper, Ph.D. 

Merck Research Laboratories

West Point, PA
14 Chromium Picolinate Monohydrate, NTP TR 556 

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SUMMARY OF TECHNICAL REPORTS REVIEW SUBCOMMITTEE COMMENTS

NOTE: A summary of the Technical Reports Review Subcommittee’s remarks will appear in a future draft of this

report.
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15

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CHEMICAL AND PHYSICAL PROPERTIES

Chromium picolinate monohydrate is the commercially available form of chromium picolinate. Chromium

picolinate exists as ruby red crystals. Chromium picolinate is a lipophilic compound (Merck, 1996), with a greater

solubility in organic solvents (greater than 6 g/L in dimethyl sulfoxide) than in water (1 ppm at 25° C and neutral

pH) (ATSDR, 2000). The partition coefficient (logKow) is 1.753 (ATSDR, 2000). Chromium picolinate is one of

a number of compounds that contain chromium in the trivalent state (Cr III). Other examples of Cr III compounds

are chromium acetate, chromium nitrate, chromium chloride, ferrochromite, chromium oxide, chromium

phosphate, chromium sulfate, and sodium chromite. These compounds, with the exception of chromium acetate,

chromium chloride, and chromium nitrate, are generally insoluble in water. 

Chromium is a group 6 transition metal and occurs in the Earth’s crustal rock at a concentration averaging

122 ppm. It has six oxidation states. The most stable states are Cr III, hexavalent chromium (Cr VI) and metallic

INTRODUCTION

CHROMIUM PICOLINATE MONOHYDRATE

CAS No. 27882-76-4

Chemical Formula: C18H12CrN3O6@H2O Molecular Weight: 436

Synonyms: Chromium 2-pyridine-carboxylate; chromium tripicolinate; chromium tris (picolinato)-; 

chromium, tris (2-pyridinecarboxylato-N(1), O(2))-(9CI); picolinate acid, chromium salt

Trade name: Chromax®

N

O

N

N

O

O

Cr3+

O

O

O

H2O @
16 Chromium Picolinate Monohydrate, NTP TR 556 

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chromium (Cr 0). Cr VI is easily reduced to Cr III in acidic solutions containing organic molecules such as

proteins, DNA, or glutathione. Glutathione is also capable of reducing Cr VI at neutral pH at a slower rate than

under acidic conditions (Zhitkovich, 2005).

PRODUCTION, USE, AND HUMAN EXPOSURE

Cr III is the predominant form of chromium in nature, occurring in ores such as ferrochromite (FeCr2O4). Cr III

ores are used in the production of Cr VI (Hartford, 1979; Westbrook, 1979) and Cr 0. Cr 0 is used in the

metallurgical industry for the production of stainless steel and ferrous and nonferrous alloys. The major uses of

chromium in the chemical and manufacturing industries include the production of chromium pigments (Cr III and

Cr VI) and in metal finishing (Cr VI), leather tanning (Cr III), and wood preservation (Cr VI) (Barnhart, 1997).

Chromium enters the environment from combustion processes and ore processing mainly as chromium (III) oxide.

Both Cr III and Cr VI enter water resources by leaching from soil or from industrial contamination (Pellerin and

Booker, 2000) as well as from atmospheric fallout. Exposure of the public to chromium occurs through food,

water, and air. In many occupational settings, workers are exposed both to Cr III and Cr VI. Workplace exposure

is typically by inhalation or dermal contact. Dermal exposure to chromium compounds can result in irritation and

ulceration of the skin and causes allergic contact dermatitis in sensitized individuals. 

Cr III is the biologically active form of chromium and an essential trace element. Humans ingest Cr III in food

and dietary supplements. Typical serving sizes of a variety of foods and beverages, including broccoli, grape juice,

whole wheat English muffins, mashed potatoes, dried garlic, dried basil, beef cubes, orange juice, turkey breast,

whole wheat bread, red wine, unpeeled apple, banana, and green beans, provide 1 to 13 μg Cr III (NIH, 2007). An

estimated safe and adequate daily dietary intake of 50 to 200 μg Cr III for adults and adolescents was established

by the National Research Council in 1989 (NAS, 1989). In 2001, the Institute of Medicine of the NAS determined

an adequate intake, which is generally set at a level that healthy people typically consume, of 20 to 45 μg Cr III for

adolescents and adults (IOM, 2001). An adequate intake is set when there is not enough data to set a
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recommended daily allowance, which is the average daily intake that meets a nutrient requirement of nearly all

(97% to 98%) healthy individuals. Chromium appears to increase sensitivity to insulin and thus participates in

carbohydrate protein and lipid metabolism. The mechanism involves increased insulin binding through increasing

the number of insulin receptors and increasing insulin receptor phosphorylation when the chromium is bound to a

low molecular weight chromium binding substance (LMWCr; also referred to as chromodulin) and insulin is

present (Anderson, 1998). In the blood, Cr III is bound to and transported to tissues by transferrin, a process

regulated, at least in part, by insulin (Clodfelder et al., 2001). Cr III deficiency contributes to glucose intolerance

and diabetes mellitus (Type 2). 

Chromium picolinate is widely used as a dietary supplement, primarily because of claims of increased metabolic

(weight reducing) and antidiabetic effects. Chromium picolinate is produced by formation of a coordination

complex between Cr III and picolinate (picolinic acid), which occurs because picolinate is a bidentate chelating

ligand that coordinates with Cr III through the pyridine nitrogen and the carboxyl oxygen (Evans and Pouchnik,

1993). Picolinate is a metabolite of tryptophan formed during endogenous metabolism and is normally found in

the liver and kidneys (Mehler and May, 1956). Chromium-containing supplements, which are available over the

counter as pills, chewing gums, sports drinks, and nutrition bars, have become very popular, generating an

estimated $500,000,000 in retail sales in 1999 (Mirasol, 2000). Typical daily doses of chromium picolinate

monohydrate contain 200 to 600 μg Cr III. Recent reviews examining the effectiveness of chromium picolinate

indicate that the supplement has little effect on body composition (Campbell et al., 2002; Pittler et al., 2003;

Vincent, 2003). The essentiality of Cr III and the ability of Cr III to increase insulin sensitivity have been

questioned (Stearns, 2000; Stallings and Vincent, 2006). 

ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Chromium and chromium compounds are absorbed after oral, dermal, or inhalation exposure (Wahlberg and Skog,

1965; Wahlberg, 1970; Kerger et al., 1997; Mancuso, 1997). Current analytical procedures cannot differentiate
18 Chromium Picolinate Monohydrate, NTP TR 556 

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between the oxidation states of chromium in biological tissues, so measurements in excreta and tissues represent

total chromium content. Most studies of absorption of Cr III or Cr VI after oral administration to rodents find that

only 1% or 2% of the administered dose is bioavailable, whereas similar studies with humans report somewhat

higher numbers (ATSDR, 2000), particularly for Cr VI. Cr III enters cells by passive diffusion or phagocytosis of

precipitates, while Cr VI is transported into cells by anion carriers (ATSDR, 2000). Following uptake by red blood

cells and tissues, Cr VI is thought to undergo reduction to Cr III intracellularly, primarily by ascorbate (Sugiyama,

1992). In a study of 51Cr (VI) uptake by human red blood cells and rat hepatocytes, Alexander and Aaseth (1995)

found the Michaelis constant (Km) for uptake to be very low. The authors attributed the low Km to efficient

intracellular reduction of Cr VI to Cr III inside the cell as opposed to high affinity. The transport was of the same

magnitude as the physiological substrates, lactate and sulfate. The uptake rate was increased when the pH was

lowered from 7.4 to 6, consistent with bichromate (HOCrO3

–1) as the transported species. Absorption and

retention of chromium thus depends on a number of factors: the rate of reduction of Cr VI to Cr III outside the

cells, the pH of the milieu, the rate of transport of Cr VI into the cells, the rate of reduction of Cr VI to Cr III

inside the cells, and the rate of diffusion of Cr III from the cells. 

Chromium picolinate is a chelated form of chromium expected to increase chromium absorption. Gargas et al.

(1994) reported that ingestion of 400 μg chromium picolinate per day in humans resulted in 2.80% ± 1.14%

(standard deviation) absorption. No comparison to unchelated Cr III was reported. A study in rats compared the

uptake of 51Cr (III) as chromium chloride, chromium picolinate, and chromium nicotinate, a chelated form of

chromium similar to picolinate (Olin et al., 1994). Male and female CD rats were gavaged with 2.7 nmol of one of

the three forms of Cr III. Tissue and blood concentrations were determined 1, 3, 6, and 12 hours after dosing.

There was little difference among the three groups. At 1 hour after dosing, absorption was 0.287% (chloride),

0.291% (nicotinate), and 0.542% (picolinate). After 12 hours, absorption was 0.710% (chloride), 0.616%

(nicotinate), and 0.406% (picolinate).
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Absorbed Cr III is widely distributed to tissues. Concentrations of chromium in the kidney, liver, spleen, heart, and

lung of weanling Sprague-Dawley rats fed a diet containing chromium picolinate or one of nine other formulations

of Cr III (5,000 ng chromium/g of diet) for 3 weeks were compared to concentrations in tissues of control rats fed

basal diet (30 ng chromium/g of diet) (Anderson et al., 1996). Of the 10 formulations, exposure to chromium

picolinate produced the highest chromium concentrations in the liver (50 ng/g versus 5 ng/g in controls), lung

(60 ng/g versus 20 ng/g in controls), and heart (30 ng/g versus 12 ng/g in controls). Overall, chromium

concentrations were highest in the kidney; Cr III uptake by the kidney was enhanced to the greatest extent by a

chromium dinicotinate-diglycine-cysteine-glutamic acid complex. That complex resulted in a kidney concentration

of 850 ng/g, compared to 368 ng/g for chromium picolinate and 23 ng/g for controls. Chromium concentrations

were not increased over controls in the kidney or liver following exposure to chromium chloride. After 20 weeks

of exposure to 5, 25, 50, or 100 mg chromium/kg of diet, concentrations of chromium in the liver and kidney were

two to sixfold higher following treatment with chromium picolinate than with chromium chloride (Anderson et al.,

1997). Distribution studies in Sprague-Dawley rats following intravenous dosing with a single dose of 51Cr (III)

picolinate (Hepburn and Vincent, 2003) or 51Cr (III) 3H-picolinate for 2 weeks (Hepburn and Vincent, 2002)

revealed that 51Cr accumulated in the liver, kidney, and blood and most of the chromium in blood was bound to

chromodulin. Higher tissue chromium levels were found in rats receiving Cr VI orally than in those receiving an

equivalent dose of Cr III (Costa, 1997; NTP, 2008). 

Ingested chromium is excreted primarily in the feces because of its poor absorption. Absorbed chromium is

primarily excreted in the urine (Donaldson and Barreras, 1966; Sayato et al., 1980). Distribution studies in

Sprague-Dawley rats following intravenous dosing with a single dose of 51Cr (III) picolinate (Hepburn and

Vincent, 2003) or 51Cr (III) 3H-picolinate for 2 weeks (Hepburn and Vincent, 2002) revealed that the most 51Cr

was excreted in urine in the first 12 hours after dosing. However, the loss was only about 10% of the administered

dose. Picolinate-derived tritium appears in urine and feces at a much greater extent than 51Cr, with about 20 times

more in urine than feces implying that the chelating agent may become separated from the chromium in the gut.

The material containing the tritium, a single chromatographic peak, was isolated but could not be identified.
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TOXICITY

Experimental Animals

Cr III displays very little evidence of toxicity in animals (ATSDR, 2000). There was no indication of local or

systemic toxicity in male Harlan Sprague-Dawley rats fed diets containing chromium picolinate at concentrations

up to 100 mg chromium/kg diet (9 mg chromium/kg per day) for 20 weeks (Anderson et al., 1997). This lack of

toxicity was also observed in a number of studies with other Cr III compounds following oral administration.

These studies include Becton Dickinson (BD) rats fed diets of 5% chromium oxide, 5 days per week for 90 days

(1,806 mg chromium/kg per day) or 2 years (2,040 mg chromium/kg per day) (Ivankovic and Preussmann, 1975);

Harlan Sprague-Dawley rats fed diets containing chromium chloride at concentrations up to 100 mg chromium/kg

diet (9 mg chromium/kg per day) for 20 weeks (Anderson et al., 1997); rats exposed to 25 ppm chromium chloride

(2.7 mg chromium/kg per day) in drinking water for 1 year (MacKenzie et al., 1958); and Swiss mice (0.48 mg

chromium/kg per day) (Schroeder et al., 1964) or Long-Evans rats (0.46 mg chromium/kg per day) (Schroeder

et al., 1965) exposed to 5 mg/L chromium acetate in the drinking water for 2 to 3 years. Following exposure of

CDF rats to 30 mg chromium/m3 (6.6 mg chromium/kg per day) as chromium oxide (44 mg/m3) or basic

chromium sulfate (168 mg/m3) for 13 weeks by inhalation, effects on the respiratory system were typical of inhaled

particles; however, there was no evidence of systemic toxicity (Derelanko et al., 1999). 

Cr VI is significantly more toxic than Cr III. The National Toxicology Program (NTP) conducted studies on

Cr VI, as sodium dichromate dihydrate, administered to male and female F344/N rats and B6C3F1 mice in

drinking water at concentrations of up to 1,000 mg/L for 3 months or 516 mg/L (257.4 mg/L for male mice) for

2 years (NTP, 2007, 2008). In the 3-month and 2-year studies, there were reduced body weights, decreased water

consumption due to decreased palatability, a microcytic hypochromic anemia, and histiocytic cell infiltration in the

liver, duodenum, and pancreatic and mesenteric lymph nodes. In the 3-month studies, focal ulceration,

hyperplasia, and metaplasia were observed in the glandular stomach of rats, while hyperplasia was observed in the

duodenum of mice. In the 2-year studies, in addition to squamous cell neoplasms of the oral cavity in rats and
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epithelial neoplasms of the small intestinal in mice, diffuse epithelial hyperplasia of the duodenum and jejunum

was observed in mice.

Humans

A small number of case reports provide limited data on the toxicity of chromium picolinate in humans; adverse

effects following ingestion of chromium picolinate supplements have been reviewed elsewhere (Lamson and Plaza,

2002). Dermal effects, including contact dermatitis and acute generalized exanthematous pustulosis, were

observed in two separate cases (Young, et al., 1999; Fowler, 2000). Renal effects, including acute tubular necrosis,

chronic active interstitial nephritis, and acute renal failure have been observed in three separate cases (Wasser

et al., 1997; Cerulli et al., 1998; Wani et al., 2006). Other effects, such as liver dysfunction (Cerulli et al., 1998),

rhabdomyolysis (Martin and Fuller, 1998) or cognitive, perceptual, and motor changes (Huszonek, 1993), have

been observed in isolated cases.

REPRODUCTIVE AND DEVELOPMENTAL TOXICITY

Experimental Animals

A series of studies was conducted to determine the effects of chromium chloride hexahydrate on reproduction in

rats and mice following exposure in drinking water (Bataineh et al., 1997; Elbetieha and Al-Hamood, 1997;

Al-Hamood et al., 1998). Histopathology was not performed in animals from these studies. Fertility was assessed

by mating exposed animals of each sex with unexposed animals of the other sex. In general, there were decreased

body weights, changes (primarily decreases) in reproductive organ weights, and decreases in fertility in male and

female mice at exposure concentrations of up to 5,000 mg/L (Elbetieha and Al-Hamood, 1997) and in male mice

following exposure during gestation and lactation through exposure of dams to 1,000 mg/L (Al-Hamood et al.,

1998); delayed vaginal opening was observed in female mice exposed during gestation and lactation. Effects on

body and organ weights and sexual behavior and aggressiveness, without a decrease in fertility, were observed in

male Sprague-Dawley rats at exposure concentrations of up to 1,000 mg/L for 12 weeks during adulthood

(Bataineh et al., 1997). A significant increase in the incidence of bifurcated cervical arches was observed, in the
22 Chromium Picolinate Monohydrate, NTP TR 556 

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absence of maternal toxicity or effect on maternal fertility, in the offspring of pregnant CD-1 mice fed diets

containing 200 mg chromium picolinate/kg (25 mg chromium/kg per day) from gestation days 6 to 17 (Bailey

et al., 2006). The incidence of bifurcated cervical arches was elevated following treatment with 174 mg picolinic

acid/kg; however, this increase was not significant. Developmental effects were not observed following treatment

with 200 mg chromium chloride hexahydrate/kg (39 mg chromium/kg per day).

Other studies have failed to show evidence of reproductive or developmental toxicity. There were no changes in

testis or epididymis weights in rats following treatment with chromium picolinate or chromium chloride (9 mg

Cr III/kg per day) in the diet for 20 weeks (Anderson et al., 1997). There was no evidence of reproductive or

developmental toxicity in male or female rats following dietary exposure to chromium oxide (1,806 mg

chromium/kg per day) for 60 days prior to gestation and during gestation (Ivankovic and Preussmann, 1975).

There were no effects on testis or ovary weights or histopathology and no alterations in sperm parameters

following exposure of rats to 30 mg chromium/m3 as chromium oxide (44 mg/m3) or basic chromium sulfate

(168 mg/m3) for 13 weeks by inhalation (Derelanko et al., 1999). Following intraperitoneal injection of up to

4 mg/kg chromium chloride (1.3 mg chromium/kg), there was no histopathologic evidence of damage or changes

in epididymal sperm (Ernst, 1990).

Humans

No studies on reproductive or developmental toxicity of Cr III in humans were located in a review of the literature. 

CARCINOGENICITY

No studies examining the carcinogenic potential of chromium picolinate in animals or humans were identified in a

review of the literature. Cr III exposure is not considered to pose a significant cancer risk in humans (ATSDR,

2000). Both Cr III and Cr 0 are classified as Group 3, not classifiable as to their carcinogenicity to humans, by the

International Agency for Research on Cancer (1990). The United States Environmental Protection Agency (1998)

concluded that there are inadequate data to determine the potential carcinogenicity of Cr III. In contrast to Cr III,
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exposure to Cr VI compounds by inhalation has long been recognized as carcinogenic to humans. The

United States Department of Health and Human Services, the United States Environmental Protection Agency, and

the International Agency for Research on Cancer classified Cr VI compounds as human carcinogens based on

increased incidences of lung cancers in workers in the chromium industry and in experimental animals exposed to

these compounds by inhalation (IARC, 1990; Cohen et al., 1993; NTP, 1998). In industrial settings, workers are

often exposed to both Cr III and Cr VI. However, there was no excess cancer in workers exposed only to Cr III

during leather tanning (ATSDR, 2000). 

Carcinogenicity studies in which animals were dosed directly with Cr III compounds were uniformly negative.

There was no evidence of carcinogenicity in male or female BD rats fed diets containing up to 5% chromium oxide

(2,040 mg Cr III/kg per day), 5 days per week for 2 years (Ivankovic and Preussmann, 1975); in the offspring of

rats treated for 60 days prior to gestation and during gestation, after 600 days of observation (Ivankovic and

Preussmann, 1975); in Swiss mice (0.46 mg chromium/kg per day) (Schroeder et al., 1964), or Long-Evans rats

(0.48 mg chromium/kg per day) (Schroeder et al., 1965) dosed with 5 mg/L chromium acetate in drinking water

for 2 to 3 years. Implantation of an intrabronchial pellet containing a 50:50 mix of chromium oxide:cholesterol

binder in rats for 136 weeks failed to produce lung tumors, while bronchial carcinomas were increased in rats

similarly exposed to Cr VI as calcium chromate (Laskin et al., 1970). Use of the same experimental design with

2 mg of Cr III/pellet failed to produce local squamous carcinomas, carcinoma in situ, or increases in squamous

metaplasia following exposure to chromium oxide, chromium chloride hexahydrate, or chrome tan (basic

chromium sulfate) (Levy and Venitt, 1986). There was no increase in lung tumor frequency over controls in

strain A mice given 24 intraperitoneal injections of 2,400 mg chromium sulfate/kg (650 mg chromium/kg), over

8 weeks and killed 30 weeks after the initiation of dosing (Stoner et al., 1976). Urethane, the positive control in

this study, did increase lung tumor frequency. There was no increase in renal tumors in male F344 rats treated with

N-ethyl-N-hydroxyethylnitrosamine followed by 600 mg chromium chloride hexahydrate/L (11 mg chromium/kg

per day) in drinking water for 25 weeks, although dysplastic foci were significantly increased (Kurokawa et al.,

1985). 
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A 2-year study revealed clear evidence of carcinogenicity of Cr VI (as sodium dichromate dihydrate) in both male

and female F344/N rats and B6C3F1 mice following exposures up to 516 mg/L (257.4 mg/L in male mice) in

drinking water, based on increased incidences of squamous cell neoplasms of the oral cavity in rats and increased

incidences of neoplasms of the small intestine (duodenum, jejunum, or ileum) in mice (NTP, 2008). 

GENETIC TOXICITY

Cr III has been shown to be genotoxic in acellular test systems that permit direct contact with DNA (Snow et al.,

1991; Snow, 1994; Bridgewater et al., 1994). However, it often gives negative results in standard genetic toxicity

assays. This lack of genotoxicity is attributed to the low level of absorption of most Cr III salts. For chromium

picolinate, the published mutagenicity test data indicate that the compound may be detected as mutagenic in some

in vitro mammalian test systems, but not in standard bacterial mutation assays. For example, no mutagenicity was

observed in Salmonella typhimurium strains TA98, TA100, TA1535, or TA1537 treated with chromium picolinate

(up to 10,000 μg/plate) with or without exogenous metabolic activation enzymes (liver S9 fraction) (Whittaker

et al., 2005). However, results of a mutagenicity assay conducted with chromium picolinate (6.2 to 124 μg/mL) in

mouse L5178Y tk+/– cells demonstrated a clear increase in mutant cell colonies, with and without S9; no increases

in mutant colonies were seen with picolinic acid (Whittaker et al., 2005). Particulate chromium picolinate

(suspended in acetone, 440 μg/mL, corresponding to 52 μg/mL Cr III) was reported to induce hprt mutations in

Chinese hamster ovary (CHO) cells after a 48-hour exposure period (Stearns et al., 2002); picolinic acid was

negative in this assay. Analysis of the hprt mutations induced by chromium picolinate suggested that most were

deletions, predominantly one exon in size (Coryell and Stearns, 2006). In contrast to these results, a second CHO

cell study conducted with a commercial preparation of chromium picolinate (Chromax®

; up to 500 μg/mL in

dimethylsulfoxide) found no significant increases in hprt mutations using a 5-hour exposure period with and

without S9 activation, in addition to the 48-hour exposure used in the earlier study (Slesinski et al., 2005). 
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Both soluble or particulate forms of chromium picolinate were reported to induce highly significant increases in

chromosomal aberrations in CHO AA8 cells treated for 24 hours with concentrations ranging from 0.05 to 1.0 mM

in acetone, in the absence of S9 (Stearns et al., 1995). In this study, picolinic acid (1.5 and 2.0 mM) also induced

chromosomal aberrations, although at a much lower level than was seen with chromium picolinate; no

chromosomal damage was induced by two other Cr III salts, chromium nicotinate or chromium chloride (Stearns

et al., 1995). A subsequent study of chromosomal aberration induction in CHO cells using Chromax® (96.25 to

770 μg/mL in dimethylsulfoxide) gave negative results, with and without S9, after 4 or 20 hours of exposure (Gudi

et al., 2005). Recently, studies for induction of DNA damage in cultured mouse lymphoma L5178Y tk+/– cells and

human lymphocytes, measured by the comet assay, showed no increase in DNA damage after a 3-hour exposure to

500 μM chromium picolinate dissolved in dimethylsulfoxide, or dissolved directly in medium containing serum

(lymphocytes only); when lymphocyte exposures were carried out in medium without serum, a small but

statistically significant increase in DNA damage was noted (Andersson et al., 2007). These observations led the

authors to speculate that serum components may have bound chromium picolinate, preventing it from entering the

cells.

Salivary gland chromosomal aberrations were reported in Drosophila melanogaster larvae whose male parents

were reared on medium containing 260 μg/L chromium picolinate (Stallings et al., 2006). In contrast to the

observations in Drosophila, no significant increases in micronucleus frequencies in peripheral blood erythrocytes

or DNA damage in lymphocytes or hepatocytes, measured by the comet assay, were seen in male CBA/Ca mice

administered a single intraperitoneal injection of 3 mg/kg chromium picolinate (Andersson et al., 2007). 

Additional mutagenicity test data are available for other trivalent chromium compounds, and although limited, the

data appear to be consistent with what has been shown for chromium picolinate. No mutagenicity was detected

with chromium carbonyl in S. typhimurium (Zeiger et al., 1992), but in CHO cells, chromium chloride induced

significant increases in hprt mutations, although the increases were less than those observed with chromium

picolinate under the same treatment conditions and equivalent concentrations (Stearns et al., 2002). In contrast,
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chromium chloride did not produce significant increases in the frequencies of TK mutations in mouse L5178Y

TK+/– cells under the same treatment conditions and at higher concentrations than were tested with chromium

picolinate (Whittaker et al., 2005). Chromium chloride (1 to 5 μM) was reported to increase the frequency of

micronuclei in cultured human fibroblasts (Seoane and Dulout, 2001), but no increase in chromosomal aberrations

was observed in CHO cells treated with chromium carbonyl (NTP unpublished data). In vivo, no increase in

micronucleated erythrocytes was seen in male B6C3F1 mice administered chromium carbonyl (0.51 to 255 μg)

once daily by intraperitoneal injection for 4 weeks (Witt et al., 2000). In addition, no significant increases in

mutant clones were observed in the Drosophila wing spot test after exposure of flies to 1.0 to 10.0 mM chromium

chloride in medium from day 3 until adulthood (Amrani et al., 1999). 

In a study examining the comparative genotoxicity of Cr VI and Cr III, Kirpnick-Sobol et al. (2006) reported that

exposure of pregnant mice (C57BL/6J-p

un/

pun) to either potassium dichromate (Cr VI; 62.5 or 125.0 mg/L,

calculated to yield an average daily dose of 12.5 or 25 mg/kg) or chromium (III) chloride (1,875 or 3,750 mg/L,

calculated to yield an average daily dose of 375 or 750 mg/kg) in drinking water during gestational days 10 to 20

resulted in significant increases in the frequencies of large-scale DNA deletions in pups examined at 20 days of

age. Furthermore, Kirpnick-Sobol et al. (2006) reported that in mouse fetuses exposed to Cr III and examined on

gestational day 17.5, significant increases in DNA deletions were seen at threefold lower chromium tissue

concentrations than in fetuses exposed to Cr VI. These authors conducted additional studies in the yeast strain

RS 112 and found similar effects: dose-related increases in DNA deletions following exposure in medium to

concentrations ranging from 0.7 to 2.1 mM chromium. Comparing intracellular chromium concentrations in the

yeast with DNA deletion frequency showed that Cr III was a more potent inducer of DNA deletions than was

Cr VI. The authors concluded that the data from both the mouse studies and the yeast studies indicated that

although only small amounts of Cr III were absorbed, Cr III was highly effective at inducing DNA damage.
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STUDY RATIONALE

Chromium picolinate was nominated by the National Cancer Institute and a private individual for testing based on

the potential for widespread consumer exposure from use as a dietary supplement. The monohydrate form was

selected for testing because it is the commercially available form of chromium picolinate. Dietary exposure was

chosen because humans ingest chromium picolinate in supplements. 

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29

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PROCUREMENT AND CHARACTERIZATION

OF CHROMIUM PICOLINATE MONOHYDRATE

Chromium picolinate monohydrate was obtained from TCI America (Portland, OR) in one lot (OGJ01) and from

Sigma-Aldrich (St. Louis, MO) in one lot (CHESS0204DFCI). Lot OGJ01 was used in the 3-month studies; the

unused remainder of lot OGJ01 was combined with lot CHESS0204DFCI by the analytical chemistry laboratory,

Battelle Toxicology Northwest (Richland, WA), and assigned lot number 672002, which was used in the 2-year

studies. Identity, purity, and stability analyses were performed by the analytical chemistry laboratory, the study

laboratory at Southern Research Institute (Birmingham, AL), PIXE Analytical Laboratories (Tallahassee, FL),

Element Analysis Corporation (Lexington, KY), Galbraith Laboratories, Inc. (Knoxville, TN), and Oneida

Research Services, Inc. (Whitesboro, NY) (Appendix I). Reports on analyses performed in support of the

chromium picolinate monohydrate studies are on file at the National Institute of Environmental Health Sciences.

Lots OGJ01 and 672002 of the chemical, a reddish-purple crystalline powder, were identified as chromium

picolinate monohydrate by infrared and proton nuclear magnetic resonance spectroscopy, X-ray diffraction, and

electrospray ionization-mass spectrometry (EI-MS). 

The moisture contents of lots OGJ01 and 672002 were determined using Karl Fischer titration, and weight loss on

drying was determined for lot 672002. Purity of the test chemical was determined by elemental analyses

(lots OGJ01 and 672002), proton-induced X-ray emission (PIXE) spectroscopy (lots OGJ01 and 672002),

inductively coupled plasma-atomic emission spectroscopy (ICP-AES) (lots OGJ01 and 672002), high-performance

liquid chromatography (HPLC) with diode-array detection (HPLC-DAD) (lot OGJ01), ultraviolet-visible detection

(HPLC-UV/Vis) (lot 672002), UV detection (HPLC-UV) (lots OGJ01 and 672002), or ICP-mass spectrometric

detection (HPLC-ICP-MS) (lots OGJ01 and 672002).

MATERIALS AND METHODS
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For lot OGJ01, the results of Karl Fischer titration for water content, elemental analyses for carbon, hydrogen, and

nitrogen, and ICP-AES analysis for total chromium were all consistent with the theoretical values for chromium

picolinate monohydrate. PIXE analysis indicated the absence of significant metallic impurities and a total

chromium content of 117% of the theoretical value. HPLC-DAD revealed a major component at 96%, one

impurity at 2.1%, and five additional impurities at greater than or equal to 0.1% each. HPLC-UV by one system

followed by HPLC-ICP-MS indicated that the maximum concentrations of free (uncomplexed) Cr(III) or Cr(VI)

were less than 0.025%. The overall purity of lot OGJ01 was determined to be greater than 96%.

For lot 672002, Karl Fischer titration and weight loss on drying assays indicated the presence of approximately

1 mole of water in the test chemical complex. Results of elemental analyses for carbon, hydrogen, and nitrogen

and of ICP-AES analysis for total chromium content were consistent with the theoretical values for chromium

picolinate monohydrate. PIXE analyses indicated a chromium content consistent with the theoretical value and

absence of significant metallic impurities. HPLC-UV/Vis indicated one major peak with an area percent purity of

approximately 95%. HPLC-UV by one system coupled with HPLC-ICP-MS indicated that the maximum

concentrations of free Cr III or Cr VI were less than 0.025%. The overall purity of lot 672002 was determined to

be greater than 95%.

In an attempt to identify the impurities indicated by HPLC-DAD in lot OGJ01, preparations of

chromium:picolinate complexes were made and analyzed using HPLC-DAD by a second system and

HPLC-EI-MS. The results were inconclusive due to insufficient assay sensitivity and lack of authentic reference

standards; however, these analyses provided evidence that the impurities in the test chemical were probably

chromium:picolinate complexes, although the exact structures and ratios were uncertain. 

Stability studies of the bulk chemical were performed using ICP-AES and HPLC-UV. These studies indicated that

chromium picolinate monohydrate was stable as a bulk chemical for at least 2 weeks when stored in sealed amber

glass containers at temperatures up to 60° C. To ensure stability, the bulk chemical was stored at room
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temperature, protected from light, in sealed plastic buckets. Periodic reanalyses of the bulk chemical were

performed during the 3-month and 2-year studies using HPLC-UV, and no degradation of the bulk chemical was

detected. 

PREPARATION AND ANALYSIS OF DOSE FORMULATIONS

The dose formulations were prepared by mixing chromium picolinate monohydrate with feed (Table I2).

Homogeneity studies of 82 and 50,000 ppm dose formulations and stability studies of the 82 ppm dose formulation

were performed by the analytical chemistry laboratory using ICP-AES. Additional homogeneity studies of 80 and

50,000 ppm dose formulations were performed by the study laboratory using HPLC-UV. Homogeneity was

confirmed, and the stability of the dose formulations was confirmed for at least 42 days at room temperature when

stored in double-thick sealed plastic bags, protected from light. 

Periodic analyses of the dose formulations of chromium picolinate monohydrate were conducted by the study

laboratory using HPLC-UV. For the 3-month studies, the dose formulations were analyzed at the beginning,

midpoint, and end of the studies; all 35 dose formulations analyzed for rats and mice were within 10% of the target

concentrations (Table I3). During the 2-year studies, the dose formulations were analyzed approximately every

12 weeks (Table I4). Of the dose formulations analyzed, all 167 for rats and all 99 for mice were within 10% of

the target concentrations. 

Samples of dosed feed taken from the animal rooms were analyzed periodically during the studies. During the

3-month studies, all 10 samples taken from the rat animal room and 8 of 10 samples from the mouse animal room

were within 10% of target; all samples were within 13% of target. During the 2-year studies, all 12 samples from

the rat animal room and 8 of 15 samples from the mouse animal room were within 10% of target; all samples were

within at least 27% of target. Low results in the mouse samples were attributed to contamination by urine, feces,

and bedding during the study period when animals were small enough to get into the feeders.
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3-MONTH STUDIES

Male and female F344/N rats and B6C3F1 mice were obtained from Taconic Farms, Inc. (Germantown, NY). On

receipt, the rats and mice were approximately 4 weeks old. Rats were quarantined for 13 (males) or 14 (females)

days, and mice were quarantined for 12 (males) or 11 (females) days. Animals were approximately 6 weeks old on

the first day of the studies. Before the studies began, five male and five female rats and mice were randomly

selected for parasite evaluation and gross observation for evidence of disease. At the end of the studies, serologic

analyses were performed on five male and five female control rats and mice using the protocols of the NTP

Sentinel Animal Program (Appendix L).

Groups of 10 male and 10 female rats and mice were fed diets containing 0, 80, 240, 2,000, 10,000, or 50,000 ppm

chromium picolinate monohydrate for 14 weeks. Additional groups of 10 male and 10 female clinical pathology

study rats were exposed to the same concentrations for 3 weeks. Feed and water were available ad libitum. Male

mice were housed individually, and rats and female mice were housed five per cage. Clinical findings were

recorded weekly for core study animals. Feed consumption by core study animals was recorded weekly by cage.

Core study animals were weighed initially, weekly, and at the end of the studies. Details of the study design and

animal maintenance are summarized in Table 1.

Animals were anesthetized with carbon dioxide, and blood was collected from the retroorbital sinus of clinical

pathology rats on days 3 and 21 and from core study rats and mice at the end of the study for hematology and

clinical chemistry (rats only) analyses. Blood samples for hematology were placed in tubes containing EDTA.

Hematology parameters were measured on an ADVIATM 120 hematology analyzer (Bayer, Inc., Tarrytown, NY)

using reagents provided by the manufacturer. Blood samples for clinical chemistry were placed in tubes containing

no anticoagulant and analyzed using a Hitachi 911 automated analyzer (Boehringer Mannheim, Indianapolis, IN)

with reagents provided by the manufacturer or Sigma Diagnostics (St. Louis, MO). The parameters measured are

listed in Table 1.
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At the end of the 3-month studies, samples were collected for sperm motility and vaginal cytology evaluations on

core study rats and mice exposed to 0, 2,000, 10,000, or 50,000 ppm. The parameters evaluated are listed in

Table 1. For 12 consecutive days prior to scheduled terminal sacrifice, the vaginal vaults of the females were

moistened with saline, if necessary, and samples of vaginal fluid and cells were stained. Relative numbers of

leukocytes, nucleated epithelial cells, and large squamous epithelial cells were determined and used to ascertain

estrous cycle stage (i.e., diestrus, proestrus, estrus, and metestrus). Male animals were evaluated for sperm count

and motility at terminal sacrifice. The left testis and left epididymis were isolated and weighed. The tail of the

epididymis (cauda epididymis) was then removed from the epididymal body (corpus epididymis) and weighed.

Test yolk (rats) or modified Tyrode’s buffer (mice) was applied to slides, and a small incision was made at the

distal border of the cauda epididymis. The sperm effluxing from the incision were dispersed in the buffer on the

slides, and the numbers of motile and nonmotile spermatozoa were counted for five fields per slide by two

observers. Following completion of sperm motility estimates, each left cauda epididymis was placed in buffered

saline solution. Caudae were finely minced, and the tissue was incubated in the saline solution and then heat fixed

at 65° C. Sperm density was then determined microscopically with the aid of a hemacytometer. To quantify

spermatogenesis, the testicular spermatid head count was determined by removing the tunica albuginea and

homogenizing the left testis in phosphate-buffered saline containing 10% dimethyl sulfoxide. Homogenizationresistant spermatid nuclei were counted with a hemacytometer.

Necropsies were performed on all core study animals. The heart, right kidney, liver, lung, right testis, and thymus

were weighed. Tissues for microscopic examination were fixed and preserved in 10% neutral buffered formalin

(eyes were initially fixed in Davidson’s solution), processed and trimmed, embedded in paraffin, sectioned to a

thickness of 4 to 6 μm, and stained with hematoxylin and eosin. Complete histopathologic examinations were

performed on control and 50,000 ppm rats and mice. Table 1 lists the tissues and organs routinely examined.
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2-YEAR STUDIES

Study Design

Groups of 50 male and 50 female rats and mice were fed diets containing 0, 2,000, 10,000, or 50,000 ppm

chromium picolinate monohydrate for 105 weeks. Additional tissue distribution study groups of 30 male rats and

30 female mice were exposed to the same concentrations for up to 180 days (Appendix M). 

Source and Specification of Animals

Male and female F344/N rats and B6C3F1 mice were obtained from Taconic Farms, Inc. (Germantown, NY), for

use in the 2-year studies. Rats and mice were quarantined for 12 days before the beginning of the studies. Five

male and five female rats and mice were randomly selected for parasite evaluation and gross observation of

disease. Rats and mice were approximately 5 to 6 weeks old at the beginning of the studies. The health of the

animals was monitored during the studies according to the protocols of the NTP Sentinel Animal Program

(Appendix L).

Animal Maintenance

Rats were housed three (males) or five (females) per cage, and mice were housed individually (males) or five

(females) per cage. Feed and water were available ad libitum. Feed consumption was measured weekly for the

first 13 weeks of the study and approximately monthly thereafter. Cages and racks were changed and rotated every

2 weeks. Further details of animal maintenance are given in Table 1. Information on feed composition and

contaminants is provided in Appendix K.

Clinical Examinations and Pathology

All animals were observed twice daily and were weighed initially, weekly for the first 13 weeks, monthly

thereafter, and at the end of the studies. Clinical findings were recorded monthly.

Complete necropsies and microscopic examinations were performed on all rats and mice. At necropsy, all organs

and tissues were examined for grossly visible lesions, and all major tissues were fixed and preserved in 10%
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neutral buffered formalin (eyes were initially fixed in Davidson’s solution), processed and trimmed, embedded in

paraffin, sectioned to a thickness of 4 to 6 μm, and stained with hematoxylin and eosin for microscopic

examination. For all paired organs (e.g., adrenal gland, kidney, ovary), samples from each organ were examined.

Tissues examined microscopically are listed in Table 1.

Microscopic evaluations were completed by the study laboratory pathologist, and the pathology data were entered

into the Toxicology Data Management System. The slides, paraffin blocks, and residual wet tissues were sent to

the NTP Archives for inventory, slide/block match, and wet tissue audit. The slides, individual animal data

records, and pathology tables were evaluated by an independent quality assessment laboratory. The individual

animal records and tables were compared for accuracy; the slide and tissue counts were verified, and the

histotechnique was evaluated. For the 2-year studies, a quality assessment pathologist evaluated slides from all

tumors and all potential target organs, which included the liver of all animals; the pituitary gland of male and

female rats; and the adrenal cortex, bone marrow, clitoral gland, lung, mesenteric lymph node, thyroid gland, and

uterus of female rats.

The quality assessment report and the reviewed slides were submitted to the NTP Pathology Working Group

(PWG) coordinator, who reviewed the selected tissues and addressed any inconsistencies in the diagnoses made by

the laboratory and quality assessment pathologists. Representative histopathology slides containing examples of

lesions related to chemical administration, examples of disagreements in diagnoses between the laboratory and

quality assessment pathologists, or lesions of general interest were presented by the coordinator to the PWG for

review. The PWG consisted of the quality assessment pathologist and other pathologists experienced in rodent

toxicologic pathology. This group examined the tissues without any knowledge of dose groups or previously

rendered diagnoses. When the PWG consensus differed from the opinion of the laboratory pathologist, the

diagnosis was changed. Final diagnoses for reviewed lesions represent a consensus between the laboratory

pathologist, reviewing pathologist(s), and the PWG. Details of these review procedures have been described, in

part, by Maronpot and Boorman (1982) and Boorman et al. (1985). For subsequent analyses of the pathology data,
36 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE 1

Experimental Design and Materials and Methods in the Feed Studies of Chromium Picolinate Monohydrate

3-Month Studies 2-Year Studies

Study Laboratory

Southern Research Institute (Birmingham, AL)

Strain and Species

F344/N rats

B6C3F1 mice

Animal Source

Taconic Farms, Inc. (Germantown, NY)

Time Held Before Studies

Rats: 13 (males) or 14 (females) days

Mice: 12 (males) or 11 (females) days

Average Age When Studies Began

6 weeks

Date of First Exposure

Rats: October 16 (males) or 17 (females), 2001

Mice: October 15 (males) or 14 (females), 2001

Duration of Exposure

Core studies: 14 weeks

Clinical pathology study: 3 weeks (rats)

Date of Last Exposure

Core studies:

Rats: January 16 (males) or 17 (females), 2002

Mice: January 15 (males) or 14 (females), 2002

Clinical pathology study:

November 5 (males) or 6 (females), 2001

Necropsy Dates

Rats: January 16 (males) or 17 (females), 2002

Mice: January 15 (males) or 14 (females), 2002

Average Age at Necropsy

19 weeks

Size of Study Groups

Core studies: 10 males and 10 females

Clinical pathology study: 10 male and 10 female rats

Method of Distribution

Animals were distributed randomly into groups of approximately

equal initial mean body weights.

Southern Research Institute (Birmingham, AL)

F344/N rats

B6C3F1 mice

Taconic Farms, Inc. (Germantown, NY)

12 days 

5 to 6 weeks

Rats: August 12, 2002

Mice: July 29, 2002

105 weeks

Rats: August 9-17, 2004

Mice: July 26 to August 2, 2004

Rats: August 9-17, 2004

Mice: July 26 to August 2, 2004

Rats: 109 to 111 weeks

Mice: 110 to 112 weeks

50 males and 50 females

Same as 3-month studies

the decision of whether to evaluate the diagnosed lesions for each tissue type separately or combined was generally

based on the guidelines of McConnell et al. (1986).
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TABLE 1

Experimental Design and Materials and Methods in the Feed Studies of Chromium Picolinate Monohydrate

3-Month Studies 2-Year Studies

Animals per Cage

Rats: 5

Mice: 1 (males) or 5 (females)

Method of Animal Identification

Tail tattoo

Diet

Irradiated NTP-2000 open formula meal diet (Zeigler Brothers, Inc.,

Gardners, PA), available ad libitum, changed weekly

Water

Tap water (Birmingham, AL, municipal supply) via automatic

watering system (Edstrom Industries, Waterford, WI), available

ad libitum

Cages

Polycarbonate (Lab Products, Inc., Maywood, NJ); changed once

every 2 weeks

Bedding

Irradiated, heat-treated hardwood bedding chips (P.J. Murphy Forest

Products, Inc., Montville, NJ); changed weekly (male mice) or twice

weekly

Rack Filters

Reemay® spun-bonded polyester (Andico, Birmingham, AL);

changed once every 2 weeks

Racks

Stainless steel (Lab Products, Maywood, NJ); changed once every

2 weeks

Animal Room Environment

Temperature: 72° ± 3° F

Relative humidity: 50% ± 15%

Room fluorescent light: 12 hours/day

Room air changes: 10/hour

Exposure Concentrations

0, 80, 240, 2,000, 10,000, or 50,000 ppm in feed, available

ad libitum

Type and Frequency of Observation

Observed twice daily; core study animals were weighed initially,

weekly, and at the end of the studies; clinical findings were recorded

weekly. Feed consumption by core study animals was recorded

weekly by cage.

Method of Sacrifice

Carbon dioxide asphyxiation

Necropsy

Necropsies were performed on all core study animals. Organs

weighed were heart, right kidney, liver, lung, right testis, and thymus.

Rats: 3 (males) or 5 (females)

Mice: 1 (males) or 5 (females)

Tail tattoo

Same as 3-month studies

Same as 3-month studies

Same as 3-month studies

Same as 3-month studies

Same as 3-month studies

Same as 3-month studies

Temperature: 72° ± 3° F

Relative humidity: 50% ± 15%

Room fluorescent light: 12 hours/day

Room air changes: 10/hour

0, 2,000, 10,000, or 50,000 ppm in feed, available ad libitum

Observed twice daily; animals were weighed and feed consumption

was recorded initially, weekly for 13 weeks, monthly thereafter, and

at the end of the studies. Clinical findings were recorded monthly for

core study animals.

Same as 3-month studies

Necropsies were performed on all animals. 
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STATISTICAL METHODS

Survival Analyses

The probability of survival was estimated by the product-limit procedure of Kaplan and Meier (1958) and is

presented in the form of graphs. Animals found dead of other than natural causes or missing were censored from

the survival analyses; animals dying from natural causes were not censored. Statistical analyses for possible doserelated effects on survival used Cox’s (1972) method for testing two groups for equality and Tarone’s (1975) life

table test to identify dose-related trends. All reported P values for the survival analyses are two sided.

Clinical Pathology

Blood was collected from the retroorbital sinus of clinical pathology

rats on days 3 and 21 and from core study animals at the end of the

studies for hematology or clinical chemistry (rats only).

Hematology: hematocrit; hemoglobin; erythrocyte, reticulocyte, and

platelet counts; nucleated erythrocytes; mean cell volume; mean cell

hemoglobin; mean cell hemoglobin concentration; and leukocyte

count and differentials

Clinical chemistry: urea nitrogen, creatinine, total protein, albumin,

alanine aminotransferase, alkaline phosphatase, creatine kinase,

sorbitol dehydrogenase, and bile acids

Histopathology

Complete histopathology was performed on 0 and 50,000 ppm core

study rats and mice. In addition to gross lesions and tissue masses,

the following tissues were examined: adrenal gland, bone with

marrow, brain, clitoral gland, esophagus, eyes, gallbladder (mice

only), harderian gland, heart, large intestine (cecum, colon, rectum),

small intestine (duodenum, jejunum, ileum), kidney, liver, lung,

lymph nodes (mandibular and mesenteric), mammary gland, nose,

ovary, pancreas, parathyroid gland, pituitary gland, preputial gland,

prostate gland, salivary gland, skin, spleen, stomach (forestomach

and glandular), testis with epididymis and seminal vesicle, thymus,

thyroid gland, trachea, urinary bladder, and uterus.

Sperm Motility and Vaginal Cytology

At the end of the studies, sperm samples were collected from male

animals in the 0, 2,000, 10,000, and 50,000 ppm groups for sperm

motility evaluations. The following parameters were evaluated:

spermatid heads per testis and per gram testis, and epididymal

spermatozoal motility and concentrations. The left cauda, left

epididymis, and left testis were weighed. Vaginal samples were

collected for up to 12 consecutive days prior to the end of the studies

from females exposed to 0, 2,000, 10,000, or 50,000 ppm for vaginal

cytology evaluations. The percentage of time spent in the various

estrous cycle stages and estrous cycle length were evaluated.

TABLE 1

Experimental Design and Materials and Methods in the Feed Studies of Chromium Picolinate Monohydrate

3-Month Studies 2-Year Studies

None

Complete histopathology was performed on all animals. In addition

to gross lesions and tissue masses, the following tissues were

examined: adrenal gland, bone with marrow, brain, clitoral gland,

esophagus, eyes, gallbladder (mice only), harderian gland, heart,

large intestine (cecum, colon, rectum), small intestine (duodenum,

jejunum, ileum), kidney, liver, lung, lymph nodes (mandibular and

mesenteric), mammary gland, nose, ovary, pancreas, parathyroid

gland, pituitary gland, preputial gland, prostate gland, salivary gland,

skin, spleen, stomach (forestomach and glandular), testis with

epididymis and seminal vesicle, thymus, thyroid gland, trachea,

urinary bladder, and uterus. 

None
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Calculation of Incidence

The incidences of neoplasms or nonneoplastic lesions are presented in Tables A1, A4, B1, B4, C1, C3, D1, and D3

as the numbers of animals bearing such lesions at a specific anatomic site and the numbers of animals with that site

examined microscopically. For calculation of statistical significance, the incidences of most neoplasms (Tables A2,

B2, C2, and D2) and all nonneoplastic lesions are given as the numbers of animals affected at each site examined

microscopically. However, when macroscopic examination was required to detect neoplasms in certain tissues

(e.g., harderian gland, intestine, mammary gland, and skin) before microscopic evaluation, or when neoplasms had

multiple potential sites of occurrence (e.g., leukemia or lymphoma), the denominators consist of the number of

animals on which a necropsy was performed. Tables A2, B2, C2, and D2 also give the survival-adjusted neoplasm

rate for each group and each site-specific neoplasm. This survival-adjusted rate (based on the Poly-3 method

described below) accounts for differential mortality by assigning a reduced risk of neoplasm, proportional to the

third power of the fraction of time on study, only to site-specific, lesion-free animals that do not reach terminal

sacrifice.

Analysis of Neoplasm and Nonneoplastic Lesion Incidences

The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989; Piegorsch and Bailer, 1997) was used to assess

neoplasm and nonneoplastic lesion prevalence. This test is a survival-adjusted quantal-response procedure that

modifies the Cochran-Armitage linear trend test to take survival differences into account. More specifically, this

method modifies the denominator in the quantal estimate of lesion incidence to approximate more closely the total

number of animal years at risk. For analysis of a given site, each animal is assigned a risk weight. This value is

one if the animal had a lesion at that site or if it survived until terminal sacrifice; if the animal died prior to

terminal sacrifice and did not have a lesion at that site, its risk weight is the fraction of the entire study time that it

survived, raised to the kth power.

This method yields a lesion prevalence rate that depends only upon the choice of a shape parameter for a Weibull

hazard function describing cumulative lesion incidence over time (Bailer and Portier, 1988). Unless otherwise

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specified, a value of k=3 was used in the analysis of site-specific lesions. This value was recommended by Bailer

and Portier (1988) following an evaluation of neoplasm onset time distributions for a variety of site-specific

neoplasms in control F344 rats and B6C3F1 mice (Portier et al., 1986). Bailer and Portier (1988) showed that the

Poly-3 test gave valid results if the true value of k was anywhere in the range from 1 to 5. A further advantage of

the Poly-3 method is that it does not require lesion lethality assumptions. Variation introduced by the use of risk

weights, which reflect differential mortality, was accommodated by adjusting the variance of the Poly-3 statistic as

recommended by Bieler and Williams (1993).

Tests of significance included pairwise comparisons of each exposed group with controls and a test for an overall

exposure-related trend. Continuity-corrected Poly-3 tests were used in the analysis of lesion incidence, and

reported P values are one sided. The significance of lower incidences or decreasing trends in lesions is represented

as 1–P with the letter N added (e.g., P=0.99 is presented as P=0.01N). 

Analysis of Continuous Variables

Two approaches were employed to assess the significance of pairwise comparisons between exposed and control

groups in the analysis of continuous variables. Organ and body weight data, which historically have approximately

normal distributions, were analyzed with the parametric multiple comparison procedures of Dunnett (1955) and

Williams (1971, 1972). Hematology, clinical chemistry, spermatid, and epididymal spermatozoal data, which have

typically skewed distributions, were analyzed using the nonparametric multiple comparison methods of Shirley

(1977) (as modified by Williams, 1986) and Dunn (1964). Jonckheere’s test (Jonckheere, 1954) was used to assess

the significance of the dose-related trends and to determine whether a trend-sensitive test (Williams’ or Shirley’s

test) was more appropriate for pairwise comparisons than a test that does not assume a monotonic dose-related

trend (Dunnett’s or Dunn’s test). Prior to statistical analysis, extreme values identified by the outlier test of Dixon

and Massey (1957) were examined by NTP personnel, and implausible values were eliminated from the analysis.

Because vaginal cytology data are proportions (the proportion of the observation period that an animal was in a

given estrous stage), an arcsine transformation was used to bring the data into closer conformance with a normality
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assumption. Treatment effects were investigated by applying a multivariate analysis of variance (Morrison, 1976)

to the transformed data to test for simultaneous equality of measurements across exposure concentrations.

Proportions of regular cycling females in each exposed group were compared to the control group using the Fisher

exact test (Gart et al., 1979). Tests for extended periods of estrus and diestrus were constructed based on a Markov

chain model proposed by Girard and Sager (1987). For each exposure group, a transition probability matrix was

estimated for transitions among the proestrus, estrus, metestrus, and diestrus stages, with provision for extended

stays within estrus and diestrus. Equality of transition matrices among exposure groups and between the control

group and each exposed group was tested using chi-square statistics.

Historical Control Data

The concurrent control group represents the most valid comparison to the treated groups and is the only control

group analyzed statistically in NTP bioassays. However, historical control data are often helpful in interpreting

potential treatment-related effects, particularly for uncommon or rare neoplasm types. For meaningful

comparisons, the conditions for studies in the historical database must be generally similar. One significant factor

affecting the background incidence of neoplasms at a variety of sites is diet. In 1995, the NTP incorporated a new

diet (NTP-2000) that contains less protein and more fiber and fat than the NIH-07 diet previously used in toxicity

and carcinogenicity studies (Rao, 1996, 1997). The NTP historical database contains all studies that use the

NTP-2000 diet with histopathology findings completed within the most recent 5-year period. A second potential

source of variability is route of administration. In general, the historical database for a given study will include

studies using the same route of administration, and the overall incidences of neoplasms for all routes of

administration are included for comparison, including the present study.

QUALITY ASSURANCE METHODS

The 3-month and 2-year studies were conducted in compliance with Food and Drug Administration Good

Laboratory Practice Regulations (21 CFR, Part 58). In addition, as records from the 2-year studies were submitted
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to the NTP Archives, these studies were audited retrospectively by an independent quality assessment contractor.

Separate audits covered completeness and accuracy of the pathology data, pathology specimens, final pathology

tables, and a draft of this NTP Technical Report. Audit procedures and findings are presented in the reports and

are on file at NIEHS. The audit findings were reviewed and assessed by NTP staff, and all comments were

resolved or otherwise addressed during the preparation of this Technical Report.

GENETIC TOXICOLOGY

The genetic toxicity of chromium picolinate monohydrate was assessed by testing the ability of the chemical to

induce mutations in various strains of Salmonella typhimurium and increases in the frequency of micronucleated

erythrocytes in mouse peripheral blood. The genetic toxicity of chromium picolinate was assessed by testing the

ability of the chemical to induce mutations in various strains of S. typhimurium and micronucleated erythrocytes in

rat bone marrow. Micronuclei (literally “small nuclei” or Howell-Jolly bodies) are biomarkers of induced

structural or numerical chromosomal alterations and are formed when acentric fragments or whole chromosomes

fail to incorporate into either of two daughter nuclei during cell division (Schmid, 1975; Heddle et al., 1983). The

protocols for these studies and the results are given in Appendix E.

The genetic toxicity studies have evolved from an earlier effort by the NTP to develop a comprehensive database

permitting a critical anticipation of a chemical’s carcinogenicity in experimental animals based on numerous

considerations, including the molecular structure of the chemical and its observed effects in short-term in vitro and

in vivo genetic toxicity tests (structure-activity relationships). The short-term tests were originally developed to

clarify proposed mechanisms of chemical-induced DNA damage based on the relationship between electrophilicity

and mutagenicity (Miller and Miller, 1977) and the somatic mutation theory of cancer (Straus, 1981; Crawford,

1985). However, it should be noted that not all cancers arise through genotoxic mechanisms.
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DNA reactivity combined with Salmonella mutagenicity is highly correlated with induction of carcinogenicity in

multiple species/sexes of rodents and at multiple tissue sites (Ashby and Tennant, 1991). A positive response in

the Salmonella test was shown to be the most predictive in vitro indicator for rodent carcinogenicity (89% of the

Salmonella mutagens are rodent carcinogens) (Tennant et al., 1987; Zeiger et al., 1990). Additionally, no battery

of tests that included the Salmonella test improved the predictivity of the Salmonella test alone. However, these

other tests can provide useful information on the types of DNA and chromosomal damage induced by the chemical

under investigation. 

The predictivity for carcinogenicity of a positive response in acute in vivo bone marrow chromosome aberration or

micronucleus tests appears to be less than that in the Salmonella test (Shelby et al., 1993; Shelby and Witt, 1995).

However, clearly positive results in long-term peripheral blood micronucleus tests have high predictivity for rodent

carcinogenicity (Witt et al., 2000); negative results in this assay do not correlate well with either negative or

positive results in rodent carcinogenicity studies. Because of the theoretical and observed associations between

induced genetic damage and adverse effects in somatic and germ cells, the determination of in vivo genetic effects

is important to the overall understanding of the risks associated with exposure to a particular chemical. Most

organic chemicals that are identified by the International Agency for Research on Cancer as human carcinogens,

other than hormones, are genotoxic. The vast majority of these are detected by both the Salmonella assay and

rodent bone marrow cytogenetics tests (Shelby, 1988; Shelby and Zeiger, 1990). 
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RATS

3-MONTH STUDY

All rats survived to the end of the study (Table 2). Final mean body weights and body weight gains of all exposed

groups were similar to those of the control groups. Feed consumption by exposed groups of males and females

was generally similar to that by the controls throughout the study. Dietary concentrations of 80, 240, 2,000,

10,000, and 50,000 ppm resulted in average daily doses of approximately 7, 20, 160, 800, and 4,240 mg chromium

picolinate monohydrate/kg body weight to males and 6, 20, 160, 780, and 4,250 mg/kg to females. There were no

RESULTS

TABLE 2

Survival, Body Weights, and Feed Consumption of Rats in the 3-Month Feed Study 

of Chromium Picolinate Monohydrate

Final Weight

Mean Body Weightb (g) Relative Feed

Concentration Survivala Initial Final Change to Controls Consumptionc

(ppm) (%) Week 1 Week 13

Male

0 10/10 92 ± 1 314 ± 8 222 ± 7 13.9 15.4

80 10/10 91 ± 2 317 ± 3 227 ± 2 101 14.8 19.0

240 10/10 91 ± 2 314 ± 4 223 ± 4 100 14.2 14.5

2,000 10/10 92 ± 1 318 ± 6 227 ± 6 101 15.3 16.2

10,000 10/10 92 ± 1 311 ± 5 218 ± 5 99 15.1 15.4

50,000 10/10 92 ± 1 294 ± 12 202 ± 12 94 15.9 17.9

Female

0 10/10 93 ± 1 184 ± 4 91 ± 3 11.4 10.1

80 10/10 93 ± 1 181 ± 3 89 ± 3 98 12.7 10.4

240 10/10 93 ± 1 181 ± 3 88 ± 4 98 12.3 10.6

2,000 10/10 93 ± 2 184 ± 3 91 ± 3 100 12.3 10.9

10,000 10/10 94 ± 1 183 ± 4 89 ± 3 99 12.3 11.5

50,000 10/10 93 ± 1 183 ± 3 91 ± 3 99 12.7 11.9

a Number of animals surviving at 14 weeks/number initially in group b Weights and weight changes are given as mean ± standard error. Differences from the control group are not significant by Dunnett’s test. c Feed consumption is expressed as grams per animal per day.
46 Chromium Picolinate Monohydrate, NTP TR 556 

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clinical findings related to exposure to chromium picolinate monohydrate; reddish-colored feces of 50,000 ppm

animals was believed to be due to excretion of the test article and was not considered a sign of toxicity.

There were minor sporadic changes in the hematology and clinical chemistry variables in rats (Table F1). All

changes were within physiological normal levels, did not demonstrate an exposure relationship, and were not

considered biologically important or toxicologically relevant. 

Absolute and relative kidney weights of all exposed groups of females were significantly greater than those of the

controls, and relative liver weights of exposed groups of females were generally greater than that of the controls

(Table G1). Since there were no significant histologic or clinical chemistry effects in the liver or kidney or

dose-related trends in kidney weights, the increases in the weights of these organs were not considered to be

biologically significant.

There were no significant changes in reproductive organ weights in male or female rats, in sperm parameters in

male rats, or in estrous cyclicity in female rats at any dose (Tables H1 and H2).

No exposure-related lesions occurred in male or female rats.

Exposure Concentration Selection Rationale: Because chromium picolinate monohydrate produced no biologically

significant changes in any of the parameters examined in male or female rats, exposure concentrations of 2,000,

10,000, and 50,000 ppm were selected for the 2-year study in rats.
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2-YEAR STUDY

Survival

Estimates of 2-year survival probabilities for male and female rats are shown in Table 3 and in the Kaplan-Meier

survival curves (Figure 1). Survival of exposed groups of females was similar to that of the control groups.

Although there was a significant trend for decreases in survival in male rats, the decreases were not significant at

any exposure concentration and were considered not related to chromium picolinate monohydrate exposure.

TABLE 3

Survival of Rats in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Male

Animals initially in study 50 50 50 50

Moribund 11 9 12 19

Natural deaths 2 5 3 3

Animals surviving to study termination 37 36 35d 28

Percent probability of survival at end of studya 74 72 70 56

Mean survival (days)b 705 683 705 683

Survival analysisc P=0.041 P=0.897 P=0.783 P=0.077

Female

Animals initially in study 50 50 50 50

Moribund 11 13 11 8

Natural deaths 3 2 3 2

Animals surviving to study termination 36e 35 36 40e

Percent probability of survival at end of study 72 70 72 80

Mean survival (days) 699 704 696 713

Survival analysis P=0.294N P=0.981 P=1.000 P=0.453N

a Kaplan-Meier determinations b Mean of all deaths (uncensored, censored, and terminal sacrifice) c The result of the life table trend test (Tarone, 1975) is in the control column, and the results of the life table pairwise comparisons 

(Cox, 1972) with the controls are in the exposed group columns. A negative trend or lower mortality in an exposure group is 

indicated by N. d Includes two animals that died during the last week of the study e Includes one animal that died during the last week of the study
48 Chromium Picolinate Monohydrate, NTP TR 556 

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FIGURE 1

Kaplan-Meier Survival Curves for Male and Female Rats Exposed 

to Chromium Picolinate Monohydrate in Feed for 2 Years
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Body Weights, Feed and Compound Consumption, and Clinical Findings

Mean body weights of exposed groups of males and females were similar to those of the controls throughout the

study (Tables 4 and 5; Figure 2). Feed consumption by exposed groups of males and females was generally similar

to that of the controls throughout the study (Tables J1 and J2). Dietary concentrations of 2,000, 10,000, and

50,000 ppm resulted in average daily doses of approximately 90, 460, and 2,400 mg chromium picolinate

monohydrate/kg body weight to males and 100, 510, and 2,630 mg/kg to females. No clinical findings were

attributed to chromium picolinate monohydrate exposure.
50 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE 4

Mean Body Weights and Survival of Male Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

Days 0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

on Av. Wt. No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of

Study (g) Survivors (g) controls) Survivors (g) controls) Survivors (g) controls) Survivors

1 114 50 114 100 50 113 99 50 113 99 50

8 143 50 141 98 50 139 97 50 138 97 50

15 170 50 166 98 50 166 98 50 162 95 50

22 199 50 192 97 50 193 97 50 188 95 50

29 208 50 206 99 50 205 98 50 198 95 50

36 235 50 232 99 50 232 99 50 226 96 50

43 251 50 246 98 50 248 99 50 244 97 50

50 269 50 264 98 49 267 99 50 263 98 50

57 271 50 269 99 49 276 102 50 271 100 50

64 286 50 286 100 49 292 102 50 287 100 50

71 301 50 298 99 49 301 100 50 296 98 50

78 311 50 309 99 49 312 100 50 307 99 50

85 318 50 315 99 49 320 101 50 315 99 50

113 350 50 344 98 49 351 100 50 346 99 50

141 367 50 364 99 49 370 101 50 366 100 50

169 385 50 380 99 49 387 100 50 379 99 50

197 400 50 392 98 48 386 96 50 392 98 50

225 414 50 406 98 48 413 100 50 409 99 50

253 423 50 415 98 48 424 100 50 419 99 50

281 435 50 428 98 48 434 100 50 431 99 50

309 439 50 433 99 48 439 100 50 437 99 50

337 443 50 435 98 48 445 101 50 443 100 50

365 448 50 443 99 48 448 100 50 443 99 49

393 448 50 443 99 48 448 100 50 445 99 49

400 451 50 443 98 48 449 100 50 446 99 49

421 447 50 438 98 48 444 99 50 438 98 49

449 453 50 445 98 48 449 99 50 444 98 49

477 450 50 444 99 48 451 100 49 448 100 48

505 451 49 443 98 48 451 100 49 448 99 48

533 451 47 445 99 48 452 100 48 448 99 46

561 447 47 442 99 48 449 101 48 445 100 45

568 449 46 442 98 47 448 100 48 448 100 44

589 445 46 435 98 45 450 101 47 444 100 43

617 444 46 437 98 44 445 100 47 445 100 43

645 443 45 439 99 41 451 102 45 447 101 40

673 443 43 436 98 40 443 100 44 441 100 38

701 439 41 438 100 37 439 100 38 428 98 32

Mean for weeks

1-13 237 234 99 236 99 231 98

14-52 406 400 98 405 100 402 99

53-101 447 441 99 448 100 444 99
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TABLE 5

Mean Body Weights and Survival of Female Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

Days 0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

on Av. Wt. No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of

Study (g) Survivors (g) controls) Survivors (g) controls) Survivors (g) controls) Survivors

1 95 50 95 100 50 95 100 50 96 100 50

11 116 50 115 99 50 113 98 50 115 100 50

18 133 50 132 99 50 130 98 50 132 99 50

25 142 50 142 100 50 141 99 50 141 99 50

32 147 50 147 100 50 146 99 50 148 101 50

39 153 50 156 102 50 153 100 50 156 102 50

46 161 50 162 101 50 160 99 50 162 100 50

53 165 50 166 101 50 163 99 50 165 100 50

60 169 50 170 100 50 166 98 50 169 100 50

67 173 50 175 101 50 170 98 50 173 100 50

74 175 50 179 102 50 174 100 50 177 101 50

81 177 50 181 102 50 177 100 50 180 102 50

88 181 50 184 102 50 179 99 50 182 101 50

116 191 50 193 101 50 189 99 50 192 101 50

144 198 50 203 102 50 198 100 50 201 101 50

172 204 50 207 102 50 203 100 50 206 101 50

200 210 50 213 102 50 205 98 50 212 101 50

228 218 50 224 103 50 217 100 50 221 101 50

256 226 50 230 102 50 223 99 50 226 100 50

284 234 50 241 103 50 232 99 50 236 101 50

312 237 50 247 104 50 236 99 50 240 101 50

340 243 50 253 104 50 241 99 50 246 101 50

368 252 50 260 103 50 250 99 50 254 101 50

396 258 49 268 104 50 257 100 50 260 101 50

403 260 49 270 104 50 259 100 50 263 101 50

424 267 48 275 103 50 262 98 50 267 100 50

452 277 48 284 103 50 271 98 49 275 99 50

480 284 48 292 103 50 279 98 47 283 100 50

508 294 48 303 103 49 289 98 47 289 98 50

536 296 48 306 103 48 291 98 47 295 100 50

571 301 48 315 105 48 299 100 47 303 101 50

592 307 46 315 103 48 305 99 47 306 100 49

620 315 45 322 102 47 308 98 45 310 99 46

648 309 44 317 103 44 307 99 42 307 100 45

676 312 40 316 101 39 309 99 37 313 101 42

704 313 38 318 101 36 311 99 37 315 101 41

Mean for weeks

1-13 153 154 101 151 99 154 100

14-52 218 223 103 216 99 220 101

53-101 289 297 103 286 99 289 100
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FIGURE 2

Growth Curves for Male and Female Rats Exposed to Chromium Picolinate Monohydrate 

in Feed for 2 Years
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Pathology and Statistical Analyses

This section describes the statistically significant or biologically noteworthy changes in the incidences of

neoplasms and nonneoplastic lesions of the preputial gland and clitoral gland. Summaries of the incidences of

neoplasms and nonneoplastic lesions, statistical analyses of primary neoplasms that occurred with an incidence of

at least 5% in at least one animal group, and historical incidences for the neoplasms mentioned in this section are

presented in Appendix A for male rats and Appendix B for female rats.

Preputial and Clitoral Glands: The incidence of preputial gland adenoma was significantly increased in

10,000 ppm males compared to that in the control and exceeded the historical control ranges for feed studies and

for all routes combined (Tables 6, A2, A3). The incidence of clitoral gland adenoma was significantly decreased in

2,000 ppm females (Tables 6 and B2). There was no incidence of preputial gland or clitoral gland carcinoma.

There were no differences in the incidences of preputial gland hyperplasia or clitoral gland hyperplasia between

exposed and control groups of rats (Tables 6, A4, and B4). 

Preputial gland hyperplasia was focal, characterized either by an increase in stratified squamous epithelium of the

ducts or by increased numbers of sebaceous cells and possibly basal cells. Preputial gland adenomas were wellcircumscribed masses that grew by expansion with compression of the surrounding parenchyma. The neoplastic

glands retained some resemblance of acinar structure, although there was some fusion of the acini to form solid

clusters of cells (Copeland-Haines and Eustis, 1990).

The female counterpart gland of the preputial gland in males is the clitoral gland. Proliferative lesions of the

preputial and clitoral gland comprise a morphological continuum, and separation of these into categories of

hyperplasia, adenoma, and carcinoma is based largely on cytological features and degree of altered growth pattern.

Lesions classified as hyperplasia are considered to be preneoplastic (Copeland-Haines and Eustis, 1990).
54 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE 6

Incidences of Neoplasms and Nonneoplastic Lesions of the Preputial Gland in Male Rats 

and Clitoral Gland in Female Rats in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Male

Number Examined Microscopically 50 50 50 50

Hyperplasiaa 3 (2.7)b 1 (4.0) 0 2 (2.5)

Adenomac

Overall rated 1/50 (2%) 1/50 (2%) 7/50 (14%) 4/50 (8%)

Adjusted ratee 2.2% 2.3% 14.9% 9.3%

Terminal ratef 0/37 (0%) 1/36 (3%) 5/35 (14%) 2/28 (7%)

First incidence (days) 653 729 (T) 528 582

Poly-3 testg P=0.206 P=0.750 P=0.031 P=0.158

Female

Number Examined Microscopically 50 50 50 50

Hyperplasia 8 (2.4) 10 (2.6) 11 (2.6) 4 (2.3)

Adenoma, Bilateral 0001

Adenoma (includes bilateral)h

Overall rate 10/50 (20%) 2/50 (4%) 8/50 (16%) 11/50 (22%)

Adjusted rate 21.9% 4.4% 17.7% 23.4%

Terminal rate 9/36 (25%) 1/35 (3%) 6/36 (17%) 11/40 (28%)

First incidence (days) 666 698 647 729 (T)

Poly-3 test P=0.109 P=0.013N P=0.405N P=0.534

(T)Terminal sacrifice

a Number of animals with lesion b Average severity grade of lesions in affected animals: 1=minimal, 2=mild, 3=moderate, 4=marked c Historical incidence for 2-year feed studies with controls given NTP-2000 diet (mean ± standard deviation): 

8/250 (3.2% ± 4.2%), range 0%-10%; all routes: 43/1,193 (3.6% ± 3.5%), range 0%-10% d Number of animals with neoplasm per number of animals with tissue examined microscopically e Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality f Observed incidence at terminal kill

g Beneath the control incidence is the P value associated with the trend test. Beneath the exposed group incidences are the P values 

corresponding to pairwise comparisons between the controls and that exposed group. The Poly-3 test accounts for differential mortality 

in animals that do not reach terminal sacrifice. A lower incidence in an exposed group is indicated by N. h Historical incidence: 26/200 (13.0% ± 6.2%), range 6%-20%; all routes: 104/1,096 (9.5% ± 8.6%), range 0%-34%
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MICE

3-MONTH STUDY

All mice survived to the end of the study (Table 7). Final mean body weights and body weight gains of all

exposed groups were similar to those of the control group. Feed consumption by exposed groups of males and

females was generally similar to that by the controls throughout the study. Dietary concentrations of 80, 240,

2,000, 10,000, and 50,000 ppm resulted in average daily doses of approximately 17, 50, 450, 2,300, and 11,900 mg

chromium picolinate monohydrate/kg body weight to males and 14, 40, 370, 1,775, and 9,140 mg/kg to females.

There were no clinical findings related to exposure to chromium picolinate monohydrate; reddish-colored feces of

50,000 ppm animals was believed to be due to excretion of the test article and was not considered a sign of

toxicity.

TABLE 7

Survival, Body Weights, and Feed Consumption of Mice in the 3-Month Feed Study 

of Chromium Picolinate Monohydrate

Final Weight

Mean Body Weightb (g) Relative Feed

Concentration Survivala Initial Final Change to Controls Consumptionc

(ppm) (%) Week 1 Week 13

Male

0 10/10 19.6 ± 0.2 28.4 ± 0.5 8.8 ± 0.4 5.9 5.0

80 10/10 19.5 ± 0.3 28.7 ± 1.1 9.2 ± 0.9 101 5.5 5.1

240 10/10 19.5 ± 0.3 29.4 ± 0.8 9.9 ± 0.6 104 6.3 4.8

2,000 10/10 19.4 ± 0.3 27.5 ± 0.6 8.0 ± 0.4 97 4.9 5.5

10,000 10/10 19.4 ± 0.3 27.8 ± 0.5 8.4 ± 0.4 98 5.6 5.8

50,000 10/10 19.6 ± 0.3 28.0 ± 0.6 8.4 ± 0.5 99 5.8 5.8

Female

0 10/10 16.9 ± 0.2 24.3 ± 0.3 7.4 ± 0.4 3.0 3.9

80 10/10 16.7 ± 0.4 24.8 ± 0.4 8.2 ± 0.4 102 3.1 3.5

240 10/10 16.8 ± 0.2 24.7 ± 0.3 7.9 ± 0.2 102 2.8 3.8

2,000 10/10 16.7 ± 0.3 25.1 ± 0.4 8.4 ± 0.4 103 3.0 3.7

10,000 10/10 16.8 ± 0.3 23.5 ± 0.5 6.7 ± 0.5 97 2.7 3.6

50,000 10/10 16.9 ± 0.4 25.0 ± 0.5 8.2 ± 0.4 103 3.3 4.0

a Number of animals surviving at 14 weeks/number initially in group b Weights and weight changes are given as mean ± standard error. Differences from the control group are not significant by Dunnett’s test. c Feed consumption is expressed as grams per animal per day.
56

There were no hematological effects in mice administered chromium picolinate monohydrate (Table F2). 

There were no biologically significant differences in organ weights between exposed and control groups of mice

(Table G2). 

There were no significant changes in reproductive organ weights in male or female mice or in sperm parameters in

male mice (Table H3). Female mice exposed to 10,000 ppm had significantly longer estrous cycles than the

controls (Table H4); however, this was considered to be likely the result of sampling bias since there were only

three females with regular cycles, and therefore, not biologically significant.

No exposure-related lesions occurred in male or female mice.

Exposure Concentration Selection Rationale: Because chromium picolinate monohydrate produced no biologically

significant changes in any of the parameters examined in male or female mice, exposure concentrations of 2,000,

10,000, and 50,000 ppm were selected for the 2-year study in mice.

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TABLE 8

Survival of Mice in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Male

Animals initially in study 50 50 50 50

Moribund 3 3 6 1

Natural deaths 1 4 6 4

Animals surviving to study termination 46 43 38 45d

Percent probability of survival at end of studya 92 86 76 90

Mean survival (days)b 715 701 704 705

Survival analysisc P=0.783N P=0.532 P=0.062 P=0.983

Female

Animals initially in study 50 50 50 50

Missinge 010 0

Moribund 1 2 2 6

Natural deaths 4 3 4 5

Animals surviving to study termination 45 44 44 39

Percent probability of survival at end of study 90 90 88 78

Mean survival (days) 695 720 715 705

Survival analysis P=0.063 P=1.000 P=1.000 P=0.179

a Kaplan-Meier determinations b Mean of all deaths (uncensored, censored, and terminal sacrifice) c The result of the life table trend test (Tarone, 1975) is in the control column, and the results of the life table pairwise comparisons 

(Cox, 1972) with the controls are in the exposed group columns. A negative trend is indicated by N. d Includes one animal that died during the last week of the study e Censored from survival analyses

2-YEAR STUDY

Survival

Estimates of 2-year survival probabilities for male and female mice are shown in Table 8 and in the Kaplan-Meier

survival curves (Figure 3). Survival of exposed groups of males and females was similar to that of the control

groups.
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FIGURE 3

Kaplan-Meier Survival Curves for Male and Female Mice Exposed 

to Chromium Picolinate Monohydrate in Feed for 2 Years
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Body Weights, Feed and Compound Consumption, and Clinical Findings

Mean body weights of exposed groups of males were generally similar to those of the controls throughout the

study; mean body weights of exposed groups of females were generally decreased during the middle of the study

but recovered to control values by the end of the study (Tables 9 and 10; Figure 4). Feed consumption by exposed

groups of males and females was similar to that by the controls throughout the study (Tables J3 and J4). Dietary

concentrations of 2,000, 10,000, and 50,000 ppm resulted in average daily doses of approximately 250, 1,200, and

6,565 mg chromium picolinate monohydrate/kg body weight to males and 240, 1,200, and 6,100 mg/kg to females.

No clinical findings were attributed to chromium picolinate monohydrate exposure.
60 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE 9

Mean Body Weights and Survival of Male Mice in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

Days 0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

on Av. Wt. No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of

Study (g) Survivors (g) controls) Survivors (g) controls) Survivors (g) controls) Survivors

1 20.3 50 20.5 101 50 20.4 101 50 20.5 101 50

10 21.5 50 21.4 100 50 21.2 99 50 21.8 102 50

17 22.8 50 22.9 101 50 22.7 100 50 22.9 100 50

24 23.5 50 23.5 100 50 23.1 99 50 23.6 100 50

31 23.7 50 24.2 103 50 23.7 100 50 24.5 103 49

38 25.0 50 25.3 101 50 25.1 100 50 25.4 102 49

45 26.2 50 25.9 99 50 25.7 98 50 25.8 98 49

52 26.2 50 26.9 103 49 26.1 100 50 26.6 102 49

59 27.3 50 27.5 101 49 26.9 99 50 27.5 101 49

66 27.4 50 27.0 99 49 26.9 98 50 27.3 100 49

73 28.7 50 28.9 101 49 28.5 99 50 29.0 101 49

80 29.6 50 29.8 101 49 29.2 99 50 29.7 101 49

87 30.5 50 31.0 102 49 30.4 100 50 31.2 102 49

115 33.4 50 34.1 102 49 33.0 99 49 33.6 101 49

143 35.1 50 36.1 103 49 35.0 100 49 36.0 103 49

171 37.3 50 38.4 103 49 37.1 100 49 38.0 102 49

199 39.5 50 40.8 103 49 38.8 98 49 40.1 101 49

227 42.4 50 43.3 102 49 40.8 96 49 42.0 99 49

255 45.2 50 46.0 102 49 44.0 97 49 44.8 99 49

283 46.6 49 47.1 101 49 45.3 97 49 45.9 98 49

311 48.8 49 48.9 100 49 47.2 97 49 48.1 99 49

339 49.4 49 49.7 101 49 48.4 98 49 49.0 99 49

367 49.3 49 49.4 100 49 48.2 98 49 48.5 98 49

402 49.5 49 49.5 100 48 48.7 98 49 49.6 100 49

423 49.6 49 47.3 95 48 46.7 94 49 47.7 96 49

451 47.2 49 46.5 99 47 45.5 96 49 46.4 98 49

479 49.1 49 47.7 97 47 47.1 96 49 48.1 98 49

507 49.5 49 49.3 100 47 47.8 97 49 49.3 100 48

535 49.7 49 49.2 99 47 48.3 97 49 49.8 100 48

563 48.8 49 49.1 101 47 47.4 97 49 48.9 100 48

591 49.2 49 49.2 100 47 47.7 97 48 48.5 99 48

619 50.0 47 49.7 99 47 47.7 95 48 48.7 97 47

647 48.6 47 48.2 99 47 46.2 95 47 47.0 97 47

675 48.1 47 47.0 98 47 46.1 96 44 46.1 96 46

703 45.8 46 44.3 97 46 44.5 97 41 43.4 95 45

Mean for weeks

1-13 25.6 25.8 101 25.4 99 25.8 101

14-52 42.0 42.7 102 41.1 98 41.9 100

53-101 48.8 48.2 99 47.1 96 47.8 98
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TABLE 10

Mean Body Weights and Survival of Female Mice in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

Days 0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

on Av. Wt. No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of Av. Wt. Wt. (% of No. of

Study (g) Survivors (g) controls) Survivors (g) controls) Survivors (g) controls) Survivors

1 16.6 50 16.7 101 50 16.6 100 50 16.5 100 50

9 17.4 50 17.0 98 50 16.9 97 50 17.2 99 50

16 18.2 50 18.5 101 50 18.3 101 50 18.1 99 50

23 19.5 48 19.4 100 50 19.1 98 50 19.2 99 50

30 19.5 48 20.1 103 50 20.3 104 50 20.1 103 50

36 21.2 48 21.2 100 50 20.8 98 50 21.0 99 50

44 22.1 48 21.3 97 50 22.1 100 50 21.6 98 50

51 22.8 48 22.4 98 50 22.9 101 50 22.8 100 50

58 23.2 48 23.0 99 50 22.3 96 50 22.8 98 50

65 24.3 48 23.9 99 50 23.1 95 50 23.9 98 50

72 24.9 48 24.3 98 50 24.2 97 50 24.3 98 50

79 24.0 48 24.1 100 50 23.8 99 50 23.7 98 50

86 25.7 48 25.7 100 50 25.7 100 50 25.6 100 50

114 29.2 48 28.4 97 50 28.0 96 50 28.6 98 50

142 30.7 48 29.8 97 50 30.3 99 50 30.0 98 50

170 33.1 48 32.0 97 50 31.4 95 50 32.1 97 50

198 36.0 48 34.8 97 50 33.8 94 50 34.3 95 50

226 37.4 48 34.3 92 50 33.9 91 50 35.1 94 50

254 39.3 48 37.1 95 50 36.7 93 50 37.3 95 49

282 40.2 48 37.8 94 50 38.5 96 50 38.1 95 49

310 42.2 48 39.6 94 50 39.7 94 49 39.7 94 49

338 42.8 48 39.5 92 50 40.5 95 49 39.8 93 49

366 42.6 48 39.8 94 50 40.7 96 49 38.3 90 49

401 45.2 48 43.2 96 50 43.3 96 49 42.6 94 49

422 45.1 48 42.2 94 50 42.5 94 49 42.1 93 49

450 43.3 48 41.2 95 50 41.3 96 49 41.1 95 49

478 44.2 48 40.6 92 50 42.2 96 49 41.7 94 49

506 46.2 47 42.7 93 50 44.8 97 49 43.9 95 49

534 48.8 47 44.7 92 50 46.6 96 49 46.0 94 49

562 49.9 47 45.9 92 49 47.6 95 49 46.8 94 49

590 49.8 47 46.7 94 49 48.3 97 49 47.3 95 49

618 50.3 47 46.5 92 49 48.8 97 49 48.2 96 46

646 49.3 47 47.0 95 48 48.6 99 47 46.7 95 45

674 48.4 47 46.9 97 45 48.4 100 47 47.6 99 41

702 46.4 47 45.4 98 44 46.4 100 45 45.7 99 41

Mean for weeks

1-13 21.5 21.4 100 21.2 99 21.3 99

14-52 36.8 34.8 95 34.8 95 35.0 95

53-101 46.9 44.1 94 45.3 97 44.5 95
62 Chromium Picolinate Monohydrate, NTP TR 556 

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FIGURE 4

Growth Curves for Male and Female Mice Exposed to Chromium Picolinate Monohydrate 

in Feed for 2 Years
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Pathology and Statistical Analyses

This section describes the statistically significant or biologically noteworthy changes in the incidences of

neoplasms and/or nonneoplastic lesions of the liver and lung. Summaries of the incidences of neoplasms and

nonneoplastic lesions and statistical analyses of primary neoplasms that occurred with an incidence of at least 5%

in at least one animal group are presented in Appendix C for male mice and Appendix D for female mice.

Liver: In males, the incidences of hepatoblastoma occurred with a positive trend (p#0.05) (0 ppm, 0/50;

2,000 ppm, 1/50; 10,000 ppm, 0/50; 50,000 ppm, 3/50; Table C2). Hepatoblastoma is considered a variant of

hepatocellular carcinoma. The incidences of hepatocellular adenoma (21/50, 22/50, 21/50, 22/50), hepatocellular

carcinoma (15/50, 18/50, 20/50, 16/50), and hepatocellular adenoma, hepatocellular carcinoma, or hepatoblastoma

(combined) (32/50, 32/50, 33/50, 33/50) were similar between control and exposed males (Table C2). Because of

the lack of an increase in the incidences of hepatocellular adenoma, hepatocellular carcinoma, or hepatocellular

adenoma, hepatocellular carcinoma, or hepatoblastoma (combined) and the low incidences of hepatoblastoma in

exposed animals, the incidences of hepatoblastoma were not considered to be related to chromium picolinate

monohydrate exposure. There were no treatment-related effects in females.

Lung: In males, the incidences of alveolar/bronchiolar carcinoma occurred with a positive trend (P#0.05) (3/50,

2/50, 5/50, 8/50; Table C2). The incidences of alveolar/bronchiolar adenoma (13/50, 10/50, 7/50, 8/50) and

alveolar/bronchiolar adenoma or carcinoma (combined) (16/50, 12/50, 12/50, 12/50) were decreased in exposed

males (Table C2). Because of the decrease in alveolar/bronchiolar adenoma and alveolar/bronchiolar adenoma or

carcinoma (combined), the incidences of alveolar/bronchiolar carcinoma were not considered related to chromium

picolinate monohydrate exposure. There were no treatment-related effects in females.
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GENETIC TOXICOLOGY

Under the conditions of the present studies, chromium picolinate monohydrate showed no clear evidence of

genotoxicity in standard assays. Over a concentration range of 100 to 10,000 μg/plate, no evidence of

mutagenicity was observed in Salmonella typhimurium strains TA100 or TA98 or Escherichia coli strain WP2

uvrA/pKM101 when chromium picolinate monohydrate was tested with or without exogenous metabolic activation

(S9) (Table E1). In addition, no increase in the frequency of micronucleated normochromatic erythrocytes (NCEs)

was observed in male B6C3F1 mice administered chromium picolinate monohydrate (80 to 50,000 ppm) in feed

for 3 months, indicating no potential for chromium picolinate monohydrate to induce chromosomal alterations in

dividing cell populations in this test system (Table E2). In female mice, however, the small increase in

micronucleated NCEs noted in the highest exposure concentration group (50,000 ppm) was not significant at

P=0.0396, but it resulted in a significant trend test (P=0.005) and, therefore,the test with chromium picolinate

monohydrate was judged to be equivocal in female mice (Table E2). No significant alterations in the percentage of

polychromatic erythrocytes (PCEs) among total erythrocytes was observed in exposed mice, indicating that these

exposure concentrations of chromium picolinate monohydrate did not induce bone marrow toxicity (Table E2).

Additional genotoxicity testing was conducted with chromium picolinate (not the monohydrate form of the

compound), and results were negative. No induction of gene mutations was observed in two independent studies

conducted with chromium picolinate (up to 10,000 μg/plate) in several strains of S. typhimurium with and without

hamster or rat liver S9 (Table E3). No induction of micronucleated PCEs was observed in bone marrow of male

F344/N rats treated with chromium picolinate (156 to 2,500 mg/kg) by oral gavage three times at 24-hour intervals,

and no significant alterations in the percentage of PCEs among total erythrocytes was observed in dosed rats,

indicating that these doses of chromium picolinate did not induce bone marrow toxicity (Table E4).
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ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION

Absorption, distribution, metabolism, and excretion studies were conducted using [14C]-chromium picolinate

monohydrate in rats and mice; the [14C]-label allowed the fate of the chelating agent to be determined. Chromium

picolinate monohydrate is soluble in propylene glycol, but has limited solubility in water. The studies compared

the fate of chromium picolinate monohydrate dosed as a solution, in propylene glycol, or as an aqueous slurry

following doses of 15 to 20 mg/kg; urine was collected for 48 hours. Dosing of rats with the solution resulted in

urinary excretion of 1.25% ± 0.24% of the chromium and 56.3% ± 0.9% of the 14C, while dosing with the aqueous

slurry resulted in urinary excretion of 1.53% ± 0.51% of the chromium and 40.9% ± 3.6% of the 14C. Dosing of

mice with the solution resulted in urinary excretion of 3.9% ± 1.1% of the chromium and 42.0% ± 7.8% of the 14C,

while dosing with the aqueous slurry resulted in urinary excretion of 1.1% ± 0.7% of the chromium and

25.5% ± 3.0% of the 14C. Urinary excretion of radiolabel in both rats and mice was nearly all in the form of a

single metabolite that was identified as N-picolinoylglycine. In both rats and mice, 90% or more of the chromium

was excreted in feces, while [14C]-picolinate-derived radioactivity was excreted about equally in feces and urine,

implying that most of picolinate was absorbed without the chromium. 

CHROMIUM TISSUE DISTRIBUTION

As part of the 2-year study, total chromium concentrations in selected tissues and excreta were determined in

additional groups of animals (tissue distribution groups) composed of male rats and female mice (Appendix M).

These animals were treated the same as the animals in the core study groups with respect to exposure, housing, and

handling. Two days prior to scheduled tissue collection, up to 10 animals of each species in each exposure group

were placed in individual metabolism cages for the separate collection of urine and feces. Animals were provided

undosed feed during this time to allow unabsorbed chromium to be excreted. After 48 hours, the animals were

euthanized and erythrocytes, plasma, liver, kidney, glandular stomach, and forestomach were removed for

chromium analysis. The 48-hour washout period was based on an elimination half-life of 8 to 21 hours (Bragt and

van Dura, 1983; Vanoirbeek et al., 2003). The analytical procedure could not differentiate between the oxidation

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states of chromium, so the values reported are for total chromium irrespective of oxidation state. Increased

chromium concentrations in plasma after the washout period should represent the chromium entering plasma from

the tissues. Due to interference with the analytical method and only partial success of efforts to correct for this

interference, high concentrations of chloride in the stomach tissue may have contributed to the relatively high

control tissue concentrations seen in these samples. Insufficient mouse urine was collected for analysis at most

time points; incomplete separation of urine and feces and low urine volumes for mice are typical in metabolism

cages. The complete data sets for rats and mice are presented in Tables M1 and M2, respectively.

Chromium concentrations in liver, kidney, and plasma from the 2,000, 10,000, and 50,000 ppm rats and mice were

significantly increased over their respective controls on days 6, 13, and 182. Concentrations were also increased at

all exposure concentrations on days 6 and 182 in rat forestomach and at 10,000 and 50,000 ppm in rat erythrocytes

on day 182. In rat glandular stomach, chromium concentrations were not significantly increased over controls at

any exposure concentration or duration. In mice, there were significant increases in chromium concentrations on

day 182, in mouse forestomach at 2,000 ppm and in mouse erythrocytes at all exposure concentrations. Significant

increases were found in mouse glandular stomach on day 13 at 10,000 ppm and on day 182 at 2,000 ppm. In both

rats and mice, chromium tissue concentrations were generally not proportional to exposure concentration

(Figure 5). This pattern appears to reflect either absorption-limited uptake of trivalent chromium, or a

concentration-mediated clearance of chromium from the plasma, prior to it being available to tissues. In kidney

and liver, chromium concentrations increased with increasing exposure duration, at all exposure concentrations, in

both rats and mice; however, the increases were not proportional to the duration of exposure. Tissue chromium

concentrations did not increase with exposure duration at all doses in erythrocytes, forestomach, or glandular

stomach of rats and mice. These observations can be interpreted as the tissues approaching equilibrium between

Cr III uptake and Cr III loss via diffusion from the tissues and from cell turnover. In both rats and mice, increases

with increasing exposure concentration and exposure duration were most apparent in the kidney and liver. In rats,

mean concentrations in the kidney were higher than those in the liver at all exposure durations and increased

dramatically between day 13 and day 182. In mice, concentrations in the liver were higher than those in the

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FIGURE 5

Dependence of Tissue Chromium Concentrations on Exposure Concentration and Duration 

in Male Rats and Female Mice Exposed to Chromium Picolinate Monohydrate

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68 Chromium Picolinate Monohydrate, NTP TR 556 

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kidney at all exposure durations. Most of the ingested chromium was excreted in the feces of both species.

Chromium exposure per day appeared to increase throughout the study in both rats and mice, as reflected in the

higher amounts of chromium found in feces on day 182 compared to earlier exposure durations, except in mice on

day 6. At 50,000 ppm on day 182, the 48-hour feces collection contained up to 59.3 mg chromium in rats and

5.6 mg chromium in mice; the 48-hour urine collection from rats contained 30.0 mg chromium, but urine collection

from mice was unsuccessful as noted previously.
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Cr III, a natural dietary constituent, is the biologically active form of chromium and an essential trace element,

which participates in carbohydrate and lipid metabolism. Cr III is complexed with picolinate for use in dietary

supplements in an attempt to increase absorption; these supplements are marketed primarily for weight loss and

antidiabetic effects. Chromium picolinate was nominated for study because of widespread consumer exposure

from its use as a dietary supplement. The monohydrate form was selected for testing because it is the

commercially available form of chromium picolinate. Humans typically ingest 20 to 45 μg Cr III per day in the

diet, which corresponds to a daily dose of 0.29 to 0.64 μg Cr III/kg body weight (IOM, 2001). Recommended

doses of supplements typically contain 200 to 600 μg Cr III, which corresponds to a daily dose of 2.86 to 8.57 μg

Cr III/kg. Thus, human exposures though consumption of supplements are approximately an order of magnitude

higher than exposures through the diet. 

The exposure concentrations selected for the 3-month studies (80, 240, 2,000, 10,000 and 50,000 ppm) were

widely spaced to extend the exposure-response curve. The three highest exposure concentrations used in the

3-month studies (2,000, 10,000 and 50,000 ppm) were selected for testing in the 2-year studies because of the lack

of chemical-related toxic effects at any exposure concentration in the 3-month studies. The highest exposure

concentration of 50,000 ppm (5%) used in these studies is considered to be a limit dose in feed studies because

higher concentrations would alter the nutritional content of the diet. 

Average daily doses of Cr III, resulting from chromium picolinate monohydrate exposure in the present 3-month

studies, ranged from approximately 1 to 500 mg/kg in rats and 2 to 1,500 mg/kg in mice. Average daily doses in

the 2-year studies ranged from approximately 10 to 300 mg/kg in rats and 30 to 800 mg/kg in mice. These doses

DISCUSSION AND CONCLUSIONS
70

were up to five orders of magnitude higher than those consumed by humans ingesting typical doses of

supplements. 

Despite the use of a much higher exposure to chromium compared to that ingested by humans, there was very little

evidence of adverse effects in F344/N rats or B6C3F1 mice following exposure to chromium picolinate

monohydrate in the present 3-month and 2-year studies. There were no biologically significant changes in survival

or body weight, or clinical signs of toxicity associated with chromium picolinate monohydrate exposure. Feed

consumption was similar between control and exposed animals. There were no biologically significant alterations

in hematology, clinical chemistry, organ weights, sperm morphology, vaginal cytology, or nonneoplastic lesions

associated with exposure to chromium picolinate monohydrate. These results are in general agreement with

previous studies on compounds containing Cr III, including chromium picolinate (MacKenzie et al., 1958;

Schroeder et al., 1964, 1965; Laskin, et al., 1970; Ivankovic and Preussmann, 1975; Stoner, et al., 1976; Kurokowa

et al., 1985; Levy and Venitt, 1986; Anderson et al., 1997; Derelanko et al., 1999; ATSDR, 2000). Based on the

SMVCE results, the reproductive organ weights, and histopathology of the reproductive organs, there was no

evidence of toxicity to the reproductive system in these 3-month studies in rats and mice. Chromium picolinate is

marketed as a dietary supplement primarily for weight loss in humans; however, no effect on body weight was

observed in rats or mice in the present studies. 

In male rats in the 2-year study, there was an exposure-related significant increase in the incidence of preputial

gland adenoma at 10,000 ppm; the incidence exceeded the historical control ranges for feed studies and for all

routes of exposure. Proliferative lesions of the preputial and clitoral glands (the clitoral gland is the corresponding

accessory sex gland in females) constitute a morphological continuum, and separation of these into categories of

hyperplasia, adenoma, and carcinoma is based largely on cytological features and degree of altered growth pattern

(Copeland-Haines and Eustis, 1990). Lesions classified as hyperplasia are considered preneoplastic. Although the

increase in the incidence of preputial gland adenoma at 10,000 ppm appeared to be treatment-related, this increase

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was considered to be equivocal evidence of carcinogenic activity because of the lack of an exposure concentrationresponse, absence of increased incidences in neoplasms in the corresponding tissue in females, lack of progression

to carcinoma, and lack of preneoplastic lesions. There were no biologically significant increases in the incidences

of neoplasms in any other tissue in male rats, or in any tissue in female rats or male or female mice. 

Evidence of carcinogenic activity was based solely on exposure-related increases in preputial and/or clitoral gland

neoplasms in NTP carcinogenesis bioassays with five other chemicals: p-anisidine hydrochloride (NCI, 1978),

turmeric oleoresin (curcumin) (NTP, 1993), p-nitrobenzoic acid (NTP, 1994), p-nitrotoluene (NTP, 2002) and

nalidixic acid (NTP, 1989). In each of these studies, animals were exposed to the test chemical mixed in feed.

Increases in preputial or clitoral gland neoplasms were observed in rats and were not accompanied by hyperplasia,

as was observed in the present study. Although some structural similarities are present among the six test

chemicals that produce preputial or clitoral gland neoplasms in rats, none are shared in common by all six or with

chromium picolinate monohydrate in particular.

Absorption, distribution, metabolism, and excretion studies of [14C]-chromium picolinate monohydrate were

conducted to aid in the design and interpretation of the present studies (Appendix N); the 14C-label allowed the fate

of the picolinic acid to be determined. Urinary excretion of the label, which was primarily in the form of

N-picolinoylglycine, was much higher than that of chromium and while urinary and fecal excretion were similar

for picolinic acid, most of the chromium was excreted in the feces. These data indicate that the absorption of

picolinic acid, which accounts for 88% of the mass of the complex, is much higher than the absorption of

chromium and suggest that most of the picolinic acid is not bound to chromium during absorption. There is,

however, little evidence for accumulation of picolinate-derived material in tissues. The low absorption of Cr III is

in good agreement with previous studies (ATSDR, 2000). Chromium picolinate and picolinic acid have been

tested for developmental or genetic toxicity in the same test system (Stearns et al., 1995, 2002; Bailey et al., 2006);

the observed effects in these studies were more apparent with chromium picolinate than with picolinic acid. Based

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on the relatively high absorption of picolinic acid compared to chromium and the near complete lack of adverse

effects in the present studies, it appears that picolinic acid is essentially nontoxic.

As part of the present 2-year studies, total chromium content in excreta and selected tissues was determined in

additional groups of male rats and female mice on days 6, 13, and 182 of exposure (Appendix M). Animals were

removed from chromium picolinate monohydrate exposure and urine and feces were collected for 2 days before the

time of tissue collection. In the liver and kidney of rats and mice, chromium accumulated with exposure

concentration and time, indicating that Cr III is taken up by these tissues; this pattern was less apparent with other

tissues. The observed tissue concentrations of chromium were higher than would be expected based only on the

presence of erythrocytes in the blood of the tissues. In both rats and mice, chromium tissue concentrations were

generally not proportional to exposure concentration. As a result, tissue chromium concentrations in animals

exposed to 50,000 ppm chromium picolinate monohydrate were similar to those in animals exposed to lower doses.

These data suggest that the maximum achievable tissue chromium concentrations were reached in these studies,

and may explain the lack of an exposure concentration-response relationship for the increased incidence of

preputial gland adenomas in male rats. 

In contrast to the relative lack of exposure-related adverse effects following exposure to chromium picolinate

monohydrate (Cr III), the toxic and carcinogenic effects observed following oral administration of hexavalent

chromium (Cr VI) in rodents were significant. NTP studies of Cr VI (sodium dichromate dihydrate) concluded

that there was clear evidence of carcinogenic activity in male and female rats and mice exposed to Cr VI (sodium

dichromate dihydrate) in the drinking water for 2 years, based on increased incidences of squamous neoplasms of

the oral cavity in male and female rats, and increased incidences of epithelial neoplasms of the small intestine in

male and female mice (NTP, 2008). The observations of histiocytic infiltration in several tissues and effects on the

erythron in rats and mice, demonstrated that Cr VI is absorbed systemically following oral exposure. 

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The measurement of total chromium in male rats and female mice in the present studies on chromium picolinate

monohydrate and following exposure to sodium dichromate dihdyrate (NTP, 2008) allowed for tissue chromium

concentrations to be compared in animals exposed to Cr III or Cr VI. Collectively, the results of chromium tissue

concentration studies demonstrate that although both Cr III and Cr VI were widely distributed to tissues, uptake of

chromium was much greater following exposure to Cr VI, which is consistent with previous reports (Costa, 1997;

Costa and Klein, 2006). For example, on day 182, tissue chromium concentrations were five to 16 times lower in

animals administered chromium as Cr III than in animals administered chromium as Cr VI (NTP, 2008). Greater

uptake of Cr VI is thought to occur because Cr VI can enter cells via the sulfate channel (Wiegand et al., 1988),

while Cr III is not a substrate for transport (Gray and Sterling, 1950) and is thought to enter cells via diffusion. 

Cr III compounds give negative or conflicting results in standard assays for genetic toxicity. Chromium picolinate

is not mutagenic in the Ames assay (Whittaker et al., 2005; Table E1), but some laboratories have reported

increases in gene mutations or chromosomal aberrations in cultured mammalian cells treated with chromium

picolinate (Stearns et al., 1995, 2002; Whittaker et al., 2005). In contrast, other laboratories reported no increases

in these lesions in similar studies with chromium picolinate (Gudi et al., 2005; Slesinski et al., 2005). In the

present studies, there was equivocal evidence of the ability of chromium picolinate to induce micronucleated

erythrocytes in female B6C3F1 mice, but no evidence of an increase in male mice (Table E2). Results of recent

in vitro and in vivo DNA damage studies with chromium picolinate, which included analysis of micronucleated

erythrocytes in mice, were negative (Andersson et al., 2007). Other Cr III compounds have given negative results

in a number of in vitro and in vivo assays (Zeiger et al., 1992; Amrani et al., 1999; Witt et al., 2000; Whittaker

et al., 2005) and the genotoxicity test results for picolinic acid, although extremely limited, show little evidence of

activity (Stearns et al., 1995, 2002; Whittaker et al., 2005). 

In contrast to the limited evidence of genetic toxicity of Cr III, the genotoxicity of Cr VI is well-documented

(De Flora et al., 1990; IARC, 1990; O’Brien et al., 2003) and likely occurs as a result of the intracellular reduction

of Cr VI to Cr III (Zhitkovich et al., 2001, 2002; O’Brien et al., 2003; Quievryn et al., 2003, 2006; Zhitkovich,

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2005; Kirpnick-Sobol et al., 2006; Reynolds et al., 2007). The lack of observed genotoxicity of Cr III in most

standard assays may be due in large part to the relative inability of Cr III to enter cells, because interaction of

Cr III with DNA has been shown to result in the formation of DNA adducts, DNA-protein crosslinks, and DNAinterstrand crosslinks, which are thought to contribute to the induction of chromosomal alterations and mutational

changes. 

CONCLUSIONS

Under the conditions of these 2-year feed studies there was equivocal evidence of carcinogenic activity* of

chromium picolinate monohydrate in male F344/N rats based on an increase in the incidence of preputial gland

adenoma. There was no evidence of carcinogenic activity of chromium picolinate monohydrate in female F344/N

rats or in male or female B6C3F1 mice.

__________

* Explanation of Levels of Evidence of Carcinogenic Activity is on page 12.

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Agency for Toxic Substances and Disease Registry (ATSDR) (2000). Toxicological Profile for Chromium. U.S.

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role of the anion carrier. Analyst 120, 931-933.

Al-Hamood, M.H., Elbetieha, A., and Bataineh, H. (1998). Sexual maturation and fertility of male and female

mice exposed prenatally and postnatally to trivalent and hexavalent chromium compounds. Reprod. Fertil. Dev.

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Amrani, S., Rizki, M., Creus, A., and Marcos, R. (1999). Genotoxic activity of different chromium compounds in

larval cells of Drosophila melanogaster, as measured in the wing spot test. Environ. Mol. Mutagen. 34, 47-51.

Anderson, R.A. (1998). Chromium, glucose intolerance and diabetes. J. Am. Coll. Nutr. 17, 548-555. 

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A-1

Board Draft 

APPENDIX A

SUMMARY OF LESIONS IN MALE RATS

IN THE 2-YEAR FEED STUDY

OF CHROMIUM PICOLINATE MONOHYDRATE

TABLE A1 Summary of the Incidence of Neoplasms in Male Rats

in the 2-Year Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . A-2

TABLE A2 Statistical Analysis of Primary Neoplasms in Male Rats

in the 2-Year Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . A-6

TABLE A3 Historical Incidence of Preputial Gland Adenoma in Untreated Male F344/N Rats . . . . . . A-9

TABLE A4 Summary of the Incidence of Nonneoplastic Lesions in Male Rats

in the 2-Year Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . A-10
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A-2 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE A1

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of Chromium Picolinate Monohydratea

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Disposition Summary

Animals initially in study 50 50 50 50

Early deaths

Moribund 11 9 12 19

Natural deaths 2 5 3 3

Survivors

Died last week of study 2

Terminal sacrifice 37 36 33 28

Animals examined microscopically 50 50 50 50

Alimentary System

Intestine large, cecum (48) (48) (48) (50)

Intestine large, colon (48) (46) (46) (48)

Adenoma 1 (2%)

Intestine large, rectum (48) (48) (48) (50)

Intestine small, duodenum (49) (48) (48) (50)

Carcinoma 1 (2%)

Intestine small, ileum (49) (45) (47) (45)

Liver (50) (50) (50) (50)

Cholangioma 1 (2%)

Hepatocellular adenoma 1 (2%) 1 (2%)

Hepatocellular adenoma, multiple 1 (2%) 1 (2%) 1 (2%) 1 (2%)

Mesentery (15) (10) (8) (9)

Hemangiosarcoma 1 (7%)

Oral mucosa (1) (0) (1) (0)

Squamous cell carcinoma 1 (100%)

Pancreas (49) (50) (50) (50)

Acinus, adenoma 1 (2%) 1 (2%) 1 (2%)

Salivary glands (50) (50) (50) (50)

Schwannoma malignant 1 (2%)

Stomach, forestomach (49) (49) (50) (50)

Sarcoma 1 (2%)

Stomach, glandular (49) (49) (50) (50)

Squamous cell carcinoma, metastatic, lung 1 (2%)

Tongue (1) (2) (0) (1)

Squamous cell papilloma 1 (100%)

Cardiovascular System

Heart (50) (50) (50) (50)

Schwannoma malignant 1 (2%) 1 (2%) 1 (2%)

Squamous cell carcinoma, metastatic, lung 1 (2%)

Endocrine System

Adrenal cortex (50) (49) (50) (50)

Adenoma 1 (2%) 1 (2%)

Adrenal medulla (50) (49) (50) (50)

Pheochromocytoma benign 8 (16%) 9 (18%) 7 (14%) 7 (14%)

Pheochromocytoma complex 1 (2%)

Pheochromocytoma malignant 3 (6%) 3 (6%) 3 (6%)

Bilateral, pheochromocytoma benign 1 (2%)
Chromium Picolinate Monohydrate, NTP TR 556 A-3

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TABLE A1

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Endocrine System (continued)

Islets, pancreatic (50) (50) (50) (50)

Adenoma 1 (2%) 3 (6%) 1 (2%) 3 (6%)

Carcinoma 1 (2%) 1 (2%)

Parathyroid gland (45) (48) (49) (47)

Adenoma 1 (2%) 1 (2%)

Pituitary gland (50) (49) (50) (50)

Pars distalis, adenoma 10 (20%) 11 (22%) 15 (30%) 14 (28%)

Thyroid gland (50) (50) (50) (50)

C-cell, adenoma 5 (10%) 6 (12%) 1 (2%) 2 (4%)

C-cell, carcinoma 4 (8%) 1 (2%) 1 (2%) 2 (4%)

Follicular cell, adenoma 2 (4%) 1 (2%)

General Body System

Tissue NOS (2) (1) (2) (1)

Hemangiosarcoma 1 (50%)

Squamous cell carcinoma, metastatic, lung 1 (50%)

Genital System

Epididymis (49) (50) (50) (50)

Preputial gland (50) (50) (50) (50)

Adenoma 1 (2%) 1 (2%) 7 (14%) 4 (8%)

Prostate (50) (50) (50) (50)

Seminal vesicle (50) (50) (50) (50)

Testes (50) (50) (50) (50)

Schwannoma malignant 1 (2%)

Bilateral, interstitial cell, adenoma 37 (74%) 37 (74%) 26 (52%) 30 (60%)

Interstitial cell, adenoma 10 (20%) 8 (16%) 15 (30%) 9 (18%)

Hematopoietic System

Bone marrow (50) (50) (50) (50)

Squamous cell carcinoma, metastatic, lung 1 (2%)

Lymph node (15) (15) (13) (17)

Axillary, carcinoma, metastatic, thyroid gland 1 (6%)

Mediastinal, carcinoma, metastatic, thyroid gland 1 (6%)

Pancreatic, squamous cell carcinoma, metastatic, lung 1 (7%)

Lymph node, mandibular (2) (0) (1) (2)

Lymph node, mesenteric (50) (50) (50) (50)

Spleen (50) (50) (50) (50)

Hemangiosarcoma 1 (2%)

Leiomyosarcoma 1 (2%)

Thymus (48) (45) (48) (48)

Integumentary System

Mammary gland (49) (50) (50) (49)

Carcinoma 1 (2%)

Fibroadenoma 2 (4%) 1 (2%) 2 (4%)
Board Draft 

A-4 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION

TABLE A1

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Integumentary System (continued)

Skin (50) (50) (50) (50)

Basal cell adenoma 1 (2%) 2 (4%)

Basal cell carcinoma 1 (2%)

Hemangiopericytoma 1 (2%)

Keratoacanthoma 3 (6%) 2 (4%) 2 (4%) 1 (2%)

Osteosarcoma 1 (2%)

Squamous cell carcinoma, metastatic, lung 1 (2%)

Squamous cell papilloma 2 (4%) 1 (2%)

Pinna, neural crest tumor 1 (2%)

Subcutaneous tissue, fibroma 4 (8%) 3 (6%) 2 (4%) 4 (8%)

Subcutaneous tissue, lipoma 1 (2%) 1 (2%)

Subcutaneous tissue, sarcoma 1 (2%)

Musculoskeletal System

Bone (50) (50) (50) (50)

Skeletal muscle (2) (1) (0) (3)

Hemangioma 1 (33%)

Nervous System

Brain (50) (50) (50) (50)

Granular cell tumor benign 2 (4%) 1 (2%) 1 (2%)

Respiratory System

Lung (50) (50) (50) (50)

Alveolar/bronchiolar adenoma 1 (2%) 1 (2%) 1 (2%)

Alveolar/bronchiolar carcinoma 1 (2%) 1 (2%)

Alveolar/bronchiolar carcinoma, multiple 1 (2%)

Carcinoma, metastatic, thyroid gland 1 (2%) 1 (2%)

Hemangioma 1 (2%)

Sarcoma, metastatic, skin 1 (2%)

Schwannoma malignant, metastatic, salivary glands 1 (2%)

Squamous cell carcinoma 1 (2%)

Nose (50) (50) (50) (50)

Trachea (50) (50) (50) (50)

Carcinoma, metastatic, thyroid gland 1 (2%)

Special Senses System

Ear (1) (0) (0) (0)

Eye (50) (50) (50) (50)

Harderian gland (50) (50) (50) (50)

Zymbal’s gland (0) (1) (0) (0)

Carcinoma 1 (100%)

Urinary System

Kidney (50) (49) (50) (50)

Lipoma 1 (2%)

Squamous cell carcinoma, metastatic, lung 1 (2%)

Renal tubule, carcinoma 1 (2%)

Urethra (0) (1) (1) (0)

Urinary bladder (50) (50) (50) (50)
Chromium Picolinate Monohydrate, NTP TR 556 A-5

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION

TABLE A1

Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Systemic Lesions

Multiple organsb (50) (50) (50) (50)

Histiocytic sarcoma 2 (4%)

Leukemia mononuclear 19 (38%) 16 (32%) 22 (44%) 16 (32%)

Lymphoma malignant 1 (2%) 1 (2%)

Mesothelioma benign 2 (4%)

Mesothelioma malignant 1 (2%) 3 (6%) 1 (2%) 2 (4%)

Neoplasm Summary

Total animals with primary neoplasmsc 50 49 49 49

Total primary neoplasms 126 121 123 112

Total animals with benign neoplasms 49 48 46 46

Total benign neoplasms 91 90 85 87

Total animals with malignant neoplasms 27 25 27 23

Total malignant neoplasms 34 31 36 25

Total animals with metastatic neoplasms 2 2 3

Total metastatic neoplasms 9 2 4

Total animals with uncertain neoplasmsbenign or malignant 1

Total uncertain neoplasms 1

a Number of animals examined microscopically at the site and the number of animals with neoplasm b Number of animals with any tissue examined microscopically c Primary neoplasms: all neoplasms except metastatic neoplasms
Board Draft 

A-6 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION

TABLE A2

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Adrenal Medulla: Benign Pheochromocytoma

Overall ratea 8/50 (16%) 9/49 (18%) 8/50 (16%) 7/50 (14%)

Adjusted rateb 17.1% 20.8% 17.4% 16.3%

Terminal ratec 6/37 (16%) 8/36 (22%) 7/35 (20%) 6/28 (21%)

First incidence (days) 565 720 696 662

Poly-3 testd P=0.453N P=0.429 P=0.594 P=0.574N

Adrenal Medulla: Malignant Pheochromocytoma

Overall rate 3/50 (6%) 3/49 (6%) 3/50 (6%) 0/50 (0%)

Adjusted rate 6.4% 6.8% 6.5% 0.0%

Terminal rate 2/37 (5%) 1/36 (3%) 2/35 (6%) 0/28 (0%)

First incidence (days) 528 569 704 —e

Poly-3 test P=0.094N P=0.635 P=0.656 P=0.136N

Adrenal Medulla: Benign or Malignant Pheochromocytoma

Overall rate 11/50 (22%) 13/49 (27%) 10/50 (20%) 7/50 (14%)

Adjusted rate 23.2% 29.2% 21.6% 16.3%

Terminal rate 8/37 (22%) 9/36 (25%) 8/35 (23%) 6/28 (21%)

First incidence (days) 528 569 696 662

Poly-3 test P=0.159N P=0.336 P=0.528N P=0.292N

Lung: Alveolar/bronchiolar Adenoma or Carcinoma

Overall rate 0/50 (0%) 3/50 (6%) 2/50 (4%) 1/50 (2%)

Adjusted rate 0.0% 6.8% 4.4% 2.3%

Terminal rate 0/37 (0%) 3/36 (8%) 2/35 (6%) 1/28 (4%)

First incidence (days) — 729 (T) 729 (T) 729 (T)

Poly-3 test P=0.558N P=0.111 P=0.237 P=0.485

Pancreatic Islets: Adenoma

Overall rate 1/50 (2%) 3/50 (6%) 1/50 (2%) 3/50 (6%)

Adjusted rate 2.2% 6.8% 2.2% 7.0%

Terminal rate 1/37 (3%) 3/36 (8%) 1/35 (3%) 2/28 (7%)

First incidence (days) 729 (T) 729 (T) 729 (T) 628

Poly-3 test P=0.321 P=0.290 P=0.760 P=0.281

Pancreatic Islets: Adenoma or Carcinoma

Overall rate 2/50 (4%) 3/50 (6%) 2/50 (4%) 3/50 (6%)

Adjusted rate 4.3% 6.8% 4.4% 7.0%

Terminal rate 2/37 (5%) 3/36 (8%) 2/35 (6%) 2/28 (7%)

First incidence (days) 729 (T) 729 (T) 729 (T) 628

Poly-3 test P=0.463 P=0.481 P=0.693 P=0.469

Pituitary Gland (Pars Distalis): Adenoma

Overall rate 10/50 (20%) 11/49 (22%) 15/50 (30%) 14/50 (28%)

Adjusted rate 21.4% 24.7% 32.2% 32.1%

Terminal rate 8/37 (22%) 9/36 (25%) 13/35 (37%) 10/28 (36%)

First incidence (days) 565 564 618 622

Poly-3 test P=0.207 P=0.451 P=0.172 P=0.180

Preputial Gland: Adenoma

Overall rate 1/50 (2%) 1/50 (2%) 7/50 (14%) 4/50 (8%)

Adjusted rate 2.2% 2.3% 14.9% 9.3%

Terminal rate 0/37 (0%) 1/36 (3%) 5/35 (14%) 2/28 (7%)

First incidence (days) 653 729 (T) 528 582

Poly-3 test P=0.206 P=0.750 P=0.031 P=0.158
Chromium Picolinate Monohydrate, NTP TR 556 A-7

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION

TABLE A2

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Skin: Keratoacanthoma

Overall rate 3/50 (6%) 2/50 (4%) 2/50 (4%) 1/50 (2%)

Adjusted rate 6.5% 4.5% 4.4% 2.3%

Terminal rate 3/37 (8%) 2/36 (6%) 2/35 (6%) 0/28 (0%)

First incidence (days) 729 (T) 729 (T) 729 (T) 676

Poly-3 test P=0.318N P=0.519N P=0.501N P=0.332N

Skin: Keratoacanthoma or Squamous Cell Papilloma

Overall rate 3/50 (6%) 4/50 (8%) 3/50 (6%) 1/50 (2%)

Adjusted rate 6.5% 9.0% 6.5% 2.3%

Terminal rate 3/37 (8%) 3/36 (8%) 2/35 (6%) 0/28 (0%)

First incidence (days) 729 (T) 674 698 676

Poly-3 test P=0.192N P=0.480 P=0.662N P=0.332N

Skin: Squamous Cell Papilloma, Keratoacanthoma, Basal Cell Adenoma, or Basal Cell Carcinoma 

Overall rate 4/50 (8%) 5/50 (10%) 3/50 (6%) 3/50 (6%)

Adjusted rate 8.7% 11.3% 6.5% 7.0%

Terminal rate 3/37 (8%) 4/36 (11%) 2/35 (6%) 2/28 (7%)

First incidence (days) 698 674 698 676

Poly-3 test P=0.422N P=0.475 P=0.501N P=0.541N

Skin (Subcutaneous Tissue): Fibroma

Overall rate 4/50 (8%) 3/50 (6%) 2/50 (4%) 4/50 (8%)

Adjusted rate 8.7% 6.7% 4.3% 9.3%

Terminal rate 4/37 (11%) 2/36 (6%) 1/35 (3%) 1/28 (4%)

First incidence (days) 729 (T) 628 646 628

Poly-3 test P=0.445 P=0.518N P=0.335N P=0.608

Skin (Subcutaneous Tissue): Fibroma or Sarcoma

Overall rate 4/50 (8%) 4/50 (8%) 2/50 (4%) 4/50 (8%)

Adjusted rate 8.7% 9.0% 4.3% 9.3%

Terminal rate 4/37 (11%) 3/36 (8%) 1/35 (3%) 1/28 (4%)

First incidence (days) 729 (T) 628 646 628

Poly-3 test P=0.517 P=0.625 P=0.335N P=0.608

Testes: Adenoma

Overall rate 46/50 (92%) 45/50 (90%) 41/50 (82%) 39/50 (78%)

Adjusted rate 94.8% 94.8% 86.0% 84.0%

Terminal rate 36/37 (97%) 34/36 (94%) 32/35 (91%) 25/28 (89%)

First incidence (days) 486 569 618 509

Poly-3 test P=0.040N P=0.690N P=0.108N P=0.060N

Thyroid Gland (C-cell): Adenoma

Overall rate 5/50 (10%) 6/50 (12%) 1/50 (2%) 2/50 (4%)

Adjusted rate 10.7% 13.6% 2.2% 4.7%

Terminal rate 3/37 (8%) 6/36 (17%) 1/35 (3%) 2/28 (7%)

First incidence (days) 639 729 (T) 729 (T) 729 (T)

Poly-3 test P=0.175N P=0.462 P=0.105N P=0.254N

Thyroid Gland (C-cell): Carcinoma

Overall rate 4/50 (8%) 1/50 (2%) 1/50 (2%) 2/50 (4%)

Adjusted rate 8.7% 2.2% 2.2% 4.7%

Terminal rate 3/37 (8%) 0/36 (0%) 1/35 (3%) 2/28 (7%)

First incidence (days) 715 628 729 (T) 729 (T)

Poly-3 test P=0.578N P=0.189N P=0.180N P=0.374N
Board Draft 

A-8 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION

TABLE A2

Statistical Analysis of Primary Neoplasms in Male Rats in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Thyroid Gland (C-cell); Adenoma or Carcinoma

Overall rate 9/50 (18%) 7/50 (14%) 2/50 (4%) 4/50 (8%)

Adjusted rate 19.3% 15.7% 4.4% 9.4%

Terminal rate 6/37 (16%) 6/36 (17%) 2/35 (6%) 4/28 (14%)

First incidence (days) 639 628 729 (T) 729 (T)

Poly-3 test P=0.194N P=0.433N P=0.027N P=0.153N

All Organs: Mononuclear Cell Leukemia

Overall rate 19/50 (38%) 16/50 (32%) 22/50 (44%) 16/50 (32%)

Adjusted rate 39.5% 34.1% 46.1% 35.1%

Terminal rate 12/37 (32%) 8/36 (22%) 12/35 (34%) 7/28 (25%)

First incidence (days) 486 195 618 360

Poly-3 test P=0.403N P=0.369N P=0.328 P=0.409N

All Organs: Malignant Mesothelioma

Overall rate 1/50 (2%) 3/50 (6%) 1/50 (2%) 2/50 (4%)

Adjusted rate 2.2% 6.8% 2.2% 4.7%

Terminal rate 1/37 (3%) 2/36 (6%) 1/35 (3%) 0/28 (0%)

First incidence (days) 729 (T) 720 729 (T) 622

Poly-3 test P=0.566 P=0.291 P=0.760 P=0.476

All Organs: Benign or Malignant Mesothelioma

Overall rate 1/50 (2%) 3/50 (6%) 3/50 (6%) 2/50 (4%)

Adjusted rate 2.2% 6.8% 6.5% 4.7%

Terminal rate 1/37 (3%) 2/36 (6%) 2/35 (6%) 0/28 (0%)

First incidence (days) 729 (T) 720 701 622

Poly-3 test P=0.622 P=0.291 P=0.305 P=0.476

All Organs: Benign Neoplasms

Overall rate 49/50 (98%) 48/50 (96%) 46/50 (92%) 46/50 (92%)

Adjusted rate 99.2% 100.0% 94.9% 96.5%

Terminal rate 37/37 (100%) 36/36 (100%) 35/35 (100%) 28/28 (100%)

First incidence (days) 486 564 528 509

Poly-3 test P=0.293N P=0.954 P=0.207N P=0.382N

All Organs: Malignant Neoplasms

Overall rate 27/50 (54%) 25/50 (50%) 27/50 (54%) 23/50 (46%)

Adjusted rate 54.7% 52.0% 56.0% 48.9%

Terminal rate 18/37 (49%) 14/36 (39%) 16/35 (46%) 10/28 (36%)

First incidence (days) 486 195 575 360

Poly-3 test P=0.331N P=0.475N P=0.531 P=0.356N

All Organs: Benign or Malignant Neoplasms

Overall rate 50/50 (100%) 49/50 (98%) 49/50 (98%) 49/50 (98%)

Adjusted rate 100.0% 100.0% 99.5% 99.2%

Terminal rate 37/37 (100%) 36/36 (100%) 35/35 (100%) 28/28 (100%)

First incidence (days) 486 195 528 360

Poly-3 test P=0.810N P=1.000N P=0.996N P=0.947N

(T) Terminal sacrifice

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal medulla, lung, 

pancreatic islets, pituitary gland, preputial gland, testes, and thyroid gland; for other tissues, denominator is number of animals necropsied. b Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality c Observed incidence at terminal kill d Beneath the control incidence is the P value associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise 

comparisons between the controls and that exposed group. The Poly-3 test accounts for the differential mortality in animals that do not reach terminal 

sacrifice. A negative trend or a lower incidence in an exposure group is indicated by N. e Not applicable; no neoplasms in animal group
Chromium Picolinate Monohydrate, NTP TR 556 A-9

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TABLE A3

Historical Incidence of Preputial Gland Adenoma in Untreated Male F344/N Rats

Study Incidence of Controls

Historical Incidence in Feed Controls Given NTP-2000 Diet

Benzophenone 0/50

Chromium Picolinate Monohydrate 1/50

Cresols 5/50

2-Methylimidazole 0/50

4-Methylimidazole 2/50

Overall Historical Incidence: Feed Studies

Total (%) 8/250 (3.2%)

Mean ± standard deviation 3.2% ± 4.2%

Range 0%-10%

Overall Historical Incidence: All Routes

Total (%) 43/1,193 (3.6%)

Mean ± standard deviation 3.6% ± 3.5%

Range 0%-10%

a Data as of October 4, 2007
Board Draft 

A-10 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION

TABLE A4

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydratea

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Disposition Summary

Animals initially in study 50 50 50 50

Early deaths

Moribund 11 9 12 19

Natural deaths 2 5 3 3

Survivors

Died last week of study 2

Terminal sacrifice 37 36 33 28

Animals examined microscopically 50 50 50 50

Alimentary System

Intestine large, cecum (48) (48) (48) (50)

Intestine large, colon (48) (46) (46) (48)

Ulcer 1 (2%)

Intestine large, rectum (48) (48) (48) (50)

Hemorrhage 1 (2%)

Intestine small, duodenum (49) (48) (48) (50)

Intestine small, ileum (49) (45) (47) (45)

Liver (50) (50) (50) (50)

Angiectasis 2 (4%)

Basophilic focus 27 (54%) 32 (64%) 29 (58%) 27 (54%)

Clear cell focus 32 (64%) 32 (64%) 27 (54%) 26 (52%)

Degeneration, cystic 1 (2%) 1 (2%) 1 (2%)

Eosinophilic focus 1 (2%) 3 (6%) 5 (10%) 1 (2%)

Eosinophilic focus, multiple 1 (2%)

Hemorrhage 2 (4%) 3 (6%) 3 (6%) 1 (2%)

Hepatodiaphragmatic nodule 7 (14%) 8 (16%) 6 (12%) 3 (6%)

Inflammation, chronic 1 (2%) 1 (2%)

Mixed cell focus 4 (8%) 3 (6%)

Necrosis, focal 5 (10%) 1 (2%) 4 (8%) 1 (2%)

Vacuolization cytoplasmic 1 (2%)

Bile duct, hyperplasia 23 (46%) 11 (22%) 14 (28%) 9 (18%)

Centrilobular, bile stasis 1 (2%)

Centrilobular, necrosis 4 (8%) 3 (6%) 2 (4%) 3 (6%)

Centrilobular, hepatocyte, atrophy 1 (2%)

Hepatocyte, vacuolization cytoplasmic 8 (16%) 4 (8%) 9 (18%) 6 (12%)

Portal vein, thrombosis, focal 1 (2%)

Serosa, fibrosis 1 (2%)

Serosa, pigmentation 1 (2%)

Mesentery (15) (10) (8) (9)

Accessory spleen 3 (20%) 1 (13%)

Hemorrhage 1 (7%)

Fat, necrosis 10 (67%) 7 (70%) 5 (63%) 6 (67%)

Oral mucosa (1) (0) (1) (0)

Hyperplasia 1 (100%)

Pancreas (49) (50) (50) (50)

Atrophy 37 (76%) 40 (80%) 35 (70%) 27 (54%)

Acinus, hyperplasia, focal 6 (12%) 2 (4%) 2 (4%)

Salivary glands (50) (50) (50) (50)

Atrophy 5 (10%) 6 (12%) 7 (14%) 5 (10%)

Necrosis 1 (2%)

a Number of animals examined microscopically at the site and the number of animals with lesion
Chromium Picolinate Monohydrate, NTP TR 556 A-11

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TABLE A4

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Alimentary System (continued)

Stomach, forestomach (49) (49) (50) (50)

Edema 1 (2%) 2 (4%) 4 (8%) 1 (2%)

Erosion 1 (2%) 1 (2%) 1 (2%)

Hemorrhage 1 (2%)

Hyperkeratosis 1 (2%) 1 (2%)

Inflammation, acute 1 (2%)

Inflammation, chronic active 1 (2%) 1 (2%)

Ulcer 2 (4%) 3 (6%) 3 (6%)

Vacuolization cytoplasmic 1 (2%)

Epithelium, hyperplasia 3 (6%) 5 (10%) 4 (8%) 3 (6%)

Stomach, glandular (49) (49) (50) (50)

Edema 1 (2%)

Erosion 1 (2%) 1 (2%) 1 (2%) 3 (6%)

Hyperplasia, focal 2 (4%)

Ulcer 3 (6%) 1 (2%) 2 (4%) 1 (2%)

Tongue (1) (2) (0) (1)

Epithelium, hyperplasia 2 (100%) 1 (100%)

Cardiovascular System

Heart (50) (50) (50) (50)

Cardiomyopathy 48 (96%) 47 (94%) 47 (94%) 45 (90%)

Fibrosis 1 (2%)

Thrombosis 3 (6%) 2 (4%) 4 (8%) 2 (4%)

Artery, mineralization 1 (2%)

Endocrine System

Adrenal cortex (50) (49) (50) (50)

Accessory adrenal cortical nodule 5 (10%) 22 (45%) 23 (46%) 15 (30%)

Angiectasis 1 (2%)

Degeneration, fatty 12 (24%) 7 (14%) 8 (16%) 8 (16%)

Hematopoietic cell proliferation 1 (2%)

Hemorrhage 1 (2%) 2 (4%)

Hyperplasia, focal 18 (36%) 22 (45%) 19 (38%) 21 (42%)

Hypertrophy, focal 1 (2%)

Necrosis 1 (2%) 1 (2%)

Capsule, hyperplasia 1 (2%)

Adrenal medulla (50) (49) (50) (50)

Angiectasis 1 (2%)

Hyperplasia 10 (20%) 2 (4%) 2 (4%) 4 (8%)

Islets, pancreatic (50) (50) (50) (50)

Hyperplasia 6 (12%) 4 (8%) 3 (6%) 2 (4%)

Parathyroid gland (45) (48) (49) (47)

Pituitary gland (50) (49) (50) (50)

Cyst 1 (2%)

Pars distalis, angiectasis 1 (2%)

Pars distalis, cyst 2 (4%) 1 (2%) 2 (4%) 3 (6%)

Pars distalis, hemorrhage 1 (2%)

Pars distalis, hyperplasia, focal 15 (30%) 13 (27%) 17 (34%) 18 (36%)

Pars distalis, necrosis 1 (2%)

Pars intermedia, cyst 1 (2%)

Rathke’s cleft, hemorrhage 1 (2%)
Board Draft 

A-12 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE A4

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Endocrine System (continued)

Thyroid gland (50) (50) (50) (50)

Ultimobranchial cyst 1 (2%)

C-cell, hyperplasia 10 (20%) 5 (10%) 13 (26%) 6 (12%)

Follicular cell, hyperplasia 1 (2%)

General Body System

Tissue NOS (2) (1) (2) (1)

Hemorrhage 1 (50%)

Necrosis, focal 1 (50%)

Genital System

Epididymis (49) (50) (50) (50)

Granuloma sperm 1 (2%) 1 (2%)

Preputial gland (50) (50) (50) (50)

Atrophy 1 (2%)

Cyst 4 (8%) 3 (6%) 3 (6%) 2 (4%)

Hyperplasia 3 (6%) 1 (2%) 2 (4%)

Inflammation, chronic 26 (52%) 35 (70%) 30 (60%) 25 (50%)

Prostate (50) (50) (50) (50)

Cyst 1 (2%) 1 (2%) 1 (2%)

Inflammation, chronic 8 (16%) 12 (24%) 10 (20%) 11 (22%)

Epithelium, hyperplasia 4 (8%) 9 (18%) 11 (22%) 4 (8%)

Seminal vesicle (50) (50) (50) (50)

Inflammation, chronic 1 (2%)

Bilateral, cyst 1 (2%)

Epithelium, hyperplasia 1 (2%)

Testes (50) (50) (50) (50)

Mineralization 1 (2%)

Thrombosis, focal 1 (2%)

Germinal epithelium, atrophy 11 (22%) 9 (18%) 12 (24%) 11 (22%)

Interstitial cell, hyperplasia 3 (6%) 4 (8%) 5 (10%)

Hematopoietic System

Bone marrow (50) (50) (50) (50)

Depletion cellular 1 (2%)

Myelofibrosis 3 (6%) 1 (2%) 3 (6%)

Lymph node (15) (15) (13) (17)

Hyperplasia, lymphoid 1 (7%)

Mediastinal, ectasia 2 (13%) 1 (8%) 1 (6%)

Mediastinal, hemorrhage 2 (13%) 2 (13%) 1 (8%) 1 (6%)

Mediastinal, hyperplasia, lymphoid 1 (7%) 1 (8%) 1 (6%)

Mediastinal, pigmentation 1 (7%)

Pancreatic, ectasia 1 (7%) 1 (7%) 2 (15%) 4 (24%)

Pancreatic, hemorrhage 2 (13%) 1 (8%) 1 (6%)

Pancreatic, hyperplasia, lymphoid 1 (7%) 1 (6%)

Pancreatic, infiltration cellular, histiocyte 1 (6%)

Renal, ectasia 1 (7%)

Renal, hyperplasia, lymphoid 1 (7%)

Lymph node, mandibular (2) (0) (1) (2)
Chromium Picolinate Monohydrate, NTP TR 556 A-13

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TABLE A4

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Hematopoietic System (continued)

Lymph node, mesenteric (50) (50) (50) (50)

Atrophy 1 (2%)

Ectasia 2 (4%) 2 (4%) 5 (10%)

Hemorrhage 1 (2%) 1 (2%)

Hyperplasia, reticulum cell 1 (2%) 1 (2%) 1 (2%)

Spleen (50) (50) (50) (50)

Fibrosis 1 (2%) 1 (2%)

Hematopoietic cell proliferation 3 (6%) 1 (2%) 2 (4%) 4 (8%)

Hemorrhage 1 (2%)

Metaplasia, osseous 1 (2%)

Necrosis 2 (4%)

Pigmentation 26 (52%) 29 (58%) 28 (56%) 33 (66%)

Thrombosis 1 (2%)

Lymphoid follicle, hyperplasia 20 (40%) 9 (18%) 8 (16%) 14 (28%)

Thymus (48) (45) (48) (48)

Atrophy 37 (77%) 40 (89%) 36 (75%) 35 (73%)

Hemorrhage 1 (2%)

Integumentary System

Mammary gland (49) (50) (50) (49)

Cyst 3 (6%) 1 (2%) 3 (6%) 6 (12%)

Hyperplasia 8 (16%) 13 (26%) 20 (40%) 9 (18%)

Skin (50) (50) (50) (50)

Cyst epithelial inclusion 1 (2%) 4 (8%)

Hyperkeratosis 2 (4%) 3 (6%) 3 (6%) 1 (2%)

Inflammation, chronic 1 (2%)

Ulcer 1 (2%)

Epidermis, hyperplasia 3 (6%) 3 (6%) 1 (2%)

Pinna, ulcer 1 (2%)

Musculoskeletal System

Bone (50) (50) (50) (50)

Osteopetrosis 2 (4%) 1 (2%)

Skeletal muscle (2) (1) (0) (3)

Hemorrhage 1 (50%)

Necrosis 1 (50%)

Nervous System

Brain (50) (50) (50) (50)

Compression 5 (10%) 4 (8%) 4 (8%) 5 (10%)

Edema, focal 1 (2%)

Hemorrhage 6 (12%) 8 (16%) 5 (10%) 1 (2%)

Metaplasia, lipocyte, focal 1 (2%)

Necrosis 1 (2%) 2 (4%) 2 (4%)

Thrombosis 1 (2%) 1 (2%)
Board Draft 

A-14 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION

TABLE A4

Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Respiratory System

Lung (50) (50) (50) (50)

Fibrosis 1 (2%)

Hemorrhage 2 (4%) 3 (6%) 3 (6%)

Infiltration cellular, eosinophil 1 (2%)

Infiltration cellular, histiocyte 43 (86%) 47 (94%) 41 (82%) 43 (86%)

Inflammation, chronic 1 (2%) 3 (6%) 2 (4%)

Metaplasia, osseous 5 (10%) 4 (8%) 3 (6%) 2 (4%)

Metaplasia, squamous, focal 1 (2%)

Alveolar epithelium, hyperplasia 13 (26%) 11 (22%) 15 (30%) 19 (38%)

Alveolar epithelium, metaplasia, squamous 1 (2%)

Nose (50) (50) (50) (50)

Foreign body 12 (24%) 9 (18%) 6 (12%) 3 (6%)

Inflammation, chronic 11 (22%) 16 (32%) 11 (22%) 7 (14%)

Thrombosis 1 (2%)

Goblet cell, hyperplasia 1 (2%) 1 (2%)

Respiratory epithelium, hyperplasia 1 (2%)

Respiratory epithelium, metaplasia, squamous 2 (4%)

Trachea (50) (50) (50) (50)

Special Senses System

Ear (1) (0) (0) (0)

Hemorrhage 1 (100%)

External ear, inflammation, acute 1 (100%)

Eye (50) (50) (50) (50)

Cataract 1 (2%) 3 (6%) 3 (6%) 2 (4%)

Degeneration 1 (2%)

Pigmentation, hemosiderin, focal 1 (2%)

Retinal detachment, focal 1 (2%)

Retina, degeneration 1 (2%) 1 (2%)

Retina, necrosis, focal 1 (2%)

Harderian gland (50) (50) (50) (50)

Hyperplasia, focal 1 (2%)

Zymbal’s gland (0) (1) (0) (0)

Urinary System

Kidney (50) (49) (50) (50)

Cyst 1 (2%) 1 (2%)

Infarct 2 (4%) 1 (2%)

Inflammation, suppurative 1 (2%)

Nephropathy 50 (100%) 47 (96%) 47 (94%) 48 (96%)

Thrombosis 1 (2%)

Pelvis, dilatation 2 (4%)

Renal tubule, hyperplasia 3 (6%) 5 (10%) 3 (6%)

Renal tubule, necrosis 1 (2%)

Transitional epithelium, hyperplasia 1 (2%) 3 (6%) 1 (2%) 1 (2%)

Urethra (0) (1) (1) (0)

Angiectasis 1 (100%)

Bulbourethral gland, cyst 1 (100%)

Urinary bladder (50) (50) (50) (50)

Edema 1 (2%)

Hemorrhage 1 (2%) 1 (2%)

Inflammation, chronic 1 (2%) 1 (2%) 1 (2%)
NOT FOR DISTRIBUTION OR ATTRIBUTION

B-1

Board Draft 

APPENDIX B

SUMMARY OF LESIONS IN FEMALE RATS

IN THE 2-YEAR FEED STUDY

OF CHROMIUM PICOLINATE MONOHYDRATE

TABLE B1 Summary of the Incidence of Neoplasms in Female Rats

in the 2-Year Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . B-2

TABLE B2 Statistical Analysis of Primary Neoplasms in Female Rats

in the 2-Year Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . B-5

TABLE B3 Historical Incidence of Clitoral Gland Adenoma in Untreated Female F344/N Rats . . . . . . B-8

TABLE B4 Summary of the Incidence of Nonneoplastic Lesions in Female Rats

in the 2-Year Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . B-9
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B-2 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE B1

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of Chromium Picolinate Monohydratea

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Disposition Summary

Animals initially in study 50 50 50 50

Early deaths

Moribund 11 13 11 8

Natural deaths 3 2 3 2

Survivors

Died last week of study 1 1

Terminal sacrifice 35 35 36 39

Animals examined microscopically 50 50 50 50

Alimentary System

Esophagus (50) (50) (50) (50)

Intestine large, colon (47) (48) (50) (48)

Adenoma 1 (2%)

Schwannoma malignant 1 (2%)

Intestine small, ileum (46) (49) (50) (47)

Liver (50) (50) (50) (50)

Hepatocellular adenoma 2 (4%) 1 (2%) 1 (2%)

Hepatocellular adenoma, multiple 1 (2%)

Mesentery (11) (6) (7) (13)

Oral mucosa (0) (0) (1) (0)

Squamous cell carcinoma 1 (100%)

Pancreas (49) (50) (50) (50)

Salivary glands (50) (50) (50) (50)

Stomach, forestomach (49) (50) (50) (50)

Stomach, glandular (49) (50) (50) (50)

Tongue (1) (0) (3) (2)

Squamous cell papilloma 1 (33%)

Cardiovascular System

Heart (50) (50) (50) (50)

Schwannoma malignant 1 (2%) 1 (2%) 1 (2%)

Endocrine System

Adrenal cortex (50) (50) (50) (50)

Adenoma 2 (4%) 1 (2%)

Adrenal medulla (50) (49) (50) (50)

Pheochromocytoma benign 1 (2%) 3 (6%) 2 (4%) 1 (2%)

Pheochromocytoma malignant 2 (4%)

Islets, pancreatic (49) (50) (50) (50)

Adenoma 2 (4%) 1 (2%)

Parathyroid gland (48) (49) (48) (43)

Adenoma 1 (2%)

Pituitary gland (49) (50) (50) (50)

Carcinoma 1 (2%)

Pars distalis, adenoma 24 (49%) 22 (44%) 22 (44%) 25 (50%)

Pars distalis, carcinoma 1 (2%) 2 (4%)

Thyroid gland (49) (50) (50) (50)

C-cell, adenoma 3 (6%) 4 (8%) 6 (12%) 4 (8%)

C-cell, carcinoma 1 (2%) 1 (2%) 2 (4%)

Follicular cell, carcinoma 1 (2%)
Chromium Picolinate Monohydrate, NTP TR 556 B-3

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TABLE B1

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

General Body System

None

Genital System

Clitoral gland (50) (50) (50) (50)

Adenoma 10 (20%) 2 (4%) 8 (16%) 10 (20%)

Bilateral, adenoma 1 (2%)

Ovary (50) (50) (50) (50)

Granulosa cell tumor malignant 1 (2%)

Granulosa-theca tumor benign 1 (2%) 1 (2%)

Uterus (50) (50) (50) (50)

Adenoma 1 (2%)

Polyp stromal 10 (20%) 8 (16%) 13 (26%) 12 (24%)

Vagina (5) (6) (1) (3)

Polyp 1 (33%)

Hematopoietic System

Bone marrow (50) (50) (50) (50)

Lymph node (9) (10) (11) (7)

Mediastinal, carcinoma, metastatic, thyroid gland 1 (11%)

Lymph node, mandibular (1) (3) (1) (0)

Lymph node, mesenteric (50) (50) (50) (50)

Spleen (50) (50) (50) (49)

Lipoma 1 (2%)

Thymus (46) (48) (49) (48)

Integumentary System

Mammary gland (50) (49) (50) (50)

Adenoma 1 (2%) 1 (2%) 1 (2%) 1 (2%)

Carcinoma 1 (2%) 2 (4%)

Fibroadenoma 29 (58%) 29 (59%) 32 (64%) 31 (62%)

Fibroadenoma, multiple 1 (2%)

Fibrosarcoma 1 (2%)

Skin (50) (50) (50) (50)

Basal cell adenoma 1 (2%) 1 (2%)

Basal cell carcinoma 1 (2%)

Keratoacanthoma 2 (4%) 1 (2%)

Trichoepithelioma 1 (2%)

Subcutaneous tissue, fibroma 2 (4%) 1 (2%) 2 (4%)

Subcutaneous tissue, fibrosarcoma 2 (4%)

Musculoskeletal System

Bone (50) (50) (50) (50)

Nervous System

Brain (50) (50) (50) (50)

Granular cell tumor benign 1 (2%)

Oligodendroglioma malignant 1 (2%)

Spinal cord (2) (4) (3) (1)
Board Draft 

B-4 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE B1

Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Respiratory System

Lung (50) (50) (50) (50)

Alveolar/bronchiolar adenoma 1 (2%) 2 (4%) 1 (2%)

Alveolar/bronchiolar carcinoma 1 (2%)

Pheochromocytoma malignant, metastatic,

adrenal medulla 1 (2%)

Nose (50) (50) (50) (50)

Special Senses System

Eye (47) (49) (50) (50)

Harderian gland (50) (50) (50) (50)

Adenoma 1 (2%)

Zymbal’s gland (1) (0) (0) (0)

Carcinoma 1 (100%)

Urinary System

Kidney (47) (50) (50) (50)

Urinary bladder (50) (50) (49) (50)

Systemic Lesions

Multiple organsb (50) (50) (50) (50)

Histiocytic sarcoma 1 (2%)

Leukemia mononuclear 9 (18%) 10 (20%) 15 (30%) 6 (12%)

Lymphoma malignant 1 (2%)

Neoplasm Summary

Total animals with primary neoplasmsc 47 47 49 49

Total primary neoplasms 101 96 119 106

Total animals with benign neoplasms 45 44 45 48

Total benign neoplasms 86 81 94 92

Total animals with malignant neoplasms 15 13 21 13

Total malignant neoplasms 15 15 25 14

Total animals with metastatic neoplasms 3

Total metastatic neoplasms 9

a Number of animals examined microscopically at the site and the number of animals with neoplasm b Number of animals with any tissue examined microscopically c Primary neoplasms: all neoplasms except metastatic neoplasms
Chromium Picolinate Monohydrate, NTP TR 556 B-5

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TABLE B2

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Adrenal Medulla: Benign Pheochromocytoma

Overall ratea 1/50 (2%) 3/49 (6%) 2/50 (4%) 1/50 (2%)

Adjusted rateb 2.2% 6.7% 4.5% 2.1%

Terminal ratec 1/36 (3%) 3/35 (9%) 2/36 (6%) 1/40 (3%)

First incidence (days) 729 (T) 729 (T) 729 (T) 729 (T)

Poly-3 testd P=0.386N P=0.303 P=0.494 P=0.752N

Adrenal Medulla: Benign or Malignant Pheochromocytoma

Overall rate 3/50 (6%) 3/49 (6%) 2/50 (4%) 1/50 (2%)

Adjusted rate 6.6% 6.7% 4.5% 2.1%

Terminal rate 3/36 (8%) 3/35 (9%) 2/36 (6%) 1/40 (3%)

First incidence (days) 729 (T) 729 (T) 729 (T) 729 (T)

Poly-3 test P=0.216N P=0.659 P=0.507N P=0.292N

Clitoral Gland: Adenoma

Overall rate 10/50 (20%) 2/50 (4%) 8/50 (16%) 11/50 (22%)

Adjusted rate 21.9% 4.4% 17.7% 23.4%

Terminal rate 9/36 (25%) 1/35 (3%) 6/36 (17%) 11/40 (28%)

First incidence (days) 666 698 647 729 (T)

Poly-3 test P=0.109 P=0.013N P=0.405N P=0.534

Mammary Gland: Adenoma or Carcinoma

Overall rate 1/50 (2%) 1/50 (2%) 2/50 (4%) 3/50 (6%)

Adjusted rate 2.2% 2.2% 4.5% 6.4%

Terminal rate 1/36 (3%) 0/35 (0%) 1/36 (3%) 3/40 (8%)

First incidence (days) 729 (T) 666 708 729 (T)

Poly-3 test P=0.218 P=0.758N P=0.495 P=0.319

Mammary Gland: Fibroadenoma

Overall rate 29/50 (58%) 30/50 (60%) 32/50 (64%) 31/50 (62%)

Adjusted rate 62.0% 62.5% 68.6% 64.3%

Terminal rate 22/36 (61%) 21/35 (60%) 26/36 (72%) 26/40 (65%)

First incidence (days) 579 608 600 603

Poly-3 test P=0.507 P=0.564 P=0.321 P=0.490

Mammary Gland: Fibroadenoma or Adenoma

Overall rate 30/50 (60%) 30/50 (60%) 33/50 (66%) 31/50 (62%)

Adjusted rate 64.1% 62.5% 70.8% 64.3%

Terminal rate 23/36 (64%) 21/35 (60%) 27/36 (75%) 26/40 (65%)

First incidence (days) 579 608 600 603

Poly-3 test P=0.551N P=0.520N P=0.317 P=0.578

Mammary Gland: Fibroadenoma, Adenoma, or Carcinoma

Overall rate 30/50 (60%) 30/50 (60%) 34/50 (68%) 31/50 (62%)

Adjusted rate 64.1% 62.5% 72.8% 64.3%

Terminal rate 23/36 (64%) 21/35 (60%) 27/36 (75%) 26/40 (65%)

First incidence (days) 579 608 600 603

Poly-3 test P=0.534N P=0.520N P=0.243 P=0.578

Pituitary Gland (Pars Distalis): Adenoma

Overall rate 24/49 (49%) 22/50 (44%) 22/50 (44%) 25/50 (50%)

Adjusted rate 52.0% 46.6% 46.9% 51.1%

Terminal rate 18/36 (50%) 15/35 (43%) 16/36 (44%) 19/40 (48%)

First incidence (days) 586 646 432 586

Poly-3 test P=0.456 P=0.376N P=0.386N P=0.548N
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B-6 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE B2

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Pituitary Gland (Pars Distalis): Adenoma or Carcinoma

Overall rate 25/49 (51%) 23/50 (46%) 24/50 (48%) 25/50 (50%)

Adjusted rate 54.0% 48.3% 51.0% 51.1%

Terminal rate 18/36 (50%) 15/35 (43%) 17/36 (47%) 19/40 (48%)

First incidence (days) 586 608 432 586

Poly-3 test P=0.551N P=0.362N P=0.466N P=0.468N

Skin: Keratoacanthoma, Trichoepithelioma, Basal Cell Adenoma, or Basal Cell Carcinoma

Overall rate 0/50 (0%) 3/50 (6%) 2/50 (4%) 2/50 (4%)

Adjusted rate 0.0% 6.6% 4.5% 4.3%

Terminal rate 0/36 (0%) 3/35 (9%) 1/36 (3%) 2/40 (5%)

First incidence (days) —e 729 (T) 654 729 (T)

Poly-3 test P=0.538 P=0.120 P=0.235 P=0.246

Skin (Subcutaneous Tissue): Fibroma or Fibrosarcoma

Overall rate 0/50 (0%) 2/50 (4%) 1/50 (2%) 4/50 (8%)

Adjusted rate 0.0% 4.4% 2.2% 8.4%

Terminal rate 0/36 (0%) 1/35 (3%) 0/36 (0%) 3/40 (8%)

First incidence (days) — 647 708 646

Poly-3 test P=0.057 P=0.240 P=0.497 P=0.066

Thyroid Gland (C-cell): Adenoma

Overall rate 3/49 (6%) 4/50 (8%) 6/50 (12%) 4/50 (8%)

Adjusted rate 6.8% 8.8% 13.3% 8.5%

Terminal rate 3/35 (9%) 3/35 (9%) 5/36 (14%) 4/40 (10%)

First incidence (days) 729 (T) 707 600 729 (T)

Poly-3 test P=0.586N P=0.517 P=0.250 P=0.533

Thyroid Gland (C-cell): Adenoma or Carcinoma

Overall rate 4/49 (8%) 5/50 (10%) 6/50 (12%) 6/50 (12%)

Adjusted rate 9.0% 10.9% 13.3% 12.7%

Terminal rate 4/35 (11%) 4/35 (11%) 5/36 (14%) 6/40 (15%)

First incidence (days) 729 (T) 707 600 729 (T)

Poly-3 test P=0.428 P=0.519 P=0.380 P=0.408

Uterus: Stromal Polyp

Overall rate 10/50 (20%) 8/50 (16%) 13/50 (26%) 12/50 (24%)

Adjusted rate 22.0% 17.3% 28.9% 25.3%

Terminal rate 8/36 (22%) 7/35 (20%) 12/36 (33%) 10/40 (25%)

First incidence (days) 710 490 647 669

Poly-3 test P=0.349 P=0.379N P=0.303 P=0.451

All Organs: Mononuclear Cell Leukemia

Overall rate 9/50 (18%) 10/50 (20%) 15/50 (30%) 6/50 (12%)

Adjusted rate 19.2% 21.7% 32.5% 12.6%

Terminal rate 4/36 (11%) 7/35 (20%) 10/36 (28%) 3/40 (8%)

First incidence (days) 586 684 476 604

Poly-3 test P=0.107N P=0.482 P=0.109 P=0.275N

All Organs: Benign Neoplasms

Overall rate 45/50 (90%) 44/50 (88%) 45/50 (90%) 48/50 (96%)

Adjusted rate 93.8% 89.4% 93.7% 96.8%

Terminal rate 35/36 (97%) 31/35 (89%) 35/36 (97%) 39/40 (98%)

First incidence (days) 579 490 432 586

Poly-3 test P=0.170 P=0.336N P=0.677N P=0.396
Chromium Picolinate Monohydrate, NTP TR 556 B-7

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TABLE B2

Statistical Analysis of Primary Neoplasms in Female Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

All Organs: Malignant Neoplasms

Overall rate 15/50 (30%) 13/50 (26%) 21/50 (42%) 13/50 (26%)

Adjusted rate 31.4% 27.9% 44.1% 27.1%

Terminal rate 8/36 (22%) 8/35 (23%) 13/36 (36%) 9/40 (23%)

First incidence (days) 396 608 474 604

Poly-3 test P=0.313N P=0.441N P=0.140 P=0.407N

All Organs: Benign or Malignant Neoplasms

Overall rate 47/50 (94%) 47/50 (94%) 49/50 (98%) 49/50 (98%)

Adjusted rate 95.8% 95.2% 98.0% 98.0%

Terminal rate 35/36 (97%) 33/35 (94%) 35/36 (97%) 39/40 (98%)

First incidence (days) 396 490 432 586

Poly-3 test P=0.375 P=0.639N P=0.477 P=0.477

(T) Terminal sacrifice

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for adrenal medulla, 

clitoral gland, pituitary gland, and thyroid gland; for other tissues, denominator is number of animals necropsied. b Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality c Observed incidence at terminal kill d Beneath the control incidence is the P value associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise 

comparisons between the controls and that exposed group. The Poly-3 test accounts for the differential mortality in animals that do not reach terminal 

sacrifice. A negative trend or a lower incidence in an exposure group is indicated by N. e Not applicable; no neoplasms in animal group
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B-8 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE B3

Historical Incidence of Clitoral Gland Adenoma in Untreated Female F344/N Rats

Study Incidence of Controls

Historical Incidence in Feed Controls Given NTP-2000 Diet

Benzophenone 3/50

Chromium Picolinate Monohydrate 10/50

2-Methylimidazole 5/50

4-Methylimidazole 8/50

Overall Historical Incidence: Feed Studies

Total (%) 26/200 (13.0%)

Mean ± standard deviation 13.0% ± 6.2%

Range 6%-20%

Overall Historical Incidence: All Routes

Total (%) 104/1,096 (9.5%)

Mean ± standard deviation 9.5% ± 8.6%

Range 0%-34%

a Data as of October 4, 2007
Chromium Picolinate Monohydrate, NTP TR 556 B-9

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TABLE B4

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydratea

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Disposition Summary

Animals initially in study 50 50 50 50

Early deaths

Moribund 11 13 11 8

Natural deaths 3 2 3 2

Survivors

Died last week of study 1 1

Terminal sacrifice 35 35 36 39

Animals examined microscopically 50 50 50 50

Alimentary System

Esophagus (50) (50) (50) (50)

Infiltration cellular, lymphocyte 1 (2%)

Intestine large, colon (47) (48) (50) (48)

Intestine small, ileum (46) (49) (50) (47)

Necrosis, lymphoid 1 (2%)

Liver (50) (50) (50) (50)

Angiectasis 1 (2%)

Basophilic focus 41 (82%) 44 (88%) 46 (92%) 44 (88%)

Clear cell focus 27 (54%) 23 (46%) 22 (44%) 21 (42%)

Degeneration, cystic 1 (2%)

Eosinophilic focus 1 (2%) 1 (2%)

Eosinophilic focus, multiple 1 (2%)

Hemorrhage 1 (2%) 1 (2%) 1 (2%)

Hepatodiaphragmatic nodule 8 (16%) 8 (16%) 8 (16%) 8 (16%)

Hypertrophy, focal 1 (2%)

Infiltration cellular, lymphocyte 1 (2%)

Inflammation, chronic 3 (6%) 1 (2%) 4 (8%) 2 (4%)

Mixed cell focus 1 (2%) 4 (8%) 3 (6%)

Necrosis, focal 2 (4%) 2 (4%) 3 (6%)

Thrombosis 2 (4%) 1 (2%) 1 (2%)

Bile duct, hyperplasia 3 (6%) 6 (12%) 5 (10%) 2 (4%)

Centrilobular, necrosis 4 (8%) 1 (2%) 1 (2%) 1 (2%)

Hepatocyte, vacuolization cytoplasmic 6 (12%) 3 (6%) 6 (12%) 5 (10%)

Mesentery (11) (6) (7) (13)

Accessory spleen 1 (14%) 2 (15%)

Fibrosis 1 (14%)

Fat, necrosis 11 (100%) 6 (100%) 5 (71%) 12 (92%)

Oral mucosa (0) (0) (1) (0)

Pancreas (49) (50) (50) (50)

Atrophy 21 (43%) 27 (54%) 27 (54%) 26 (52%)

Metaplasia, hepatocyte 1 (2%)

Necrosis 1 (2%)

Acinus, hyperplasia, focal 1 (2%) 1 (2%) 1 (2%)

Salivary glands (50) (50) (50) (50)

Atrophy 11 (22%) 19 (38%) 19 (38%) 16 (32%)

Cyst 1 (2%)

Vacuolization cytoplasmic 1 (2%)

a Number of animals examined microscopically at the site and the number of animals with lesion
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B-10 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE B4

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Alimentary System (continued)

Stomach, forestomach (49) (50) (50) (50)

Edema 1 (2%) 1 (2%) 1 (2%)

Erosion 1 (2%)

Inflammation, acute 1 (2%)

Inflammation, chronic active 1 (2%)

Ulcer 1 (2%) 2 (4%) 1 (2%) 2 (4%)

Epithelium, hyperplasia 2 (4%) 1 (2%) 1 (2%) 1 (2%)

Stomach, glandular (49) (50) (50) (50)

Edema 3 (6%)

Erosion 2 (4%) 3 (6%) 1 (2%) 2 (4%)

Inflammation, acute 1 (2%)

Ulcer 1 (2%) 1 (2%)

Tongue (1) (0) (3) (2)

Hyperkeratosis 1 (33%)

Epithelium, hyperplasia 1 (100%) 2 (67%) 2 (100%)

Cardiovascular System

Heart (50) (50) (50) (50)

Cardiomyopathy 44 (88%) 44 (88%) 47 (94%) 46 (92%)

Inflammation, chronic 1 (2%)

Thrombosis 3 (6%)

Endocrine System

Adrenal cortex (50) (50) (50) (50)

Accessory adrenal cortical nodule 4 (8%) 10 (20%) 9 (18%) 9 (18%)

Angiectasis 1 (2%) 1 (2%) 2 (4%)

Degeneration, cystic 3 (6%)

Degeneration, fatty 6 (12%) 5 (10%) 2 (4%) 3 (6%)

Hematopoietic cell proliferation 1 (2%)

Hemorrhage 1 (2%) 3 (6%) 1 (2%)

Hyperplasia, focal 12 (24%) 20 (40%) 26 (52%) 24 (48%)

Hypertrophy 2 (4%) 5 (10%) 3 (6%) 7 (14%)

Hypertrophy, focal 1 (2%)

Necrosis 2 (4%) 1 (2%) 1 (2%)

Vacuolization cytoplasmic 1 (2%) 2 (4%) 1 (2%)

Adrenal medulla (50) (49) (50) (50)

Hyperplasia 4 (8%) 1 (2%)

Islets, pancreatic (49) (50) (50) (50)

Angiectasis, focal 1 (2%)

Hyperplasia 1 (2%) 2 (4%) 3 (6%) 1 (2%)

Parathyroid gland (48) (49) (48) (43)

Pituitary gland (49) (50) (50) (50)

Hemorrhage 1 (2%) 1 (2%)

Pars distalis, angiectasis 1 (2%) 1 (2%) 3 (6%) 1 (2%)

Pars distalis, cyst 11 (22%) 8 (16%) 9 (18%) 11 (22%)

Pars distalis, hyperplasia, focal 15 (31%) 7 (14%) 9 (18%) 12 (24%)

Pars distalis, infiltration cellular, mononuclear cell 1 (2%)

Pars distalis, pigmentation 1 (2%)

Pars intermedia, angiectasis 1 (2%) 1 (2%)

Pars intermedia, cyst 1 (2%) 2 (4%) 1 (2%)

Rathke’s cleft, hemorrhage 1 (2%) 1 (2%)
Chromium Picolinate Monohydrate, NTP TR 556 B-11

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TABLE B4

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Endocrine System (continued)

Thyroid gland (49) (50) (50) (50)

Ultimobranchial cyst 1 (2%) 5 (10%)

C-cell, hyperplasia 25 (51%) 29 (58%) 24 (48%) 21 (42%)

General Body System

None

Genital System

Clitoral gland (50) (50) (50) (50)

Cyst 2 (4%) 5 (10%) 2 (4%) 5 (10%)

Hyperplasia 8 (16%) 10 (20%) 11 (22%) 4 (8%)

Inflammation, chronic 4 (8%) 5 (10%) 3 (6%) 3 (6%)

Ovary (50) (50) (50) (50)

Cyst 8 (16%) 5 (10%) 6 (12%) 7 (14%)

Uterus (50) (50) (50) (50)

Angiectasis 1 (2%)

Hemorrhage 1 (2%)

Hyperplasia, cystic 17 (34%) 30 (60%) 24 (48%) 19 (38%)

Inflammation, suppurative 1 (2%) 1 (2%) 1 (2%)

Inflammation, chronic 1 (2%) 1 (2%)

Cervix, hemorrhage 1 (2%)

Vagina (5) (6) (1) (3)

Inflammation 1 (33%)

Hematopoietic System

Bone marrow (50) (50) (50) (50)

Infiltration cellular, histiocyte 1 (2%)

Myelofibrosis 2 (4%) 3 (6%) 1 (2%)

Lymph node (9) (10) (11) (7)

Deep cervical, ectasia 1 (11%)

Deep cervical, hemorrhage 1 (11%)

Deep cervical, hyperplasia, lymphoid 1 (11%)

Mediastinal, ectasia 3 (33%) 2 (20%) 1 (9%) 1 (14%)

Mediastinal, hemorrhage 2 (22%) 2 (20%) 3 (27%)

Mediastinal, hyperplasia 1 (14%)

Mediastinal, hyperplasia, lymphoid 1 (11%) 2 (18%) 1 (14%)

Mediastinal, pigmentation 2 (22%)

Mediastinal, pigmentation, hemosiderin 2 (20%)

Pancreatic, ectasia 1 (9%)

Pancreatic, hemorrhage 1 (10%) 1 (9%) 1 (14%)

Pancreatic, hyperplasia, lymphoid 1 (9%) 1 (14%)

Pancreatic, pigmentation 1 (14%)

Lymph node, mandibular (1) (3) (1) (0)

Ectasia 1 (100%) 1 (33%) 1 (100%)

Hyperplasia, lymphoid 1 (100%)

Lymph node, mesenteric (50) (50) (50) (50)

Ectasia 1 (2%)

Hemorrhage 1 (2%)

Hyperplasia, reticulum cell 1 (2%)

Pigmentation, hemosiderin 1 (2%) 1 (2%)
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B-12 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE B4

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Hematopoietic System (continued)

Spleen (50) (50) (50) (49)

Fibrosis 2 (4%) 1 (2%)

Hematopoietic cell proliferation 4 (8%) 5 (10%) 1 (2%) 2 (4%)

Hemorrhage 1 (2%)

Hyperplasia, lymphoid, focal 1 (2%) 1 (2%)

Infarct 1 (2%)

Pigmentation 40 (80%) 43 (86%) 44 (88%) 44 (90%)

Thrombosis 1 (2%)

Lymphoid follicle, hyperplasia 7 (14%) 5 (10%) 7 (14%) 6 (12%)

Thymus (46) (48) (49) (48)

Atrophy 37 (80%) 37 (77%) 45 (92%) 45 (94%)

Cyst 1 (2%)

Integumentary System

Mammary gland (50) (49) (50) (50)

Cyst 8 (16%) 2 (4%) 1 (2%)

Hyperplasia 37 (74%) 38 (78%) 33 (66%) 42 (84%)

Skin (50) (50) (50) (50)

Cyst epithelial inclusion 1 (2%) 3 (6%)

Fibrosis 1 (2%)

Hyperkeratosis 2 (4%) 2 (4%)

Inflammation, chronic 2 (4%) 2 (4%)

Ulcer 1 (2%) 1 (2%)

Epidermis, hyperplasia 1 (2%)

Sebaceous gland, hyperplasia 1 (2%)

Musculoskeletal System

Bone (50) (50) (50) (50)

Osteopetrosis 1 (2%)

Nervous System

Brain (50) (50) (50) (50)

Compression 9 (18%) 10 (20%) 11 (22%) 13 (26%)

Gliosis 1 (2%)

Hemorrhage 2 (4%) 1 (2%) 2 (4%) 3 (6%)

Spinal cord (2) (4) (3) (1)
Chromium Picolinate Monohydrate, NTP TR 556 B-13

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TABLE B4

Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Respiratory System

Lung (50) (50) (50) (50)

Congestion 1 (2%)

Foreign body 2 (4%)

Hemorrhage 3 (6%) 1 (2%) 1 (2%) 4 (8%)

Hyperplasia, lymphoid 1 (2%)

Infiltration cellular, histiocyte 49 (98%) 47 (94%) 47 (94%) 46 (92%)

Inflammation, chronic 2 (4%) 4 (8%) 3 (6%) 8 (16%)

Metaplasia, osseous 2 (4%) 2 (4%) 1 (2%)

Metaplasia, squamous 1 (2%)

Necrosis 1 (2%)

Thrombosis 1 (2%) 1 (2%)

Alveolar epithelium, hyperplasia 6 (12%) 5 (10%) 6 (12%) 4 (8%)

Alveolar epithelium, metaplasia, squamous 1 (2%)

Serosa, hyperplasia 1 (2%)

Nose (50) (50) (50) (50)

Foreign body 1 (2%) 1 (2%)

Inflammation, chronic 5 (10%) 4 (8%) 3 (6%) 3 (6%)

Nasolacrimal duct, cyst 1 (2%)

Turbinate, dysplasia 1 (2%)

Special Senses System

Eye (47) (49) (50) (50)

Cataract 2 (4%) 2 (4%) 1 (2%) 2 (4%)

Edema 1 (2%)

Hemorrhage 1 (2%)

Inflammation, chronic 1 (2%)

Retinal detachment 1 (2%)

Bilateral, retinal detachment 1 (2%)

Cornea, hyperplasia 1 (2%)

Retina, degeneration 1 (2%) 2 (4%) 1 (2%) 1 (2%)

Harderian gland (50) (50) (50) (50)

Hyperplasia, focal 1 (2%)

Zymbal’s gland (1) (0) (0) (0)

Urinary System

Kidney (47) (50) (50) (50)

Cyst 1 (2%)

Infarct, chronic 1 (2%)

Infiltration cellular, lymphocyte 1 (2%)

Nephropathy 46 (98%) 47 (94%) 48 (96%) 50 (100%)

Thrombosis 1 (2%) 1 (2%)

Papilla, inflammation, suppurative 1 (2%)

Renal tubule, hyperplasia 1 (2%) 1 (2%) 2 (4%)

Transitional epithelium, hyperplasia 1 (2%)

Transitional epithelium, mineralization, focal 1 (2%)

Urinary bladder (50) (50) (49) (50)

Edema 1 (2%) 1 (2%)

Hyperplasia, lymphoid 1 (2%)
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B-14 Chromium Picolinate Monohydrate, NTP TR 556 

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C-1

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APPENDIX C

SUMMARY OF LESIONS IN MALE MICE

IN THE 2-YEAR FEED STUDY

OF CHROMIUM PICOLINATE MONOHYDRATE

TABLE C1 Summary of the Incidence of Neoplasms in Male Mice

in the 2-Year Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . C-2

TABLE C2 Statistical Analysis of Primary Neoplasms in Male Mice

in the 2-Year Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . C-6

TABLE C3 Summary of the Incidence of Nonneoplastic Lesions in Male Mice

in the 2-Year Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . C-9
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TABLE C1

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Study of Chromium Picolinate Monohydratea

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Disposition Summary

Animals initially in study 50 50 50 50

Early deaths

Moribund 3 3 6 1

Natural deaths 1 4 6 4

Survivors

Died last week of study 1

Terminal sacrifice 46 43 38 44

Animals examined microscopically 50 50 50 50

Alimentary system

Esophagus (50) (49) (49) (50)

Gallbladder (46) (49) (45) (46)

Intestine large, cecum (50) (49) (48) (49)

Intestine large, rectum (50) (49) (50) (50)

Carcinoma 1 (2%)

Intestine small, duodenum (49) (47) (50) (49)

Adenoma 1 (2%) 2 (4%)

Carcinoma 1 (2%) 1 (2%)

Intestine small, ileum (50) (49) (46) (49)

Carcinoma 1 (2%)

Intestine small, jejunum (50) (48) (48) (49)

Carcinoma 1 (2%)

Liver (50) (50) (50) (50)

Hemangiosarcoma 2 (4%) 1 (2%) 1 (2%)

Hepatoblastoma 1 (2%) 3 (6%)

Hepatocellular adenoma 14 (28%) 10 (20%) 9 (18%) 14 (28%)

Hepatocellular adenoma, multiple 7 (14%) 12 (24%) 12 (24%) 8 (16%)

Hepatocellular carcinoma 10 (20%) 11 (22%) 14 (28%) 11 (22%)

Hepatocellular carcinoma, multiple 5 (10%) 7 (14%) 6 (12%) 5 (10%)

Mast cell tumor malignant, metastatic, bone marrow 1 (2%)

Mesentery (3) (7) (2) (4)

Hemangiosarcoma 1 (14%)

Hepatocellular carcinoma, metastatic, liver 1 (14%)

Pancreas (50) (49) (49) (50)

Hepatoblastoma, metastatic, liver 1 (2%)

Salivary glands (50) (50) (50) (50)

Stomach, forestomach (50) (50) (50) (50)

Mast cell tumor malignant 1 (2%)

Squamous cell papilloma 2 (4%) 1 (2%) 1 (2%)

Stomach, glandular (50) (49) (50) (50)

Hepatoblastoma, metastatic, liver 1 (2%)

Cardiovascular system

Heart (50) (50) (50) (50)

Hepatoblastoma, metastatic, liver 1 (2%)
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TABLE C1

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Endocrine system

Adrenal cortex (50) (50) (49) (50)

Capsule, adenoma 2 (4%) 1 (2%) 1 (2%)

Adrenal medulla (49) (50) (49) (49)

Islets, pancreatic (50) (49) (50) (50)

Adenoma 1 (2%)

Parathyroid gland (46) (48) (49) (46)

Pituitary gland (48) (49) (48) (49)

Thyroid gland (50) (50) (50) (50)

Follicular cell, adenoma 1 (2%) 1 (2%)

Follicular cell, carcinoma 1 (2%) 1 (2%)

General Body system

None

Genital system

Epididymis (50) (50) (50) (50)

Preputial gland (50) (50) (50) (50)

Prostate (50) (50) (50) (50)

Adenoma 1 (2%)

Leiomyosarcoma 1 (2%)

Seminal vesicle (50) (50) (50) (50)

Testes (50) (50) (50) (50)

Interstitial cell, adenoma 1 (2%) 1 (2%)

Hematopoietic system

Bone marrow (50) (50) (50) (50)

Hemangiosarcoma 1 (2%) 1 (2%)

Mast cell tumor malignant 1 (2%)

Lymph node (2) (2) (2) (4)

Mast cell tumor malignant, metastatic,

uncertain primary site 1 (50%)

Iliac, osteosarcoma, metastatic, bone 1 (50%)

Lymph node, mandibular (47) (46) (45) (44)

Mast cell tumor malignant, metastatic,

uncertain primary site 1 (2%)

Lymph node, mesenteric (46) (48) (48) (48)

Spleen (50) (49) (50) (50)

Hemangiosarcoma 2 (4%) 3 (6%)

Mast cell tumor malignant, metastatic,

bone marrow 1 (2%)

Thymus (41) (46) (47) (43)

Integumentary system

Skin (50) (50) (50) (50)

Subcutaneous tissue, fibrosarcoma 1 (2%)

Subcutaneous tissue, fibrous histiocytoma 1 (2%) 1 (2%)

Subcutaneous tissue, fibrous histiocytoma, multiple 1 (2%)

Subcutaneous tissue, hemangiosarcoma 1 (2%)

Subcutaneous tissue, schwannoma malignant 1 (2%)
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C-4 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE C1

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Musculoskeletal system

Bone (50) (50) (50) (50)

Osteosarcoma 1 (2%)

Nervous system

Brain (50) (50) (50) (50)

Peripheral nerve (2) (2) (1) (1)

Spinal cord (2) (2) (1) (1)

Meningioma malignant 1 (50%)

Respiratory system

Lung (50) (50) (50) (50)

Alveolar/bronchiolar adenoma 11 (22%) 9 (18%) 7 (14%) 5 (10%)

Alveolar/bronchiolar adenoma, multiple 2 (4%) 1 (2%) 3 (6%)

Alveolar/bronchiolar carcinoma 2 (4%) 2 (4%) 5 (10%) 7 (14%)

Alveolar/bronchiolar carcinoma, multiple 1 (2%) 1 (2%)

Carcinoma, metastatic, thyroid gland 1 (2%)

Hepatoblastoma, metastatic, liver 1 (2%)

Hepatocellular carcinoma, metastatic, liver 3 (6%) 3 (6%) 5 (10%) 3 (6%)

Osteosarcoma, metastatic, bone 1 (2%)

Nose (50) (50) (50) (50)

Mast cell tumor malignant 1 (2%)

Special senses system

Eye (50) (50) (50) (50)

Harderian gland (50) (50) (50) (50)

Adenoma 5 (10%) 6 (12%) 6 (12%) 7 (14%)

Carcinoma 1 (2%) 1 (2%) 1 (2%) 2 (4%)

Urinary system

Kidney (50) (50) (50) (50)

Hepatoblastoma, metastatic, liver 1 (2%)

Renal tubule, adenoma 2 (4%) 1 (2%)

Renal tubule, carcinoma 1 (2%) 1 (2%)

Urinary bladder (50) (49) (50) (50)

Systemic Lesions

Multiple organsb (50) (50) (50) (50)

Histiocytic sarcoma 1 (2%) 2 (4%)

Lymphoma malignant 2 (4%) 4 (8%) 3 (6%)
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TABLE C1

Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Neoplasm Summary

Total animals with primary neoplasmsc 40 46 41 41

Total primary neoplasms 76 74 82 78

Total animals with benign neoplasms 28 31 28 28

Total benign neoplasms 46 42 39 42

Total animals with malignant neoplasms 24 28 31 29

Total malignant neoplasms 30 32 43 36

Total animals with metastatic neoplasms 4564

Total metastatic neoplasms 5778

Total animals with malignant neoplasms

uncertain primary site 1

a Number of animals examined microscopically at the site and the number of animals with neoplasm b Number of animals with any tissue examined microscopically c Primary neoplasms: all neoplasms except metastatic neoplasms
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C-6 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE C2

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Harderian Gland: Adenoma

Overall ratea 5/50 (10%) 6/50 (12%) 6/50 (12%) 7/50 (14%)

Adjusted rateb 10.4% 12.5% 12.8% 14.6%

Terminal ratec 5/46 (11%) 5/43 (12%) 6/38 (16%) 6/45 (13%)

First incidence (days) 729 (T) 439 729 (T) 592

Poly-3 testd P=0.376 P=0.496 P=0.480 P=0.377

Harderian Gland: Adenoma or Carcinoma

Overall rate 5/50 (10%) 7/50 (14%) 7/50 (14%) 9/50 (18%)

Adjusted rate 10.4% 14.6% 15.0% 18.7%

Terminal rate 5/46 (11%) 6/43 (14%) 7/38 (18%) 7/45 (16%)

First incidence (days) 729 (T) 439 729 (T) 592

Poly-3 test P=0.216 P=0.376 P=0.360 P=0.195

Kidney (Renal Tubule): Adenoma or Carcinoma

Overall rate 3/50 (6%) 0/50 (0%) 1/50 (2%) 1/50 (2%)

Adjusted rate 6.2% 0.0% 2.1% 2.1%

Terminal rate 3/46 (7%) 0/43 (0%) 0/38 (0%) 1/45 (2%)

First incidence (days) 729 (T) —e 676 729 (T)

Poly-3 test P=0.518N P=0.123N P=0.314N P=0.310N

Liver: Hepatocellular Adenoma

Overall rate 21/50 (42%) 22/50 (44%) 21/50 (42%) 22/50 (44%)

Adjusted rate 43.7% 46.8% 44.7% 45.8%

Terminal rate 21/46 (46%) 22/43 (51%) 19/38 (50%) 20/45 (44%)

First incidence (days) 729 (T) 729 (T) 671 592

Poly-3 test P=0.520 P=0.462 P=0.542 P=0.498

Liver: Hepatocellular Carcinoma

Overall rate 15/50 (30%) 18/50 (36%) 20/50 (40%) 16/50 (32%)

Adjusted rate 30.9% 37.5% 40.9% 33.5%

Terminal rate 13/46 (28%) 15/43 (35%) 10/38 (26%) 14/45 (31%)

First incidence (days) 618 439 584 661

Poly-3 test P=0.482N P=0.317 P=0.207 P=0.478

Liver: Hepatocellular Adenoma or Carcinoma

Overall rate 32/50 (64%) 32/50 (64%) 33/50 (66%) 31/50 (62%)

Adjusted rate 65.8% 66.8% 67.4% 64.2%

Terminal rate 30/46 (65%) 29/43 (67%) 23/38 (61%) 28/45 (62%)

First incidence (days) 618 439 584 592

Poly-3 test P=0.448N P=0.547 P=0.519 P=0.518N

Liver: Hepatoblastoma

Overall rate 0/50 (0%) 1/50 (2%) 0/50 (0%) 3/50 (6%)

Adjusted rate 0.0% 2.1% 0.0% 6.3%

Terminal rate 0/46 (0%) 1/43 (2%) 0/38 (0%) 3/45 (7%)

First incidence (days) — 729 (T) — 729 (T)

Poly-3 test P=0.039 P=0.496 —f P=0.117

Liver: Hepatocellular Carcinoma or Hepatoblastoma

Overall rate 15/50 (30%) 19/50 (38%) 20/50 (40%) 19/50 (38%)

Adjusted rate 30.9% 39.6% 40.9% 39.7%

Terminal rate 13/46 (28%) 16/43 (37%) 10/38 (26%) 17/45 (38%)

First incidence (days) 618 439 584 661

Poly-3 test P=0.373 P=0.246 P=0.207 P=0.243
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TABLE C2

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Liver: Hepatocellular Adenoma, Hepatocellular Carcinoma, or Hepatoblastoma

Overall rate 32/50 (64%) 32/50 (64%) 33/50 (66%) 33/50 (66%)

Adjusted rate 65.8% 66.8% 67.4% 68.3%

Terminal rate 30/46 (65%) 29/43 (67%) 23/38 (61%) 30/45 (67%)

First incidence (days) 618 439 584 592

Poly-3 test P=0.465 P=0.547 P=0.519 P=0.482

Lung: Alveolar/bronchiolar Adenoma

Overall rate 13/50 (26%) 10/50 (20%) 7/50 (14%) 8/50 (16%)

Adjusted rate 27.0% 21.3% 15.0% 16.9%

Terminal rate 13/46 (28%) 10/43 (23%) 7/38 (18%) 8/45 (18%)

First incidence (days) 729 (T) 729 (T) 729 (T) 729 (T)

Poly-3 test P=0.238N P=0.339N P=0.117N P=0.170N

Lung: Alveolar/bronchiolar Carcinoma

Overall rate 3/50 (6%) 2/50 (4%) 5/50 (10%) 8/50 (16%)

Adjusted rate 6.2% 4.3% 10.7% 16.9%

Terminal rate 2/46 (4%) 2/43 (5%) 5/38 (13%) 8/45 (18%)

First incidence (days) 695 729 (T) 729 (T) 729 (T)

Poly-3 test P=0.028 P=0.511N P=0.340 P=0.094

Lung: Alveolar/bronchiolar Adenoma or Carcinoma

Overall rate 16/50 (32%) 12/50 (24%) 12/50 (24%) 12/50 (24%)

Adjusted rate 33.2% 25.5% 25.7% 25.3%

Terminal rate 15/46 (33%) 12/43 (28%) 12/38 (32%) 12/45 (27%)

First incidence (days) 695 729 (T) 729 (T) 729 (T)

Poly-3 test P=0.368N P=0.276N P=0.282N P=0.267N

Small Intestine (Duodenum, Ileum, or Jejunum): Adenoma or Carcinoma

Overall rate 3/50 (6%) 0/50 (0%) 1/50 (2%) 3/50 (6%)

Adjusted rate 6.2% 0.0% 2.1% 6.3%

Terminal rate 3/46 (7%) 0/43 (0%) 0/38 (0%) 3/45 (7%)

First incidence (days) 729 (T) — 635 729 (T)

Poly-3 test P=0.277 P=0.123N P=0.313N P=0.656

Spleen: Hemangiosarcoma

Overall rate 0/50 (0%) 2/49 (4%) 3/50 (6%) 0/50 (0%)

Adjusted rate 0.0% 4.3% 6.4% 0.0%

Terminal rate 0/46 (0%) 2/43 (5%) 2/38 (5%) 0/45 (0%)

First incidence (days) — 729 (T) 704 —

Poly-3 test P=0.300N P=0.228 P=0.114 —

All Organs: Hemangiosarcoma

Overall rate 2/50 (4%) 4/50 (8%) 4/50 (8%) 1/50 (2%)

Adjusted rate 4.2% 8.5% 8.5% 2.1%

Terminal rate 2/46 (4%) 4/43 (9%) 3/38 (8%) 0/45 (0%)

First incidence (days) 729 (T) 729 (T) 704 592

Poly-3 test P=0.209N P=0.328 P=0.325 P=0.501N

All Organs: Benign Neoplasms

Overall rate 28/50 (56%) 31/50 (62%) 28/50 (56%) 28/50 (56%)

Adjusted rate 57.7% 64.8% 59.6% 58.3%

Terminal rate 27/46 (59%) 30/43 (70%) 26/38 (68%) 26/45 (58%)

First incidence (days) 600 439 671 592

Poly-3 test P=0.440N P=0.306 P=0.508 P=0.558
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TABLE C2

Statistical Analysis of Primary Neoplasms in Male Mice in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

All Organs: Malignant Lymphoma

Overall rate 2/50 (4%) 0/50 (0%) 4/50 (8%) 3/50 (6%)

Adjusted rate 4.2% 0.0% 8.6% 6.2%

Terminal rate 2/46 (4%) 0/43 (0%) 3/38 (8%) 2/45 (4%)

First incidence (days) 729 (T) — 724 486

Poly-3 test P=0.312 P=0.242N P=0.325 P=0.501

All Organs: Malignant Neoplasms

Overall rate 24/50 (48%) 28/50 (56%) 31/50 (62%) 29/50 (58%)

Adjusted rate 48.0% 57.1% 63.3% 59.2%

Terminal rate 20/46 (44%) 22/43 (51%) 20/38 (53%) 25/45 (56%)

First incidence (days) 283 376 584 486

Poly-3 test P=0.310 P=0.240 P=0.092 P=0.181

All Organs: Benign or Malignant Neoplasms

Overall rate 40/50 (80%) 46/50 (92%) 41/50 (82%) 41/50 (82%)

Adjusted rate 80.0% 93.9% 83.7% 83.7%

Terminal rate 36/46 (78%) 40/43 (93%) 30/38 (79%) 37/45 (82%)

First incidence (days) 283 376 584 486

Poly-3 test P=0.430N P=0.039 P=0.417 P=0.416

(T) Terminal sacrifice

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for kidney, liver, lung, and 

spleen; for other tissues, denominator is number of animals necropsied. b Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality c Observed incidence at terminal kill d Beneath the control incidence is the P value associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise 

comparisons between the controls and that exposed group. The Poly-3 test accounts for the differential mortality in animals that do not reach terminal 

sacrifice. A negative trend or a lower incidence in an exposure group is indicated by N. e Not applicable; no neoplasms in animal group f Value of statistic cannot be computed
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TABLE C3

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Study 

of Chromium Picolinate Monohydratea

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Disposition Summary

Animals initially in study 50 50 50 50

Early deaths

Moribund 3 3 6 1

Natural deaths 1 4 6 4

Survivors

Died last week of study 1

Terminal sacrifice 46 43 38 44

Animals examined microscopically 50 50 50 50

Alimentary System

Esophagus (50) (49) (49) (50)

Infiltration cellular, polymorphonuclear 1 (2%)

Gallbladder (46) (49) (45) (46)

Intestine large, cecum (50) (49) (48) (49)

Edema 1 (2%)

Hyperplasia, lymphoid 1 (2%)

Intestine large, rectum (50) (49) (50) (50)

Inflammation, chronic active 1 (2%)

Intestine small, duodenum (49) (47) (50) (49)

Intestine small, Ileum (50) (49) (46) (49)

Hyperplasia, lymphoid 1 (2%)

Intestine small, jejunum (50) (48) (48) (49)

Hyperplasia, lymphoid 1 (2%) 1 (2%)

Inflammation, chronic 2 (4%) 1 (2%)

Liver (50) (50) (50) (50)

Angiectasis 1 (2%)

Basophilic focus 9 (18%) 10 (20%) 10 (20%) 8 (16%)

Clear cell focus 12 (24%) 7 (14%) 6 (12%) 4 (8%)

Cyst 1 (2%)

Eosinophilic focus 7 (14%) 4 (8%) 5 (10%) 5 (10%)

Hematopoietic cell proliferation 1 (2%) 1 (2%) 1 (2%) 1 (2%)

Infiltration cellular, mixed cell 1 (2%) 4 (8%) 2 (4%) 5 (10%)

Inflammation, chronic active 2 (4%) 1 (2%)

Mixed cell focus 14 (28%) 8 (16%) 6 (12%) 8 (16%)

Necrosis, focal 2 (4%) 5 (10%) 8 (16%) 4 (8%)

Tension lipidosis 1 (2%)

Bile duct, hyperplasia 1 (2%)

Centrilobular, necrosis 1 (2%)

Hepatocyte, fatty change, focal 1 (2%)

Hepatocyte, vacuolization cytoplasmic 3 (6%) 1 (2%) 4 (8%) 2 (4%)

Kupffer cell, hyperplasia 4 (8%) 1 (2%) 2 (4%)

Kupffer cell, pigmentation 1 (2%)

Mesentery (3) (7) (2) (4)

Hemorrhage 1 (33%) 4 (57%) 1 (25%)

Inflammation, granulomatous 1 (25%)

Fat, necrosis 2 (67%) 2 (29%) 2 (100%) 3 (75%)

a Number of animals examined microscopically at the site and the number of animals with lesion
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C-10 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION

TABLE C3

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Alimentary System (continued)

Pancreas (50) (49) (49) (50)

Atrophy 1 (2%) 1 (2%)

Basophilic focus 1 (2%)

Cyst 1 (2%)

Acinus, cytoplasmic alteration 1 (2%) 2 (4%)

Salivary glands (50) (50) (50) (50)

Hyperplasia, lymphoid 1 (2%) 1 (2%) 1 (2%) 1 (2%)

Stomach, forestomach (50) (50) (50) (50)

Diverticulum 1 (2%)

Erosion 1 (2%)

Inflammation, chronic 1 (2%)

Ulcer 1 (2%) 1 (2%)

Epithelium, hyperplasia 2 (4%) 2 (4%) 2 (4%) 1 (2%)

Stomach, glandular (50) (49) (50) (50)

Erosion 1 (2%) 2 (4%) 3 (6%)

Ulcer 1 (2%)

Epithelium, hyperplasia 1 (2%)

Cardiovascular System

Heart (50) (50) (50) (50)

Cardiomyopathy 1 (2%)

Inflammation, chronic 1 (2%) 1 (2%)

Mineralization 1 (2%) 1 (2%) 1 (2%)

Thrombosis 1 (2%)

Endocrine System

Adrenal cortex (50) (50) (49) (50)

Accessory adrenal cortical nodule 2 (4%) 8 (16%) 1 (2%) 2 (4%)

Hyperplasia, focal 2 (4%) 1 (2%) 1 (2%)

Hypertrophy, focal 11 (22%) 7 (14%) 8 (16%) 6 (12%)

Capsule, hyperplasia 7 (14%) 4 (8%) 5 (10%) 9 (18%)

Zona reticularis, cytoplasmic alteration 1 (2%)

Adrenal medulla (49) (50) (49) (49)

Hyperplasia 1 (2%) 1 (2%) 2 (4%) 1 (2%)

Islets, pancreatic (50) (49) (50) (50)

Hyperplasia 3 (6%) 1 (2%) 3 (6%) 2 (4%)

Parathyroid gland (46) (48) (49) (46)

Cyst 4 (9%) 2 (4%) 1 (2%)

Pituitary gland (48) (49) (48) (49)

Pars distalis, cyst 1 (2%) 2 (4%) 4 (8%)

Pars distalis, hyperplasia, focal 1 (2%) 1 (2%)

Thyroid gland (50) (50) (50) (50)

Follicle, cyst 1 (2%)

Follicle, degeneration, focal 9 (18%) 6 (12%) 11 (22%) 8 (16%)

Follicular cell, hyperplasia 3 (6%) 1 (2%) 1 (2%)

General Body System

None
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TABLE C3

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Genital System

Epididymis (50) (50) (50) (50)

Granuloma sperm 1 (2%)

Inflammation, chronic 1 (2%)

Preputial gland (50) (50) (50) (50)

Cyst 17 (34%) 12 (24%) 17 (34%) 18 (36%)

Inflammation, chronic 26 (52%) 25 (50%) 22 (44%) 16 (32%)

Prostate (50) (50) (50) (50)

Inflammation, chronic 1 (2%)

Epithelium, hyperplasia 1 (2%)

Seminal vesicle (50) (50) (50) (50)

Inflammation, chronic 1 (2%)

Testes (50) (50) (50) (50)

Germinal epithelium, atrophy 3 (6%) 1 (2%) 1 (2%) 2 (4%)

Hematopoietic System

Bone marrow (50) (50) (50) (50)

Depletion cellular 1 (2%)

Hyperplasia 19 (38%) 25 (50%) 24 (48%) 15 (30%)

Lymph node (2) (2) (2) (4)

Iliac, hyperplasia, lymphoid 1 (25%)

Inguinal, hyperplasia, lymphoid 1 (25%)

Mediastinal, hyperplasia, lymphoid 1 (50%)

Renal, hemorrhage 1 (50%)

Lymph node, mandibular (47) (46) (45) (44)

Atrophy 1 (2%) 1 (2%)

Hematopoietic cell proliferation 1 (2%) 1 (2%)

Hyperplasia, lymphoid 2 (4%) 4 (9%) 2 (4%) 2 (5%)

Pigmentation 1 (2%) 1 (2%) 1 (2%)

Lymph node, mesenteric (46) (48) (48) (48)

Atrophy 1 (2%) 1 (2%) 3 (6%)

Ectasia 1 (2%)

Hematopoietic cell proliferation 3 (7%) 3 (6%) 5 (10%) 6 (13%)

Hemorrhage 5 (11%) 9 (19%) 9 (19%) 9 (19%)

Hyperplasia, lymphoid 3 (7%) 2 (4%) 2 (4%) 2 (4%)

Infiltration cellular, polymorphonuclear 1 (2%)

Spleen (50) (49) (50) (50)

Angiectasis 1 (2%)

Hematopoietic cell proliferation 15 (30%) 13 (27%) 19 (38%) 16 (32%)

Necrosis 1 (2%)

Lymphoid follicle, atrophy 1 (2%) 1 (2%) 2 (4%)

Lymphoid follicle, hyperplasia 1 (2%)

Thymus (41) (46) (47) (43)

Atrophy 1 (2%) 4 (9%) 7 (15%) 6 (14%)

Cyst 3 (7%) 2 (4%) 2 (4%) 3 (7%)

Integumentary System

Skin (50) (50) (50) (50)

Cyst epithelial inclusion 1 (2%)

Edema 1 (2%) 2 (4%)

Inflammation, chronic 2 (4%) 1 (2%)

Epidermis, hyperplasia 1 (2%)
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C-12 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE C3

Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Musculoskeletal System

Bone (50) (50) (50) (50)

Nervous System

Brain (50) (50) (50) (50)

Cyst epithelial inclusion 1 (2%)

Hemorrhage 1 (2%)

Peripheral nerve (2) (2) (1) (1)

Atrophy 1 (50%)

Spinal cord (2) (2) (1) (1)

Respiratory System

Lung (50) (50) (50) (50)

Edema 1 (2%)

Hemorrhage 1 (2%) 2 (4%) 2 (4%)

Hyperplasia, lymphoid 2 (4%) 1 (2%) 1 (2%)

Infiltration cellular, histiocyte 3 (6%) 5 (10%) 2 (4%) 4 (8%)

Metaplasia, osseous 1 (2%)

Thrombosis 1 (2%)

Alveolar epithelium, hyperplasia 1 (2%) 1 (2%) 3 (6%) 3 (6%)

Nose (50) (50) (50) (50)

Inflammation, chronic 3 (6%) 1 (2%)

Respiratory epithelium, hyperplasia 1 (2%)

Special Senses System

Eye (50) (50) (50) (50)

Inflammation, chronic 2 (4%) 1 (2%)

Cornea, hyperplasia 1 (2%) 1 (2%)

Harderian gland (50) (50) (50) (50)

Hyperplasia, focal 2 (4%) 1 (2%)

Inflammation, chronic 1 (2%)

Urinary System

Kidney (50) (50) (50) (50)

Cyst 4 (8%) 4 (8%) 3 (6%) 3 (6%)

Glomerulosclerosis 1 (2%) 1 (2%)

Hydronephrosis 1 (2%) 1 (2%) 2 (4%) 1 (2%)

Hyperplasia, lymphoid 2 (4%) 1 (2%) 1 (2%)

Infarct 5 (10%) 2 (4%) 2 (4%) 3 (6%)

Inflammation, suppurative 1 (2%)

Inflammation, chronic 1 (2%) 1 (2%)

Metaplasia, osseous 2 (4%) 1 (2%)

Nephropathy 37 (74%) 35 (70%) 30 (60%) 34 (68%)

Papilla, necrosis 1 (2%)

Renal tubule, dilatation, diffuse 2 (4%) 1 (2%)

Renal tubule, hyperplasia 4 (8%)

Renal tubule, necrosis 1 (2%)

Urinary bladder (50) (49) (50) (50)

Edema 1 (2%)

Hemorrhage 1 (2%)

Inflammation, chronic 1 (2%)
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D-1

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APPENDIX D

SUMMARY OF LESIONS IN FEMALE MICE

IN THE 2-YEAR FEED STUDY

OF CHROMIUM PICOLINATE MONOHYDRATE

TABLE D1 Summary of the Incidence of Neoplasms in Female Mice

in the 2-Year Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . D-2

TABLE D2 Statistical Analysis of Primary Neoplasms in Female Mice

in the 2-Year Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . D-6

TABLE D3 Summary of the Incidence of Nonneoplastic Lesions in Female Mice

in the 2-Year Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . D-9
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D-2 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE D1

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of Chromium Picolinate Monohydratea

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Disposition Summary

Animals initially in study 50 50 50 50

Early deaths

Moribund 1 2 2 6

Natural deaths 4 3 4 5

Survivors

Terminal sacrifice 45 44 44 39

Missing 1

Animals examined microscopically 50 49 50 50

Alimentary System

Esophagus (48) (49) (50) (50)

Gallbladder (48) (46) (48) (49)

Intestine large, cecum (50) (48) (50) (48)

Intestine large, colon (50) (48) (50) (50)

Intestine large, rectum (50) (47) (50) (50)

Intestine small, duodenum (49) (47) (49) (49)

Intestine small, jejunum (48) (49) (50) (48)

Carcinoma 1 (2%) 1 (2%)

Liver (50) (49) (50) (50)

Carcinoma, metastatic, islets, pancreatic 1 (2%)

Hepatocellular adenoma 1 (2%) 4 (8%) 4 (8%) 5 (10%)

Hepatocellular carcinoma 3 (6%) 1 (2%) 4 (8%) 4 (8%)

Mesentery (7) (5) (4) (7)

Sarcoma, metastatic, skeletal muscle 1 (20%)

Sarcoma stromal, metastatic, uterus 1 (14%)

Schwannoma malignant, metastatic, skin 1 (14%)

Schwannoma malignant, metastatic, uterus 1 (14%)

Oral mucosa (0) (1) (0) (0)

Squamous cell carcinoma 1 (100%)

Pancreas (50) (48) (50) (50)

Hemangioma 1 (2%)

Sarcoma stromal, metastatic, uterus 1 (2%)

Salivary glands (50) (48) (49) (49)

Stomach, forestomach (50) (48) (50) (50)

Sarcoma stromal, metastatic, uterus 1 (2%)

Squamous cell papilloma 2 (4%) 1 (2%)

Stomach, glandular (50) (47) (50) (50)

Cardiovascular System

Heart (50) (49) (50) (50)
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TABLE D1

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Endocrine System

Adrenal cortex (50) (49) (50) (50)

Adrenal medulla (49) (48) (49) (50)

Pheochromocytoma benign 1 (2%)

Pheochromocytoma malignant 1 (2%)

Islets, pancreatic (50) (49) (50) (50)

Carcinoma 2 (4%)

Parathyroid gland (46) (48) (45) (46)

Pituitary gland (47) (48) (50) (50)

Pars distalis, adenoma 1 (2%) 1 (2%) 2 (4%) 2 (4%)

Pars intermedia, adenoma 1 (2%) 1 (2%)

Thyroid gland (50) (49) (49) (50)

Follicular cell, adenoma 1 (2%) 1 (2%)

General Body System

Tissue NOS (1) (0) (0) (1)

Hemangiosarcoma 1 (100%)

Genital System

Clitoral gland (44) (49) (48) (49)

Ovary (48) (49) (49) (49)

Cystadenoma 1 (2%) 1 (2%) 1 (2%)

Granulosa cell tumor benign 2 (4%)

Granulosa cell tumor malignant 1 (2%)

Uterus (50) (49) (50) (50)

Hemangiosarcoma 1 (2%)

Leiomyoma 1 (2%)

Leiomyosarcoma 2 (4%) 1 (2%)

Polyp stromal 2 (4%) 1 (2%)

Sarcoma 1 (2%)

Sarcoma stromal 1 (2%)

Schwannoma malignant 1 (2%)

Hematopoietic System

Bone marrow (50) (49) (50) (50)

Sarcoma, metastatic, skeletal muscle 1 (2%)

Lymph node (3) (8) (4) (4)

Iliac, sarcoma, metastatic, skeletal muscle 1 (25%)

Lymph node, mandibular (50) (46) (46) (48)

Hemangiosarcoma 1 (2%)

Lymph node, mesenteric (50) (48) (49) (48)

Sarcoma stromal, metastatic, uterus 1 (2%)

Spleen (50) (49) (50) (50)

Hemangiosarcoma 2 (4%) 1 (2%) 1 (2%)

Thymus (49) (48) (46) (48)
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D-4 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE D1

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Integumentary System

Mammary gland (50) (49) (50) (50)

Adenoma 1 (2%)

Carcinoma 1 (2%)

Fibroadenoma 1 (2%)

Skin (50) (49) (50) (50)

Epidermis, basosquamous tumor benign 1 (2%)

Sebaceous gland, adenoma 1 (2%)

Subcutaneous tissue, fibrosarcoma 1 (2%) 1 (2%) 1 (2%) 1 (2%)

Subcutaneous tissue, fibrous histiocytoma 1 (2%)

Subcutaneous tissue, hemangiosarcoma 1 (2%)

Subcutaneous tissue, schwannoma malignant 1 (2%)

Musculoskeletal System

Bone (50) (49) (50) (50)

Osteosarcoma 1 (2%)

Skeletal muscle (2) (2) (3) (2)

Sarcoma 2 (100%) 1 (33%) 1 (50%)

Schwannoma malignant, metastatic, skin 1 (50%)

Nervous System

Brain (50) (49) (50) (50)

Peripheral nerve (2) (3) (2) (0)

Respiratory System

Lung (50) (49) (50) (50)

Alveolar/bronchiolar adenoma 1 (2%) 1 (2%) 2 (4%)

Alveolar/bronchiolar carcinoma 1 (2%) 3 (6%) 1 (2%) 1 (2%)

Hepatocellular carcinoma, metastatic, liver 1 (2%)

Osteosarcoma, metastatic, bone 1 (2%)

Nose (50) (49) (50) (50)

Special Senses System

Eye (50) (48) (50) (50)

Harderian gland (50) (48) (50) (50)

Adenoma 7 (14%) 5 (10%) 4 (8%) 6 (12%)

Adenoma, multiple 1 (2%)

Carcinoma 2 (4%) 1 (2%) 1 (2%) 1 (2%)

Zymbal’s gland (0) (0) (1) (0)

Carcinoma 1 (100%)

Urinary System

Kidney (50) (49) (50) (50)

Urinary bladder (49) (49) (50) (50)
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TABLE D1

Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Systemic Lesions

Multiple organsb (50) (49) (50) (50)

Histiocytic sarcoma 1 (2%) 2 (4%) 3 (6%) 1 (2%)

Lymphoma malignant 5 (10%) 9 (18%) 5 (10%) 4 (8%)

Neoplasm Summary

Total animals with primary neoplasmsc 31 30 26 23

Total primary neoplasms 44 44 35 33

Total animals with benign neoplasms 16 14 14 14

Total benign neoplasms 17 18 17 17

Total animals with malignant neoplasms 22 22 17 13

Total malignant neoplasms 27 26 18 16

Total animals with metastatic neoplasms 4112

Total metastatic neoplasms 5116

a Number of animals examined microscopically at the site and the number of animals with neoplasm b Number of animals with any tissue examined microscopically c Primary neoplasms: all neoplasms except metastatic neoplasms
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D-6 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE D2

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Harderian Gland: Adenoma

Overall ratea 7/50 (14%) 6/49 (12%) 4/50 (8%) 6/50 (12%)

Adjusted rateb 14.8% 12.7% 8.3% 12.9%

Terminal ratec 6/45 (13%) 6/44 (14%) 3/44 (7%) 5/39 (13%)

First incidence (days) 704 729 (T) 726 726

Poly-3 testd P=0.585N P=0.498N P=0.252N P=0.514N

Harderian Gland: Adenoma or Carcinoma

Overall rate 9/50 (18%) 7/49 (14%) 5/50 (10%) 7/50 (14%)

Adjusted rate 19.0% 14.8% 10.4% 15.1%

Terminal rate 8/45 (18%) 7/44 (16%) 4/44 (9%) 6/39 (15%)

First incidence (days) 704 729 (T) 726 726

Poly-3 test P=0.521N P=0.391N P=0.184N P=0.408N

Liver: Hepatocellular Adenoma

Overall rate 1/50 (2%) 4/49 (8%) 4/50 (8%) 5/50 (10%)

Adjusted rate 2.1% 8.4% 8.3% 10.8%

Terminal rate 1/45 (2%) 4/44 (9%) 4/44 (9%) 5/39 (13%)

First incidence (days) 729 (T) 729 (T) 729 (T) 729 (T)

Poly-3 test P=0.194 P=0.180 P=0.185 P=0.098

Liver: Hepatocellular Carcinoma

Overall rate 3/50 (6%) 1/49 (2%) 4/50 (8%) 4/50 (8%)

Adjusted rate 6.4% 2.1% 8.3% 8.5%

Terminal rate 3/45 (7%) 1/44 (2%) 4/44 (9%) 0/39 (0%)

First incidence (days) 729 (T) 729 (T) 729 (T) 628

Poly-3 test P=0.296 P=0.304N P=0.510 P=0.501

Liver: Hepatocellular Adenoma or Carcinoma

Overall rate 4/50 (8%) 5/49 (10%) 8/50 (16%) 9/50 (18%)

Adjusted rate 8.5% 10.6% 16.6% 19.0%

Terminal rate 4/45 (9%) 5/44 (11%) 8/44 (18%) 5/39 (13%)

First incidence (days) 729 (T) 729 (T) 729 (T) 628

Poly-3 test P=0.111 P=0.503 P=0.187 P=0.117

Lung: Alveolar/bronchiolar Carcinoma

Overall rate 1/50 (2%) 3/49 (6%) 1/50 (2%) 1/50 (2%)

Adjusted rate 2.1% 6.3% 2.1% 2.2%

Terminal rate 1/45 (2%) 2/44 (5%) 1/44 (2%) 1/39 (3%)

First incidence (days) 729 (T) 642 729 (T) 729 (T)

Poly-3 test P=0.449N P=0.310 P=0.756N P=0.757

Lung: Alveolar/bronchiolar Adenoma or Carcinoma

Overall rate 2/50 (4%) 3/49 (6%) 3/50 (6%) 1/50 (2%)

Adjusted rate 4.2% 6.3% 6.2% 2.2%

Terminal rate 2/45 (4%) 2/44 (5%) 3/44 (7%) 1/39 (3%)

First incidence (days) 729 (T) 642 729 (T) 729 (T)

Poly-3 test P=0.297N P=0.505 P=0.509 P=0.506N

Ovary: Benign or Malignant Granulosa Cell Tumor

Overall rate 3/48 (6%) 0/49 (0%) 0/49 (0%) 0/49 (0%)

Adjusted rate 6.6% 0.0% 0.0% 0.0%

Terminal rate 3/43 (7%) 0/44 (0%) 0/44 (0%) 0/38 (0%)

First incidence (days) 729 (T) —e — —

Poly-3 test P=0.228N P=0.110N P=0.111N P=0.118N
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TABLE D2

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

All Organs: Hemangiosarcoma

Overall rate 3/50 (6%) 3/49 (6%) 0/50 (0%) 1/50 (2%)

Adjusted rate 6.4% 6.3% 0.0% 2.2%

Terminal rate 2/45 (4%) 3/44 (7%) 0/44 (0%) 0/39 (0%)

First incidence (days) 704 729 (T) — 726

Poly-3 test P=0.275N P=0.661N P=0.116N P=0.312N

All Organs: Hemangioma or Hemangiosarcoma

Overall rate 3/50 (6%) 3/49 (6%) 0/50 (0%) 2/50 (4%)

Adjusted rate 6.4% 6.3% 0.0% 4.3%

Terminal rate 2/45 (4%) 3/44 (7%) 0/44 (0%) 1/39 (3%)

First incidence (days) 704 729 (T) — 726

Poly-3 test P=0.522N P=0.661N P=0.116N P=0.508N

All Organs: Histiocytic Sarcoma

Overall rate 1/50 (2%) 2/49 (4%) 3/50 (6%) 1/50 (2%)

Adjusted rate 2.1% 4.2% 6.2% 2.1%

Terminal rate 1/45 (2%) 1/44 (2%) 1/44 (2%) 0/39 (0%)

First incidence (days) 729 (T) 665 628 594

Poly-3 test P=0.472N P=0.503 P=0.316 P=0.759

All Organs: Malignant Lymphoma

Overall rate 5/50 (10%) 9/49 (18%) 5/50 (10%) 4/50 (8%)

Adjusted rate 10.6% 18.7% 10.3% 8.5%

Terminal rate 5/45 (11%) 7/44 (16%) 3/44 (7%) 2/39 (5%)

First incidence (days) 729 (T) 543 629 614

Poly-3 test P=0.234N P=0.206 P=0.611N P=0.502N

All Organs: Benign Neoplasms

Overall rate 16/50 (32%) 14/49 (29%) 14/50 (28%) 14/50 (28%)

Adjusted rate 33.9% 29.6% 29.1% 29.9%

Terminal rate 15/45 (33%) 14/44 (32%) 13/44 (30%) 12/39 (31%)

First incidence (days) 704 729 (T) 726 628

Poly-3 test P=0.489N P=0.410N P=0.391N P=0.427N

All Organs: Malignant Neoplasms

Overall rate 22/50 (44%) 22/49 (45%) 17/50 (34%) 13/50 (26%)

Adjusted rate 45.8% 44.9% 34.7% 26.9%

Terminal rate 19/45 (42%) 17/44 (39%) 12/44 (27%) 5/39 (13%)

First incidence (days) 480 543 628 594

Poly-3 test P=0.028N P=0.544N P=0.181N P=0.041N
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D-8 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE D2

Statistical Analysis of Primary Neoplasms in Female Mice in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

All Organs: Benign or Malignant Neoplasms

Overall rate 31/50 (62%) 30/49 (61%) 26/50 (52%) 23/50 (46%)

Adjusted rate 64.6% 61.2% 53.0% 47.5%

Terminal rate 28/45 (62%) 25/44 (57%) 21/44 (48%) 15/39 (39%)

First incidence (days) 480 543 628 594

Poly-3 test P=0.066N P=0.448N P=0.171N P=0.068N

(T) Terminal sacrifice

a Number of neoplasm-bearing animals/number of animals examined. Denominator is number of animals examined microscopically for liver, lung, and ovary; 

for other tissues, denominator is number of animals necropsied. b Poly-3 estimated neoplasm incidence after adjustment for intercurrent mortality c Observed incidence at terminal kill d Beneath the control incidence is the P value associated with the trend test. Beneath the exposed group incidence are the P values corresponding to pairwise 

comparisons between the controls and that exposed group. The Poly-3 test accounts for the differential mortality in animals that do not reach terminal 

sacrifice. A negative trend or a lower incidence in an exposure group is indicated by N. e Not applicable; no neoplasms in animal group
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TABLE D3

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Study 

of Chromium Picolinate Monohydratea

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Disposition Summary

Animals initially in study 50 50 50 50

Early deaths

Moribund 1 2 2 6

Natural deaths 4 3 4 5

Survivors

Terminal sacrifice 45 44 44 39

Missing 1

Animals examined microscopically 50 50 50 50

Alimentary System

Esophagus (48) (49) (50) (50)

Gallbladder (48) (46) (48) (49)

Intestine large, cecum (50) (48) (50) (48)

Hyperplasia, lymphoid 1 (2%)

Necrosis 1 (2%)

Intestine large, colon (50) (48) (50) (50)

Necrosis 1 (2%)

Intestine large, rectum (50) (47) (50) (50)

Intestine small, duodenum (49) (47) (49) (49)

Intestine small, jejunum (48) (49) (50) (48)

Hyperplasia, lymphoid 1 (2%) 1 (2%) 1 (2%)

Liver (50) (49) (50) (50)

Angiectasis 1 (2%) 1 (2%)

Basophilic focus 3 (6%) 5 (10%) 4 (8%)

Cyst 1 (2%)

Eosinophilic focus 1 (2%) 1 (2%)

Hematopoietic cell proliferation 2 (4%) 3 (6%)

Hemorrhage 1 (2%) 1 (2%)

Hyperplasia, lymphoid 4 (8%) 2 (4%) 3 (6%) 1 (2%)

Infiltration cellular, mixed cell 2 (4%) 2 (4%) 3 (6%) 2 (4%)

Inflammation, chronic active 1 (2%)

Mixed cell focus 1 (2%) 3 (6%)

Necrosis, focal 4 (8%) 4 (8%) 4 (8%)

Necrosis, diffuse 1 (2%) 1 (2%)

Tension lipidosis 2 (4%)

Centrilobular, necrosis 1 (2%)

Hepatocyte, vacuolization cytoplasmic 1 (2%)

Kupffer cell, hyperplasia 1 (2%) 1 (2%) 1 (2%)

Mesentery (7) (5) (4) (7)

Hemorrhage 1 (14%) 1 (20%)

Infiltration cellular, lymphoid 1 (20%)

Fat, necrosis 5 (71%) 3 (60%) 3 (75%) 5 (71%)

Oral mucosa (0) (1) (0) (0)

Pancreas (50) (48) (50) (50)

Atrophy 2 (4%) 2 (4%) 1 (2%)

Cyst 2 (4%) 2 (4%) 1 (2%)

Inflammation, chronic 1 (2%)

Acinus, cytoplasmic alteration 1 (2%) 1 (2%) 2 (4%)

Acinus, necrosis 1 (2%)

a Number of animals examined microscopically at the site and the number of animals with lesion
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D-10 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE D3

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Alimentary System (continued)

Salivary glands (50) (48) (49) (49)

Atrophy 1 (2%)

Hyperplasia, lymphoid 10 (20%) 6 (13%) 3 (6%) 8 (16%)

Stomach, forestomach (50) (48) (50) (50)

Diverticulum 2 (4%)

Ulcer 1 (2%)

Epithelium, hyperplasia 1 (2%) 1 (2%) 1 (2%) 2 (4%)

Stomach, glandular (50) (47) (50) (50)

Cyst 1 (2%)

Edema 1 (2%)

Erosion 2 (4%)

Foreign body 1 (2%)

Inflammation, chronic active 1 (2%)

Epithelium, hyperplasia 2 (4%)

Cardiovascular System

Heart (50) (49) (50) (50)

Cardiomyopathy 1 (2%)

Inflammation, chronic 1 (2%) 2 (4%)

Mineralization 1 (2%)

Endocrine System

Adrenal cortex (50) (49) (50) (50)

Accessory adrenal cortical nodule 9 (18%) 3 (6%) 11 (22%) 5 (10%)

Hyperplasia, focal 1 (2%) 1 (2%) 1 (2%)

Capsule, hyperplasia 3 (6%) 4 (8%) 2 (4%)

Adrenal medulla (49) (48) (49) (50)

Hyperplasia 2 (4%) 1 (2%) 1 (2%)

Islets, pancreatic (50) (49) (50) (50)

Parathyroid gland (46) (48) (45) (46)

Cyst 1 (2%) 1 (2%)

Hyperplasia 1 (2%)

Pituitary gland (47) (48) (50) (50)

Pars distalis, angiectasis 3 (6%) 1 (2%) 1 (2%)

Pars distalis, cyst 3 (6%) 1 (2%) 2 (4%)

Pars distalis, hyperplasia, focal 5 (11%) 3 (6%) 5 (10%) 2 (4%)

Pars intermedia, hyperplasia 1 (2%)

Thyroid gland (50) (49) (49) (50)

Ectopic thymus 1 (2%)

Follicle, cyst 1 (2%)

Follicle, degeneration, focal 15 (30%) 17 (35%) 24 (49%) 16 (32%)

Follicular cell, hyperplasia 1 (2%) 1 (2%) 3 (6%)

General Body System

Tissue NOS (1) (0) (0) (1)

Autolysis 1 (100%)
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TABLE D3

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Genital System

Clitoral gland (44) (49) (48) (49)

Ovary (48) (49) (49) (49)

Angiectasis 1 (2%) 1 (2%)

Cyst 16 (33%) 18 (37%) 14 (29%) 18 (37%)

Hemorrhage 7 (15%) 15 (31%) 10 (20%) 10 (20%)

Metaplasia, lipocyte 1 (2%)

Granulosa cell, hyperplasia 1 (2%)

Interstitial cell, hyperplasia 4 (8%) 1 (2%)

Uterus (50) (49) (50) (50)

Angiectasis 2 (4%) 1 (2%) 3 (6%)

Hemorrhage 1 (2%) 1 (2%) 1 (2%)

Hyperplasia, cystic 47 (94%) 44 (90%) 45 (90%) 44 (88%)

Metaplasia, squamous 1 (2%)

Hematopoietic System

Bone marrow (50) (49) (50) (50)

Hyperplasia 5 (10%) 10 (20%) 8 (16%) 10 (20%)

Infiltration cellular, histiocyte 1 (2%)

Myelofibrosis 1 (2%)

Lymph node (3) (8) (4) (4)

Bronchial, hyperplasia, lymphoid 1 (13%)

Iliac, hemorrhage 1 (25%) 1 (25%)

Iliac, hyperplasia, histiocytic 1 (13%)

Iliac, hyperplasia, lymphoid 2 (25%)

Mediastinal, hyperplasia, lymphoid 1 (33%)

Renal, hemorrhage 1 (25%)

Renal, hyperplasia, lymphoid 1 (25%)

Lymph node, mandibular (50) (46) (46) (48)

Atrophy 2 (4%)

Hemorrhage 1 (2%)

Hyperplasia, lymphoid 1 (2%) 3 (7%) 6 (13%) 2 (4%)

Pigmentation 7 (14%) 5 (11%) 6 (13%) 2 (4%)

Lymph node, mesenteric (50) (48) (49) (48)

Atrophy 2 (4%)

Hematopoietic cell proliferation 3 (6%) 1 (2%) 1 (2%)

Hemorrhage 2 (4%) 2 (4%)

Hyperplasia, lymphoid 4 (8%) 3 (6%) 3 (6%)

Spleen (50) (49) (50) (50)

Accessory spleen 1 (2%) 1 (2%)

Angiectasis 2 (4%)

Hematopoietic cell proliferation 14 (28%) 12 (24%) 11 (22%) 11 (22%)

Lymphoid follicle, atrophy 2 (4%)

Lymphoid follicle, hyperplasia 5 (10%) 7 (14%) 11 (22%) 9 (18%)

Thymus (49) (48) (46) (48)

Atrophy 2 (4%) 2 (4%) 2 (4%)

Hyperplasia, lymphoid 2 (4%) 2 (4%)
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TABLE D3

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Integumentary System

Mammary gland (50) (49) (50) (50)

Hyperplasia 2 (4%) 1 (2%) 1 (2%)

Skin (50) (49) (50) (50)

Edema 3 (6%)

Inflammation, chronic 1 (2%)

Ulcer 1 (2%) 1 (2%) 1 (2%) 1 (2%)

Epidermis, hyperplasia 1 (2%) 1 (2%)

Hair follicle, atrophy 1 (2%)

Musculoskeletal System

Bone (50) (49) (50) (50)

Fibrosis 4 (8%) 9 (18%) 7 (14%) 5 (10%)

Skeletal muscle (2) (2) (3) (2)

Inflammation, chronic active 1 (50%)

Necrosis 1 (50%)

Nervous System

Brain (50) (49) (50) (50)

Compression 1 (2%) 1 (2%) 1 (2%) 1 (2%)

Gliosis 1 (2%)

Hemorrhage 1 (2%)

Peripheral nerve (2) (3) (2) (0)

Atrophy 1 (50%) 1 (33%) 1 (50%)

Respiratory System

Lung (50) (49) (50) (50)

Hemorrhage 2 (4%) 2 (4%) 1 (2%) 3 (6%)

Hyperplasia, lymphoid 1 (2%) 1 (2%) 1 (2%) 5 (10%)

Infiltration cellular, histiocyte 4 (8%) 1 (2%) 2 (4%)

Inflammation, chronic 1 (2%)

Thrombosis 1 (2%)

Alveolar epithelium, hyperplasia 2 (4%) 1 (2%) 1 (2%)

Capillary, infiltration cellular, polymorphonuclear 1 (2%) 1 (2%)

Nose (50) (49) (50) (50)

Inflammation, chronic 1 (2%)

Special Senses System

Eye (50) (48) (50) (50)

Cataract 1 (2%)

Inflammation, chronic 1 (2%) 1 (2%)

Cornea, hyperplasia 1 (2%)

Harderian gland (50) (48) (50) (50)

Atrophy 1 (2%)

Hyperplasia, focal 2 (4%)

Zymbal’s gland (0) (0) (1) (0)
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TABLE D3

Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Urinary System

Kidney (50) (49) (50) (50)

Cyst 1 (2%)

Glomerulosclerosis 1 (2%) 1 (2%)

Hyperplasia, lymphoid 2 (4%) 3 (6%) 4 (8%)

Infarct 1 (2%) 2 (4%) 1 (2%)

Inflammation, chronic 1 (2%)

Metaplasia, osseous 1 (2%) 1 (2%) 2 (4%) 4 (8%)

Nephropathy 7 (14%) 17 (35%) 13 (26%) 11 (22%)

Renal tubule, accumulation, hyaline droplet 2 (4%) 2 (4%)

Renal tubule, dilatation, diffuse 1 (2%)

Renal tubule, necrosis 1 (2%)

Renal tubule, pigmentation 1 (2%)

Urinary bladder (49) (49) (50) (50)

Hyperplasia, lymphoid 6 (12%) 3 (6%) 3 (6%) 5 (10%)

Inflammation, chronic 1 (2%)
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E-1

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APPENDIX E

GENETIC TOXICOLOGY

SALMONELLA TYPHIMURIUM MUTAGENICITY TEST PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-2

MOUSE PERIPHERAL BLOOD MICRONUCLEUS TEST PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-2

RAT BONE MARROW MICRONUCLEUS TEST PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-3

EVALUATION PROTOCOL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-3

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-3

TABLE E1 Mutagenicity of Chromium Picolinate Monohydrate in Salmonella typhimurium . . . . . . . . E-4

TABLE E2 Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice 

Following Administration of Chromium Picolinate Monohydrate in Feed 

for 3 Months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-5

TABLE E3 Mutagenicity of Chromium Picolinate in Salmonella typhimurium . . . . . . . . . . . . . . . . . . . . E-6

TABLE E4 Induction of Micronuclei in Bone Marrow Polychromatic Erythrocytes 

of Male Rats Treated with Chromium Picolinate by Gavage . . . . . . . . . . . . . . . . . . . . . . . . . E-11
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GENETIC TOXICOLOGY

SALMONELLA TYPHIMURIUM MUTAGENICITY TEST PROTOCOL

Three independent mutagenicity assays were conducted with chromium picolinate and chromium picolinate

monohydrate. The first assay, conducted with the same lot of chromium picolinate monohydrate that was tested in

the 2-year studies, used a slightly modified protocol (activation only with 10% rat liver S9) and also employed

Escherichia coli strain WP2 uvrA/pKM101 as a bacterial tester strain in addition to Salmonella typhimurium

strains. For the second and third assays, testing was performed with chromium picolinate as reported by Zeiger

et al. (1992), using strains TA97, TA98, TA100, TA102, TA104, and TA1535, tested with and without 10% and

30% hamster and rat liver S9 mix, as described below. For all tests, the compounds were sent to the testing

laboratory as coded aliquots. Test articles were incubated with the bacterial tester strains either in buffer or S9 mix

(metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat or Syrian

hamster liver) for 20 minutes at 37° C. Top agar supplemented with L-histidine and d-biotin was added, and the

contents of the tubes were mixed and poured onto the surfaces of minimal glucose agar plates. Histidineindependent mutant colonies arising on these plates were counted following incubation for 2 days at 37° C.

Each trial consisted of triplicate plates of concurrent positive and negative controls and at least five doses of

chromium picolinate or chromium picolinate monohydrate. The high dose was set at 10,000 μg/plate by

experimental design, because no toxicity was observed.

In this assay, a positive response is defined as a reproducible, dose-related increase in histidine-independent

(revertant) colonies in any one strain/activation combination. An equivocal response is defined as an increase in

revertants that is not dose related, is not reproducible, or is not of sufficient magnitude to support a determination

of mutagenicity. A negative response is obtained when no increase in revertant colonies is observed following

chemical treatment. There is no minimum percentage or fold increase required for a chemical to be judged

positive or weakly positive. 

MOUSE PERIPHERAL BLOOD MICRONUCLEUS TEST PROTOCOL

A detailed discussion of this assay is presented by MacGregor et al. (1990). At the end of the 3-month feed study

with chromium picolinate monohydrate, peripheral blood samples were obtained from male and female mice.

Smears were immediately prepared and fixed in absolute methanol. The methanol-fixed slides were stained with

acridine orange and coded. Slides were scanned to determine the frequency of micronucleated cells in

1,000 normochromatic erythrocytes (NCEs) in each of 10 animals per exposure group. In addition, the percentage

of polychromatic erythrocytes (PCEs) in a population of 1,000 erythrocytes was determined per animal as a

measure of bone marrow toxicity.

The results were tabulated as the mean of the pooled results from all animals within a treatment group plus or

minus the standard error of the mean. The frequency of micronucleated cells among NCEs was analyzed by a

statistical software package that tested for increasing trend over dose groups with a one-tailed Cochran-Armitage

trend test, followed by pairwise comparisons between each exposed group and the control group (ILS, 1990). In

the presence of excess binomial variation, as detected by a binomial dispersion test, the binomial variance of the

Cochran-Armitage test was adjusted upward in proportion to the excess variation. In the micronucleus test, an

individual trial is considered positive if the trend test P value is less than or equal to 0.025 or if the P value for any

single exposed group is less than or equal to 0.025 divided by the number of exposed groups. A final call of

positive for micronucleus induction is preferably based on reproducibly positive trials (as noted above).

Ultimately, the final call is determined by the scientific staff after considering the results of statistical analyses, the

reproducibility of any effects observed, and the magnitudes of those effects. Results of the 3-month study were

accepted without repeat tests because additional test data could not be obtained.
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RAT BONE MARROW MICRONUCLEUS TEST PROTOCOL

The standard three-exposure protocol is described in detail by Shelby et al. (1993). Male F344/N rats were

administered chromium picolinate dissolved in corn oil by oral gavage three times at 24-hour intervals. Vehicle

control animals received corn oil only. The positive control mice received a single injection of cyclophosphamide.

The rats were killed 24 hours after the final treatment, and blood smears were prepared from bone marrow cells

obtained from the femurs. The smears were air-dried, fixed, and stained; 2,000 polychromatic erythrocytes (PCEs)

were scored for the frequency of micronucleated cells in each of five rats per dose group. In addition, the

percentage of PCEs among the total erythrocyte population in the bone marrow was determined per animal as a

measure of toxicity.

The results for bone marrow PCEs were tabulated as described for NCEs in the mouse peripheral blood test.

EVALUATION PROTOCOL

These are the basic guidelines for arriving at an overall assay result for assays performed by the National

Toxicology Program. Statistical as well as biological factors are considered. For an individual assay, the statistical

procedures for data analysis have been described in the preceding protocols. There have been instances, however,

in which multiple aliquots of a chemical were tested in the same assay, and different results were obtained among

aliquots and/or among laboratories. Results from more than one aliquot or from more than one laboratory are not

simply combined into an overall result. Rather, all the data are critically evaluated, particularly with regard to

pertinent protocol variations, in determining the weight of evidence for an overall conclusion of chemical activity

in an assay. In addition to multiple aliquots, the in vitro assays have another variable that must be considered in

arriving at an overall test result. In vitro assays are conducted with and without exogenous metabolic activation.

Results obtained in the absence of activation are not combined with results obtained in the presence of activation;

each testing condition is evaluated separately. The summary table in the Abstract of this Technical Report presents

a result that represents a scientific judgement of the overall evidence for activity of the chemical in an assay.

RESULTS

In the standard screening assays conducted by the NTP, chromium picolinate monohydrate showed no clear

evidence of genotoxicity. Over a concentration range of 100 to 10,000 μg/plate, no evidence of mutagenicity was

observed in S. typhimurium strains TA100 or TA98 or E. coli strain WP2 uvrA/pKM101 when chromium picolinate

monohydrate was tested with or without exogenous metabolic activation (S9) (Table E1). In addition, no increase

in the frequency of micronucleated NCEs was observed in male B6C3F1 mice administered chromium picolinate

monohydrate (80 to 50,000 ppm) in feed for 3 months, indicating no potential for chromium picolinate

monohydrate to induce chromosomal alterations in dividing cell populations in this test system (Table E2). In

female mice, however, the small increase in micronucleated NCEs noted in the highest exposure concentration

group (50,000 ppm) was not significant at P=0.0396, but it resulted in a significant trend test (P=0.005) and,

therefore, the test with chromium picolinate monohydrate was judged to be equivocal in female mice (Table E2).

No significant alterations in the percentage of PCEs among total erythrocytes was observed in exposed mice,

indicating that these exposure concentrations of chromium picolinate monohydrate did not induce bone marrow

toxicity (Table E2).

Additional genotoxicity testing was conducted with chromium picolinate (not the monohydrate form of the

compound), and results were also negative. No induction of gene mutations was observed in two independent

studies conducted with chromium picolinate (up to 10,000 μg/plate) in several strains of S. typhimurium with and

without hamster or rat liver S9 (Table E3). No induction of micronucleated PCEs was observed in bone marrow of

male F344/N rats treated with chromium picolinate (156 to 2,500 mg/kg) by oral gavage three times at 24-hour

intervals, and no significant alterations in the percentage of PCEs among total erythrocytes was observed in dosed

rats, indicating that these doses of chromium picolinate did not induce bone marrow toxicity (Table E4).
E-4

Table E1

Mutagenicity of Chromium Picolinate Monohydrate in Salmonella typhimuriuma

Revertants/Plateb

Dose –S9 +10% rat S9

Strain (μg/plate) Trial 1 Trial 2 Trial 1 Trial 2

TA100 0 56 ± 11.0 44 ± 3.0 72 ± 7.0 65 ± 7.0

100 44 ± 7.0 37 ± 2.0 57 ± 3.0 55 ± 9.0

500 39 ± 5.0 44 ± 3.0 71 ± 11.0 46 ± 1.0

1,000 31 ± 2.0 43 ± 1.0 61 ± 1.0 55 ± 3.0

5,000 49 ± 3.0 59 ± 5.0 71 ± 4.0 58 ± 4.0

10,000 41 ± 1.0 64 ± 1.0 93 ± 3.0 60 ± 2.0

Trial summary Negative Negative Negative Negative

Positive controlc 448 ± 1.0 378 ± 5.0 598 ± 18.0 481 ± 14.0

–S9 +10% rat S9

Trial 1 Trial 2 Trial 3 Trial 1 Trial 2

TA98 0 24 ± 1.0 12 ± 2.0 15 ± 3.0 20 ± 1.0 22 ± 2.0

100 20 ± 3.0 16 ± 1.0 22 ± 3.0 27 ± 5.0 28 ± 4.0

500 20 ± 3.0 12 ± 2.0 18 ± 1.0 27 ± 4.0 20 ± 3.0

1,000 20 ± 2.0 18 ± 4.0 15 ± 3.0 29 ± 4.0 27 ± 2.0

5,000 25 ± 2.0 14 ± 1.0 13 ± 3.0 29 ± 1.0 28 ± 6.0

10,000 24 ± 2.0d 16 ± 1.0 0 ± 0.0 28 ± 4.0d 29 ± 4.0

Trial summary Negative Negative Negative Negative Negative

Positive control 316 ± 27.0 572 ± 8.0 429 ± 11.0 741 ± 41.0 649 ± 49.0

–S9 +10% rat S9

Trial 1 Trial 2 Trial 1 Trial 2

Escherichia coli WP2 uvrA/pKM101

0 108 ± 3.0 225 ± 25.0 158 ± 8.0 253 ± 23.0

100 127 ± 2.0 196 ± 28.0 169 ± 4.0 272 ± 62.0

500 147 ± 5.0 153 ± 4.0 175 ± 6.0 185 ± 5.0

1,000 126 ± 10.0 139 ± 6.0 166 ± 8.0 196 ± 4.0

5,000 164 ± 10.0 176 ± 3.0 185 ± 5.0 245 ± 2.0

10,000 204 ± 10.0 165 ± 7.0 184 ± 6.0 255 ± 6.0

Trial summary Weakly Positive Negative Negative Negative

Positive control 1,065 ± 24.0 623 ± 19.0 571 ± 30.0 659 ± 23.0

a Study was performed at SITEK Research Laboratories. 0 μg/plate was the solvent control. b Revertants are presented as mean ± standard error from three plates. c The positive controls in the absence of metabolic activation were sodium azide (TA100), 4-nitro-o-phenylenediamine (TA98), 

and methyl methanesulfonate (WP2 uvrA/pKM101). The positive control for metabolic activation with all strains was 2-aminoanthracene. d Precipitate on plate

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TABLE E2

Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice Following Administration 

of Chromium Picolinate Monohydrate in Feed for 3 Monthsa

Exposure Number of Mice

Compound Concentration with Erythrocytes Micronucleated NCEs/ P Valuec PCEsb (%)

(ppm) Scored 1,000 NCEsb

Male

NTP-2000 feedd 0 10 2.80 ± 0.44 3.740 ± 0.22

Chromium picolinate monohydrate 80 10 2.90 ± 0.57 0.4472 3.920 ± 0.13

240 10 3.20 ± 0.70 0.3025 4.660 ± 0.35

2,000 10 4.30 ± 0.30 0.0373 4.780 ± 0.23

10,000 10 3.40 ± 0.50 0.2277 4.340 ± 0.20

50,000 10 3.50 ± 034 0.1885 4.130 ± 0.29

P=0.350e

Female

NTP-2000 feed 0 10 2.10 ± 0.46 4.060 ± 0.23

Chromium picolinate monohydrate 80 10 1.30 ± 0.33 0.9151 3.980 ± 0.15

240 10 2.50 ± 0.45 0.2774 4.050 ± 0.22

2,000 10 2.20 ± 0.44 0.4393 3.970 ± 0.29

10,000 10 2.10 ± 0.31 0.5000 4.560 ± 0.52

50,000 10 3.40 ± 0.37 0.0396 3.800 ± 0.18

P=0.005

a Study was performed at ILS, Inc. The detailed protocol is presented by MacGregor et al. (1990). PCE=polychromatic erythrocyte; 

NCE=normochromatic erythrocyte. b Mean ± standard error c Pairwise comparison with the untreated control group; significant at P#0.005 (ILS, 1990) d Untreated control e Significance of micronucleated NCEs/1,000 NCEs tested by the one-tailed trend test, significant at P#0.025 (ILS, 1990)

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Table E3

Mutagenicity of Chromium Picolinate in Salmonella typhimuriuma

Dose Revertants/Plateb

Strain (μg/plate) –S9 +30% hamster S9 +30% rat S9

Study performed at BioReliance Corporation

TA102 0 206 ± 7.0 210 ± 13.0 248 ± 18.0

100 205 ± 2.0 190 ± 12.0 221 ± 14.0

333 226 ± 6.0 221 ± 20.0 235 ± 11.0

1,000 208 ± 10.0 211 ± 15.0 228 ± 11.0

3,333 214 ± 1.0 202 ± 15.0 240 ± 19.0

10,000c 202 ± 6.0 209 ± 18.0 198 ± 10.0

Trial summary Negative Negative Negative

Positive controld 1,298 ± 18.0 1,516 ± 12.0 1,298 ± 14.0

TA104 0 312 ± 6.0 214 ± 13.0 215 ± 9.0

100 314 ± 12.0 226 ± 2.0 237 ± 6.0

333 345 ± 4.0 208 ± 15.0 205 ± 15.0

1,000 343 ± 13.0 230 ± 9.0 268 ± 36.0

3,333 364 ± 6.0 239 ± 18.0 226 ± 5.0

10,000c 360 ± 7.0 230 ± 11.0 211 ± 8.0

Trial summary Negative Negative Negative

Positive control 942 ± 18.0 1,335 ± 103.0 962 ± 121.0

–S9 +hamster S9 +rat S9

Trial 1 Trial 2 10% 30% 10% 30%

TA100 0 140 ± 6.0 104 ± 5.0 94 ± 2.0 143 ± 7.0 97 ± 5.0 150 ± 1.0

33 128 ± 15.0 134 ± 2.0 155 ± 14.0

100 144 ± 6.0 125 ± 4.0 102 ± 3.0 152 ± 19.0 85 ± 8.0 155 ± 5.0

333 113 ± 5.0 114 ± 11.0 99 ± 2.0 147 ± 7.0 111 ± 7.0 144 ± 2.0

1,000 136 ± 3.0 108 ± 12.0 82 ± 3.0 133 ± 13.0 110 ± 6.0 150 ± 8.0

3,333 145 ± 2.0 111 ± 5.0 99 ± 11.0 155 ± 11.0 93 ± 9.0 142 ± 8.0

10,000c 109 ± 1.0 131 ± 3.0 100 ± 4.0

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 599 ± 29.0 590 ± 21.0 590 ± 26.0 677 ± 30.0 716 ± 65.0 1,144 ± 63.0

TA1535 0 11 ± 1.0 12 ± 1.0 12 ± 2.0 17 ± 2.0 9 ± 1.0 14 ± 2.0

100 17 ± 2.0 14 ± 1.0 11 ± 1.0 12 ± 1.0 11 ± 1.0 18 ± 1.0

333 11 ± 1.0 13 ± 1.0 13 ± 2.0 15 ± 0.0 13 ± 1.0 14 ± 3.0

1,000 9 ± 1.0 12 ± 1.0 11 ± 1.0 16 ± 1.0 13 ± 2.0 12 ± 3.0

3,333 11 ± 3.0 13 ± 1.0 11 ± 1.0 12 ± 3.0 11 ± 1.0 11 ± 2.0

10,000c 11 ± 1.0 12 ± 2.0 10 ± 1.0 14 ± 2.0 10 ± 1.0 11 ± 3.0

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 364 ± 10.0 84 ± 8.0 122 ± 13.0 237 ± 18.0 113 ± 10.0 110 ± 5.0
NOT FOR DISTRIBUTION OR ATTRIBUTION

Chromium Picolinate Monohydrate, NTP TR 556 E-7

Board Draft 

TABLE E3

Mutagenicity of Chromium Picolinate in Salmonella typhimurium

Revertants/Plate

Dose –S9 +hamster S9

Strain (μg/plate) Trial 1 Trial 2 Trial 3 10% 30%

Study performed at BioReliance Corporation (continued)

TA97 0 128 ± 3.0 89 ± 3.0 100 ± 4.0 135 ± 10.0 158 ± 24.0

100 145 ± 6.0 123 ± 8.0 137 ± 2.0 116 ± 10.0

333 161 ± 6.0 78 ± 5.0 94 ± 2.0 133 ± 8.0 120 ± 8.0

667 91 ± 7.0

1,000 171 ± 7.0 91 ± 6.0 107 ± 11.0 148 ± 9.0 117 ± 3.0

2,000 78 ± 6.0

3,333 169 ± 10.0 81 ± 6.0 130 ± 7.0 144 ± 2.0 128 ± 3.0

10,000 152 ± 7.0 122 ± 3.0c 137 ± 5.0c 153 ± 12.0c

Trial summary Equivocal Negative Negative Negative Negative

Positive control 716 ± 18.0 264 ± 88.0 725 ± 12.0 439 ± 31.0 694 ± 34.0

+rat S9

10% 30%

TA97 0 142 ± 4.0 209 ± 2.0

(continued) 100 132 ± 3.0 205 ± 5.0

333 141 ± 8.0 193 ± 6.0

1,000 140 ± 5.0 209 ± 7.0

3,333 149 ± 6.0 206 ± 4.0

10,000c 122 ± 9.0 172 ± 9.0

Trial summary Negative Negative

Positive control 721 ± 17.0 636 ± 11.0

–S9 +hamster S9 +rat S9

Trial 1 Trial 2 10% 30% 10% 30%

TA98 0 20 ± 3.0 16 ± 3.0 18 ± 2.0 29 ± 3.0 16 ± 1.0 24 ± 5.0

33 18 ± 4.0 20 ± 3.0 21 ± 1.0

100 20 ± 1.0 15 ± 2.0 14 ± 1.0 23 ± 4.0 15 ± 1.0 23 ± 3.0

333 25 ± 3.0 13 ± 1.0 16 ± 1.0 24 ± 3.0 15 ± 1.0 24 ± 2.0

1,000 21 ± 3.0 15 ± 1.0 16 ± 1.0 25 ± 2.0 13 ± 1.0 28 ± 4.0

3,333 24 ± 4.0 15 ± 1.0 16 ± 2.0 23 ± 2.0 16 ± 2.0 22 ± 3.0

10,000c 15 ± 1.0 15 ± 1.0 12 ± 1.0

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 102 ± 3.0 336 ± 20.0 231 ± 15.0 554 ± 59.0 94 ± 28.0 221 ± 7.0
E-8 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft

TABLE E3

Mutagenicity of Chromium Picolinate in Salmonella typhimurium

Revertants/Plate

Dose –S9 +hamster S9

Strain (μg/plate) Trial 1 Trial 2 10% 10% 30% 30%

Study performed at SRI International

TA102 0 233 ± 7.0 235 ± 12.0 300 ± 8.0 281 ± 10.0 388 ± 30.0 337 ± 23.0

100 213 ± 1.0 232 ± 5.0 279 ± 24.0 361 ± 21.0

333 236 ± 2.0 232 ± 21.0 276 ± 19.0 285 ± 16.0 351 ± 38.0 297 ± 9.0

666 282 ± 17.0 296 ± 17.0

1,000 240 ± 14.0 239 ± 9.0 291 ± 12.0 280 ± 6.0 421 ± 48.0 332 ± 12.0

1,666 307 ± 9.0 313 ± 10.0

3,333c 236 ± 11.0 237 ± 3.0 272 ± 25.0 275 ± 15.0 447 ± 5.0 357 ± 23.0

6,666c 356 ± 43.0 345 ± 16.0

10,000c 248 ± 19.0 259 ± 4.01 277 ± 4.0 245 ± 10.0 565 ± 35.0 390 ± 12.0

Trial summary Negative Negative Negative Negative Equivocal Negative

Positive control 1,145 ± 65.0 893 ± 6.0 878 ± 21.0 762 ± 19.0 963 ± 7.0 860 ± 9.0

+rat S9

10% 30%

TA102 0 273 ± 31.0 259 ± 13.0

(continued) 100 288 ± 11.0 253 ± 20.0

333 294 ± 5.0 253 ± 20.0

1,000 283 ± 8.0 279 ± 22.0

3,333c 287 ± 6.0 277 ± 39.0

10,000c 310 ± 3.0 267 ± 57.0

Trial summary Negative Negative

Positive control 648 ± 7.0 940 ± 19.0

–S9 +hamster S9 +rat S9

Trial 1 Trial 2 10% 30% 10% 30%

TA104 0 432 ± 3.0 254 ± 12.0 322 ± 8.0 422 ± 7.0 281 ± 17.0 359 ± 30.0

100 421 ± 17.0 293 ± 3.0 294 ± 27.0 422 ± 6.0 306 ± 5.0 418 ± 35.0

333 457 ± 8.0 267 ± 12.0 313 ± 26.0 471 ± 14.0 328 ± 2.0 405 ± 20.0

1,000 437 ± 7.0 232 ± 4.0 322 ± 3.0 427 ± 11.0 313 ± 10.0 429 ± 39.0

3,333c 444 ± 15.0 261 ± 8.0 340 ± 10.0 427 ± 13.0 320 ± 5.0 373 ± 31.0

10,000c 415 ± 10.0 280 ± 18.0 354 ± 8.0 422 ± 20.0 363 ± 21.0 377 ± 17.0

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 947 ± 45.0 1,281 ± 317.0 1,019 ± 7.0 1,124 ± 42.0 888 ± 12.0 915 ± 36.0
NOT FOR DISTRIBUTION OR ATTRIBUTION

Chromium Picolinate Monohydrate, NTP TR 556 E-9

Board Draft 

TABLE E3

Mutagenicity of Chromium Picolinate in Salmonella typhimurium

Revertants/Plate

Dose –S9 +hamster S9

Strain (μg/plate) Trial 1 Trial 2 Trial 3 10% 30% 30%

Study performed at SRI International (continued)

TA100 0 116 ± 6.0 114 ± 5.0 120 ± 2.0 122 ± 5.0 96 ± 6.0 128 ± 6.0

100 100 ± 1.0 132 ± 3.0 140 ± 8.0 117 ± 7.0

333 96 ± 5.0 126 ± 5.0 122 ± 8.0 105 ± 7.0

1,000 103 ± 4.0 114 ± 8.0 121 ± 5.0 142 ± 16.0 111 ± 5.0 119 ± 4.0

1,666 121 ± 5.0 117 ± 3.0

3,333 101 ± 6.0c 110 ± 5.0 130 ± 8.0c 127 ± 6.0c 105 ± 2.0c 119 ± 2.0

6,666c 112 ± 4.0 104 ± 6.0

10,000c 125 ± 3.0 103 ± 3.0 126 ± 10.0 133 ± 19.0 132 ± 2.0 109 ± 5.0

Trial summary Equivocal Negative Negative Negative Equivocal Negative

Positive control 867 ± 23.0 890 ± 12.0 834 ± 31.0 629 ± 23.0 644 ± 25.0 633 ± 29.0

+rat S9

10% 30% 30%

TA100 0 130 ± 8.0 110 ± 9.0 140 ± 7.0

(continued) 100 122 ± 3.0 116 ± 4.0

333 131 ± 0.0 116 ± 1.0

1,000 137 ± 5.0 115 ± 3.0 135 ± 9.0

1,666 134 ± 5.0

3,333 129 ± 18.0c 106 ± 6.0c 146 ± 6.0

6,666c 135 ± 11.0

10,000c 129 ± 8.0 130 ± 5.0 137 ± 2.0

Trial summary Negative Equivocal Negative

Positive control 555 ± 19.0 576 ± 10.0 581 ± 18.0

–S9 +hamster S9

Trial 1 Trial 2 Trial 3 10% 30% 30%

TA1535 0 9 ± 2.0 13 ± 3.0 11 ± 2.0 14 ± 1.0 11 ± 1.0 8 ± 1.0

100 10 ± 2.0 12 ± 1.0 12 ± 1.0 5 ± 1.0

333 7 ± 2.0 12 ± 1.0 11 ± 1.0 8 ± 2.0

1,000 8 ± 1.0 6 ± 1.0 14 ± 2.0 13 ± 1.0 8 ± 2.0 10 ± 4.0

1,666 12 ± 4.0 9 ± 1.0

3,333 8 ± 1.0 11 ± 1.0c 10 ± 1.0c 10 ± 1.0c 8 ± 1.0 10 ± 0.0c

6,666c 6 ± 1.0 7 ± 1.0

10,000c 7 ± 1.0 15 ± 1.0 13 ± 4.0 9 ± 1.0 7 ± 2.0 9 ± 1.0

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 909 ± 33.0 784 ± 26.0 1,107 ± 42.0 81 ± 6.0 99 ± 8.0 86 ± 8.0
E-10 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft

TABLE E3

Mutagenicity of Chromium Picolinate in Salmonella typhimurium

Revertants/Plate

Dose +rat S9

Strain (μg/plate) 10% 30% 30%

Study performed at SRI International (continued)

TA1535 0 13 ± 2.0 14 ± 2.0 13 ± 1.0

(continued) 100 9 ± 1.0 11 ± 2.0

333 10 ± 2.0 13 ± 4.0

1,000 10 ± 2.0 10 ± 3.0 10 ± 2.0

1,666 11 ± 1.0

3,333 15 ± 3.0c 9 ± 0.0 13 ± 2.0c

6,666c 9 ± 1.0

10,000c 8 ± 0.0 10 ± 2.0 11 ± 2.0

Trial summary Negative Negative Negative

Positive control 79 ± 3.0 105 ± 4.0 80 ± 4.0

–S9 +hamster S9 +rat S9

Trial 1 Trial 2 10% 30% 10% 30%

TA97 0 144 ± 9.0 135 ± 5.0 133 ± 2.0 174 ± 4.0 159 ± 11.0 206 ± 11.0

100 156 ± 8.0 137 ± 9.0 141 ± 9.0 181 ± 5.0 156 ± 5.0 206 ± 14.0

333 163 ± 5.0 137 ± 3.0 153 ± 16.0 179 ± 6.0 154 ± 11.0 205 ± 14.0

1,000 164 ± 7.0 151 ± 8.0 132 ± 13.0 174 ± 3.0 138 ± 4.0 197 ± 20.0

3,333c 159 ± 8.0 134 ± 5.0 141 ± 15.0 188 ± 7.0 152 ± 12.0 192 ± 11.0

10,000c 147 ± 5.0 125 ± 9.0 149 ± 6.0 174 ± 8.0 152 ± 7.0 202 ± 11.0

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 625 ± 14.0 490 ± 23.0 494 ± 41.0 582 ± 20.0 522 ± 6.0 521 ± 12.0

TA98 0 26 ± 3.0 13 ± 2.0 20 ± 5.0 35 ± 3.0 22 ± 2.0 35 ± 2.0

100 30 ± 3.0 14 ± 2.0 21 ± 2.0 36 ± 5.0 24 ± 3.0 33 ± 0.0

333 29 ± 5.0 15 ± 3.0 20 ± 6.0 29 ± 2.0 20 ± 3.0 40 ± 2.0

1,000 33 ± 6.0 20 ± 3.0 29 ± 3.0 38 ± 3.0 18 ± 3.0 33 ± 3.0

3,333c 26 ± 3.0 18 ± 2.0 21 ± 4.0 38 ± 1.0 21 ± 3.0 40 ± 4.0

10,000c 27 ± 5.0 19 ± 4.0 22 ± 2.0 37 ± 4.0 19 ± 5.0 36 ± 3.0

Trial summary Negative Negative Negative Negative Negative Negative

Positive control 346 ± 21.0 515 ± 33.0 439 ± 28.0 556 ± 24.0 243 ± 23.0 427 ± 34.0

a The detailed protocol is presented by Zeiger et al. (1992). 0 μg/plate was the solvent control. b Revertants are presented as mean ± standard error from three plates. c Precipitate on plate(s) d The positive controls in the absence of metabolic activation were sodium azide (TA100 and TA1535), 9-aminoacridine (TA97), 

4-nitro-o-phenylenediamine (TA98), mitomycin-C (TA102), and methyl methanesulfonate (TA104). The positive control for metabolic 

activation with all strains was 2-aminoanthracene, and 2-aminoanthracene or sterigmatocystin was used for TA102. 
NOT FOR DISTRIBUTION OR ATTRIBUTION

TABLE E4

Induction of Micronuclei in Bone Marrow Polychromatic Erythrocytes 

of Male Rats Treated with Chromium Picolinate by Gavagea

Number of Rats

Compound Dose with Erythrocytes Micronucleated PCEs/ P Valuec PCEsb (%)

(mg/kg) Scored 1,000 PCEsb

Corn oild 5 0.40 ± 0.19 53.5 ± 1.4

Chromium picolinate 156 5 0.50 ± 0.16 0.3694 54.3 ± 2.0

312 5 0.80 ± 0.12 0.1240 51.2 ± 1.0

625 5 0.50 ± 0.16 0.3694 53.5 ± 0.5

1,250 5 0.60 ± 0.10 0.2635 50.9 ± 1.9

2,500 5 0.60 ± 0.24 0.2635 51.7 ± 1.7

P=0.385e

Cyclophosphamidef 8 5 22.30 ± 1.03 0.0000

a Study was performed at SITEK Research Laboratories. The detailed protocol is presented by Shelby et al. (1993). 

PCE=polychromatic erythrocyte b Mean ± standard error c Pairwise comparison with the vehicle control group. Dosed group values significant at P=0.005; positive control value significant 

at P#0.05 (ILS, 1990) d Vehicle control e Significance of micronucleated PCEs/1,000 PCEs tested by the one-tailed trend test, significant at P#0.025 (ILS, 1990) f Positive control

Chromium Picolinate Monohydrate, NTP TR 556 E-11

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E-12 Chromium Picolinate Monohydrate, NTP TR 556 

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F-1

Board Draft 

APPENDIX F

CLINICAL PATHOLOGY RESULTS

TABLE F1 Hematology and Clinical Chemistry Data for Rats 

in the 3-Month Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . F-2

TABLE F2 Hematology Data for Mice in the 3-Month Feed Study

of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-8
F-2

TABLE F1

Hematology and Clinical Chemistry Data for Rats in the 3-Month Feed Study 

of Chromium Picolinate Monohydratea

0 ppm 80 ppm 240 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Male

Hematology

n

Day 3 10 9 10 9 10 9

Day 21 10 10 10 8 9 10

Week 14 10 10 10 10 10 9

Hematocrit (auto) (%)

Day 3 43.7 ± 0.7 43.2 ± 0.5 44.6 ± 0.9 44.0 ± 1.1 44.4 ± 0.7 44.7 ± 0.6

Day 21 46.9 ± 0.7 46.6 ± 0.3 48.0 ± 0.6 48.5 ± 0.6 47.9 ± 0.5 48.2 ± 0.6

Week 14 46.2 ± 0.4 46.4 ± 0.4 46.5 ± 0.3 45.4 ± 1.1 47.2 ± 0.7 46.7 ± 0.4

Hematocrit (spun) (%)

Day 3 44.4 ± 0.7 44.3 ± 0.5 45.0 ± 0.8 44.5 ± 1.2 45.2 ± 0.7 45.4 ± 0.5

Day 21 46.3 ± 0.7 46.0 ± 0.3 47.5 ± 0.5 47.8 ± 0.6 47.5 ± 0.4 48.0 ± 0.5*

Week 14 45.5 ± 0.3 45.5 ± 0.2 45.8 ± 0.3 44.5 ± 1.0 45.9 ± 0.8 46.2 ± 0.5

Hemoglobin (g/dL)

Day 3 14.9 ± 0.2 14.8 ± 0.2 15.1 ± 0.3 14.8 ± 0.3 15.0 ± 0.3 15.2 ± 0.3

Day 21 15.7 ± 0.2 15.6 ± 0.1 16.1 ± 0.2 16.1 ± 0.2 16.1 ± 0.1 16.1 ± 0.1

Week 14 15.5 ± 0.1 15.4 ± 0.1 15.7 ± 0.1 15.1 ± 0.4 15.7 ± 0.3 15.6 ± 0.2

Erythrocytes (106

/μL)

Day 3 7.12 ± 0.10 7.09 ± 0.08 7.27 ± 0.13 7.08 ± 0.20 7.20 ± 0.11 7.31 ± 0.13

Day 21 7.58 ± 0.10 7.52 ± 0.06 7.71 ± 0.08 7.70 ± 0.12 7.74 ± 0.09 7.75 ± 0.09

Week 14 8.99 ± 0.07 9.03 ± 0.08 9.07 ± 0.07 8.80 ± 0.22 9.15 ± 0.14 9.08 ± 0.07

Reticulocytes (106/μL)

Day 3 6.32 ± 0.27 6.16 ± 0.28 6.39 ± 0.17 6.61 ± 0.27 6.70 ± 0.28 6.41 ± 0.32

Day 21 3.55 ± 0.15 3.32 ± 0.12 3.30 ± 0.07 3.37 ± 0.17 3.06 ± 0.14 3.35 ± 0.27

Week 14 2.09 ± 0.05 2.29 ± 0.08 2.12 ± 0.07 2.35 ± 0.14* 2.51 ± 0.24* 2.38 ± 0.13*

Reticulocytes (%)

Day 3 8.84 ± 0.31 8.71 ± 0.42 8.84 ± 0.32 9.30 ± 0.18 9.33 ± 0.37 8.81 ± 0.53

Day 21 4.68 ± 0.19 4.42 ± 0.18 4.27 ± 0.09 4.38 ± 0.21 3.96 ± 0.19 4.33 ± 0.36

Week 14 2.32 ± 0.07 2.55 ± 0.09 2.34 ± 0.09 2.73 ± 0.27 2.76 ± 0.29 2.63 ± 0.15

Nucleated erythrocytes/100 leukocytes

Day 3 0.30 ± 0.21 0.22 ± 0.15 0.30 ± 0.21 0.44 ± 0.18 0.30 ± 0.15 0.11 ± 0.11

Day 21 0.30 ± 0.15 0.00 ± 0.00 0.30 ± 0.15 0.38 ± 0.26 0.11 ± 0.11 0.40 ± 0.22

Week 14 0.10 ± 0.10 0.30 ± 0.15 0.00 ± 0.00 0.20 ± 0.13 0.00 ± 0.00 0.11 ± 0.11

Mean cell volume (fL)

Day 3 61.3 ± 0.3 61.0 ± 0.3 61.4 ± 0.3 62.1 ± 0.4 61.6 ± 0.4 61.1 ± 0.4

Day 21 61.9 ± 0.4 62.0 ± 0.4 62.2 ± 0.3 63.1 ± 0.4 61.9 ± 0.5 62.3 ± 0.5

Week 14 51.4 ± 0.2 51.4 ± 0.2 51.3 ± 0.1 51.6 ± 0.2 51.6 ± 0.2 51.5 ± 0.2

Mean cell hemoglobin (pg)

Day 3 20.9 ± 0.1 20.9 ± 0.1 20.7 ± 0.1 21.0 ± 0.2 20.8 ± 0.1 20.8 ± 0.1

Day 21 20.8 ± 0.1 20.8 ± 0.1 20.8 ± 0.1 20.9 ± 0.2 20.8 ± 0.2 20.8 ± 0.2

Week 14 17.3 ± 0.1 17.1 ± 0.1 17.3 ± 0.1 17.2 ± 0.1 17.1 ± 0.1 17.1 ± 0.1

Mean cell hemoglobin concentration (g/dL)

Day 3 34.1 ± 0.1 34.2 ± 0.2 33.8 ± 0.1 33.7 ± 0.2 33.8 ± 0.2 34.0 ± 0.2

Day 21 33.6 ± 0.2 33.5 ± 0.2 33.5 ± 0.2 33.1 ± 0.1 33.6 ± 0.2 33.5 ± 0.3

Week 14 33.6 ± 0.2 33.3 ± 0.2 33.7 ± 0.2 33.3 ± 0.1 33.2 ± 0.2 33.3 ± 0.1

Platelets (103/μL)

Day 3 893.6 ± 45.2 840.0 ± 29.6 829.2 ± 34.3 946.7 ± 24.1 892.3 ± 18.9 918.7 ± 22.4

Day 21 679.9 ± 37.6 772.5 ± 24.9 789.7 ± 22.1 771.4 ± 38.2 791.9 ± 23.6 761.1 ± 13.2

Week 14 539.8 ± 24.8 554.6 ± 17.4 534.9 ± 10.4 539.1 ± 18.4 548.1 ± 18.4 577.4 ± 16.4

Chromium Picolinate Monohydrate, NTP TR 556 

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Chromium Picolinate Monohydrate, NTP TR 556 F-3

TABLE F1

Hematology and Clinical Chemistry Data for Rats in the 3-Month Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 80 ppm 240 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Male (continued)

Hematology (continued)

n

Day 3 10 9 10 9 10 9

Day 21 10 10 10 8 9 10

Week 14 10 10 10 10 10 9

Leukocytes (103/μL)

Day 3 8.86 ± 0.24 8.92 ± 0.38 8.97 ± 0.25 8.70 ± 0.47 8.56 ± 0.24 9.26 ± 0.45

Day 21 10.00 ± 0.35 9.73 ± 0.30 10.82 ± 0.26 10.03 ± 0.28 10.25 ± 0.35 10.91 ± 1.47

Week 14 8.74 ± 0.44 8.73 ± 0.45 9.57 ± 0.36 10.10 ± 0.54 9.66 ± 0.39 9.01 ± 0.48

Segmented neutrophils (103/μL)

Day 3 1.08 ± 0.05 0.97 ± 0.03 1.04 ± 0.03 1.02 ± 0.07 1.03 ± 0.03 1.21 ± 0.10

Day 21 1.18 ± 0.08 1.01 ± 0.05 1.00 ± 0.05 0.96 ± 0.04 1.08 ± 0.06 1.95 ± 0.97

Week 14 1.11 ± 0.07 1.32 ± 0.07 1.22 ± 0.05 1.29 ± 0.12 1.15 ± 0.07 1.23 ± 0.11

Lymphocytes (103/μL)

Day 3 7.36 ± 0.19 7.53 ± 0.35 7.52 ± 0.23 7.24 ± 0.40 7.14 ± 0.20 7.61 ± 0.48

Day 21 8.41 ± 0.32 8.33 ± 0.25 9.37 ± 0.23 8.68 ± 0.27 8.74 ± 0.31 8.49 ± 0.47

Week 14 7.14 ± 0.39 6.86 ± 0.39 7.80 ± 0.34 8.21 ± 0.45 7.99 ± 0.36 7.25 ± 0.40

Activated lymphocytes (103/μL)

Day 3 0.10 ± 0.01 0.10 ± 0.01 0.09 ± 0.01 0.10 ± 0.01 0.09 ± 0.01 0.12 ± 0.02

Day 21 0.09 ± 0.01 0.09 ± 0.01 0.11 ± 0.01 0.09 ± 0.01 0.10 ± 0.02 0.10 ± 0.02

Week 14 0.21 ± 0.02 0.25 ± 0.05 0.24 ± 0.02 0.28 ± 0.02* 0.22 ± 0.03 0.23 ± 0.02

Monocytes (103/μL)

Day 3 0.25 ± 0.02 0.22 ± 0.02 0.24 ± 0.01 0.25 ± 0.02 0.22 ± 0.01 0.23 ± 0.02

Day 21 0.22 ± 0.01 0.21 ± 0.01 0.24 ± 0.02 0.19 ± 0.01 0.22 ± 0.02 0.26 ± 0.08

Week 14 0.08 ± 0.01 0.10 ± 0.01 0.10 ± 0.01 0.12 ± 0.01 0.09 ± 0.01 0.09 ± 0.01

Basophils (103/μL)

Day 3 0.049 ± 0.005 0.062 ± 0.009 0.047 ± 0.004 0.066 ± 0.009 0.055 ± 0.005 0.061 ± 0.010

Day 21 0.056 ± 0.005 0.064 ± 0.006 0.071 ± 0.005 0.070 ± 0.006 0.078 ± 0.008 0.076 ± 0.012

Week 14 0.038 ± 0.004 0.046 ± 0.003 0.052 ± 0.003 0.050 ± 0.003 0.050 ± 0.005 0.047 ± 0.006

Eosinophils (103/μL)

Day 3 0.03 ± 0.00 0.02 ± 0.00 0.03 ± 0.00 0.03 ± 0.00 0.03 ± 0.00 0.03 ± 0.00

Day 21 0.04 ± 0.01 0.03 ± 0.00* 0.03 ± 0.00 0.03 ± 0.00 0.03 ± 0.00 0.04 ± 0.01

Week 14 0.16 ± 0.01 0.16 ± 0.02 0.17 ± 0.01 0.16 ± 0.01 0.17 ± 0.01 0.15 ± 0.01

Clinical Chemistry

n

Day 3 10 10 10 10 10 10

Day 21 10 10 10 8 9 10

Week 14 10 10 10 10 10 10

Urea nitrogen (mg/dL)

Day 3 13.2 ± 0.4 13.9 ± 0.5 14.1 ± 0.5 14.0 ± 0.4 14.4 ± 0.4 14.5 ± 0.5

Day 21 15.4 ± 0.7 14.0 ± 0.5 14.5 ± 0.5 12.8 ± 0.4* 15.2 ± 0.4 12.8 ± 0.6*

Week 14 16.3 ± 0.5 14.1 ± 0.5 16.0 ± 0.5 16.2 ± 0.6 14.8 ± 0.9 15.2 ± 0.7

Creatinine (mg/dL)

Day 3 0.51 ± 0.02 0.51 ± 0.01 0.51 ± 0.02 0.52 ± 0.01 0.50 ± 0.00 0.49 ± 0.02

Day 21 0.66 ± 0.02 0.66 ± 0.02 0.68 ± 0.01 0.68 ± 0.02 0.64 ± 0.03 0.64 ± 0.02

Week 14 0.65 ± 0.02 0.67 ± 0.02 0.67 ± 0.03 0.68 ± 0.02 0.70 ± 0.03 0.66 ± 0.03

Board Draft 
F-4

TABLE F1

Hematology and Clinical Chemistry Data for Rats in the 3-Month Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 80 ppm 240 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Male (continued)

Clinical Chemistry (continued)

n

Day 3 10 10 10 10 10 10

Day 21 10 10 10 8 9 10

Week 14 10 10 10 10 10 10

Total protein (g/dL)

Day 3 5.7 ± 0.1 5.6 ± 0.1 5.6 ± 0.1 5.6 ± 0.0 5.7 ± 0.1 5.7 ± 0.1

Day 21 6.4 ± 0.1 6.3 ± 0.0 6.4 ± 0.1 6.5 ± 0.1 6.4 ± 0.0 6.2 ± 0.1

Week 14 6.6 ± 0.1 6.7 ± 0.1 6.6 ± 0.1 6.5 ± 0.1 6.5 ± 0.1 6.4 ± 0.1

Albumin (g/dL)

Day 3 3.9 ± 0.1 3.9 ± 0.0 3.9 ± 0.1 3.9 ± 0.0 3.8 ± 0.1 4.0 ± 0.1

Day 21 4.3 ± 0.1 4.2 ± 0.0 4.3 ± 0.1 4.3 ± 0.0 4.2 ± 0.0 4.1 ± 0.1

Week 14 4.2 ± 0.0 4.3 ± 0.1 4.2 ± 0.0 4.2 ± 0.0 4.1 ± 0.1 4.2 ± 0.0

Alanine aminotransferase (IU/L)

Day 3 69 ± 1 57 ± 1** 58 ± 1** 61 ± 1 61 ± 2* 64 ± 3

Day 21 55 ± 2 44 ± 1** 45 ± 1** 47 ± 1 47 ± 1 46 ± 3

Week 14 138 ± 19 103 ± 13 152 ± 12 163 ± 21 137 ± 11 155 ± 22

Alkaline phosphate (IU/L)

Day 3 716 ± 16 666 ± 21 699 ± 18 715 ± 18 693 ± 17 648 ± 39

Day 21 472 ± 15 496 ± 13 508 ± 14 528 ± 13 491 ± 8 519 ± 32**

Week 14 201 ± 5 182 ± 8 198 ± 6 195 ± 7 190 ± 4 205 ± 9

Creatine kinase (IU/L)

Day 3 344 ± 38 322 ± 43 310 ± 37 369 ± 43 353 ± 63 376 ± 46

Day 21 332 ± 53 367 ± 62 289 ± 24 361 ± 40 333 ± 42 428 ± 61

Week 14 251 ± 63 230 ± 33 229 ± 40 355 ± 125 256 ± 67 259 ± 59

Sorbitol dehydrogenase (IU/L)

Day 3 16 ± 1b 14 ± 1b 16 ± 0b 17 ± 1b 15 ± 1 16 ± 1

Day 21 22 ± 1 26 ± 2 24 ± 2 27 ± 1 23 ± 1 28 ± 2

Week 14 31 ± 3 29 ± 3 33 ± 3 38 ± 4 33 ± 2 34 ± 3

Bile acids (μmol/L)

Day 3 23.1 ± 1.4 22.5 ± 1.6 20.5 ± 1.0 23.2 ± 2.3 25.9 ± 2.4 19.8 ± 1.3

Day 21 19.6 ± 1.7 28.2 ± 1.7** 22.9 ± 1.5 24.3 ± 2.4 23.7 ± 3.0 24.2 ± 1.1

Week 14 23.5 ± 1.3 25.8 ± 2.3 23.1 ± 1.0 32.3 ± 3.7 25.6 ± 3.2 28.1 ± 2.9

Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft
NOT FOR DISTRIBUTION OR ATTRIBUTION

Chromium Picolinate Monohydrate, NTP TR 556 F-5

TABLE F1

Hematology and Clinical Chemistry Data for Rats in the 3-Month Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 80 ppm 240 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Female

Hematology

n

Day 3 10 10 10 10 10 10

Day 21 10 10 10 9 10 10

Week 14 10 10 10 10 10 10

Hematocrit (auto) (%)

Day 3 45.2 ± 0.7 45.8 ± 0.8 45.4 ± 0.9 48.3 ± 1.2 46.9 ± 1.0 46.8 ± 1.0

Day 21 47.1 ± 0.6 46.6 ± 0.5 47.9 ± 0.4 47.5 ± 0.7 48.5 ± 0.9 47.8 ± 0.5

Week 14 45.5 ± 0.4 45.3 ± 0.4 45.5 ± 0.3 44.6 ± 0.5 46.0 ± 0.2 45.3 ± 0.3

Hematocrit (spun) (%)

Day 3 44.5 ± 0.7 44.7 ± 0.7 44.7 ± 0.7 47.2 ± 1.1 46.5 ± 1.0 45.7 ± 1.0

Day 21 47.8 ± 0.5 47.0 ± 0.5 48.3 ± 0.4 47.8 ± 0.7 48.8 ± 0.9 47.9 ± 0.4

Week 14 45.8 ± 0.4 45.5 ± 0.5 45.9 ± 0.2 44.6 ± 0.4 46.4 ± 0.4 45.2 ± 0.4

Hemoglobin (g/dL)

Day 3 15.0 ± 0.2 15.1 ± 0.3 15.1 ± 0.3 16.0 ± 0.4 15.7 ± 0.3 15.5 ± 0.3

Day 21 15.9 ± 0.2 15.7 ± 0.1 16.1 ± 0.2 15.8 ± 0.2 16.1 ± 0.3 16.0 ± 0.1

Week 14 15.2 ± 0.1 15.0 ± 0.1 15.1 ± 0.1 14.8 ± 0.2 15.3 ± 0.1 15.0 ± 0.1

Erythrocytes (106/μL)

Day 3 7.29 ± 0.13 7.35 ± 0.13 7.24 ± 0.15 7.80 ± 0.20 7.54 ± 0.17 7.50 ± 0.17

Day 21 7.66 ± 0.11 7.51 ± 0.06 7.72 ± 0.10 7.64 ± 0.14 7.75 ± 0.14 7.70 ± 0.09

Week 14 8.48 ± 0.07 8.46 ± 0.09 8.45 ± 0.06 8.22 ± 0.13 8.59 ± 0.04 8.44 ± 0.06

Reticulocytes (106/μL)

Day 3 5.49 ± 0.27 5.38 ± 0.24 5.46 ± 0.34 5.66 ± 0.26 5.84 ± 0.31 5.79 ± 0.18

Day 21 2.13 ± 0.08 2.04 ± 0.06 2.01 ± 0.08 2.12 ± 0.13 2.07 ± 0.06 2.04 ± 0.08

Week 14 1.67 ± 0.04 1.63 ± 0.07 1.54 ± 0.06 1.83 ± 0.22 1.64 ± 0.06 1.75 ± 0.07

Reticulocytes (%)

Day 3 7.60 ± 0.46 7.33 ± 0.35 7.61 ± 0.54 7.29 ± 0.35 7.78 ± 0.42 7.76 ± 0.34

Day 21 2.78 ± 0.13 2.71 ± 0.09 2.63 ± 0.12 2.79 ± 0.17 2.67 ± 0.10 2.65 ± 0.11

Week 14 1.96 ± 0.05 1.90 ± 0.07 1.81 ± 0.07 2.26 ± 0.34 1.90 ± 0.08 2.08 ± 0.08

Nucleated erythrocytes/100 leukocytes

Day 3 1.10 ± 0.35 0.80 ± 0.29 0.70 ± 0.34 0.80 ± 0.33 1.10 ± 0.41 0.70 ± 0.34

Day 21 0.20 ± 0.13 0.20 ± 0.13 0.00 ± 0.00 0.11 ± 0.11 0.10 ± 0.10 0.10 ± 0.10

Week 14 0.20 ± 0.13 0.10 ± 0.10 0.00 ± 0.00 0.10 ± 0.10 0.00 ± 0.00 0.00 ± 0.00

Mean cell volume (fL)

Day 3 62.0 ± 0.4 62.3 ± 0.3 62.7 ± 0.3 61.9 ± 0.3 62.3 ± 0.3 62.5 ± 0.3

Day 21 61.5 ± 0.4 62.1 ± 0.3 62.1 ± 0.3 62.3 ± 0.3 62.7 ± 0.2 62.1 ± 0.3

Week 14 53.7 ± 0.1 53.5 ± 0.1 53.8 ± 0.1 54.3 ± 0.5 53.5 ± 0.1 53.7 ± 0.1

Mean cell hemoglobin (pg)

Day 3 20.7 ± 0.1 20.6 ± 0.1 20.8 ± 0.1 20.5 ± 0.1 20.8 ± 0.1 20.7 ± 0.1

Day 21 20.7 ± 0.1 20.9 ± 0.1 20.8 ± 0.1 20.7 ± 0.2 20.8 ± 0.1 20.8 ± 0.2

Week 14 17.9 ± 0.1 17.8 ± 0.1 17.9 ± 0.0 18.0 ± 0.1 17.8 ± 0.1 17.8 ± 0.0

Mean cell hemoglobin concentration (g/dL)

Day 3 33.3 ± 0.2 33.1 ± 0.2 33.2 ± 0.1 33.1 ± 0.2 33.4 ± 0.2 33.2 ± 0.1

Day 21 33.6 ± 0.1 33.6 ± 0.1 33.5 ± 0.1 33.3 ± 0.2 33.1 ± 0.1 33.5 ± 0.2

Week 14 33.3 ± 0.1 33.2 ± 0.1 33.3 ± 0.1 33.2 ± 0.1 33.2 ± 0.1 33.2 ± 0.1

Platelets (103/μL)

Day 3 851.1 ± 26.7 825.0 ± 35.1 840.3 ± 38.2 790.5 ± 34.0 833.0 ± 22.2 803.8 ± 31.7

Day 21 718.3 ± 17.1 745.1 ± 18.5 780.3 ± 23.2 760.6 ± 26.2 755.0 ± 21.2 703.5 ± 31.2

Week 14 564.3 ± 11.0 585.4 ± 17.3 576.9 ± 15.9 567.9 ± 31.0 559.0 ± 20.3 586.8 ± 25.9

Board Draft 
F-6

TABLE F1

Hematology and Clinical Chemistry Data for Rats in the 3-Month Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 80 ppm 240 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Female (continued)

n

Day 3 10 10 10 10 10 10

Day 21 10 10 10 9 10 10

Week 14 10 10 10 10 10 10

Hematology (continued)

Leukocytes (103/μL)

Day 3 9.70 ± 0.43 9.23 ± 0.35 9.05 ± 0.49 9.31 ± 0.49 9.16 ± 0.36 9.56 ± 0.40

Day 21 9.67 ± 0.36 10.40 ± 0.28 10.53 ± 0.46 10.58 ± 0.54 10.34 ± 0.35 10.45 ± 0.48

Week 14 8.24 ± 0.30 8.79 ± 0.40 9.54 ± 0.47 9.31 ± 0.39 9.54 ± 0.48 8.87 ± 0.52

Segmented neutrophils (103/μL)

Day 3 1.06 ± 0.10 0.98 ± 0.05 0.96 ± 0.05 0.94 ± 0.04 1.00 ± 0.08 1.06 ± 0.06

Day 21 0.95 ± 0.09 0.81 ± 0.05 0.92 ± 0.07 0.92 ± 0.06 0.98 ± 0.05 1.01 ± 0.08

Week 14 1.09 ± 0.06 1.17 ± 0.07 1.20 ± 0.09 1.20 ± 0.06 1.36 ± 0.08 1.08 ± 0.07

Lymphocytes (103/μL)

Day 3 8.20 ± 0.38 7.83 ± 0.30 7.67 ± 0.45 7.98 ± 0.45 7.74 ± 0.32 8.10 ± 0.39

Day 21 8.28 ± 0.29 9.10 ± 0.26 9.10 ± 0.37 9.24 ± 0.53 8.86 ± 0.33 8.97 ± 0.42

Week 14 6.84 ± 0.27 7.30 ± 0.36 7.97 ± 0.40 7.78 ± 0.35 7.79 ± 0.40 7.45 ± 0.46

Activated lymphocytes (103/μL)

Day 3 0.11 ± 0.01 0.09 ± 0.01 0.10 ± 0.01 0.09 ± 0.01 0.09 ± 0.01 0.08 ± 0.01

Day 21 0.11 ± 0.01 0.12 ± 0.02 0.13 ± 0.02 0.10 ± 0.01 0.13 ± 0.01 0.11 ± 0.01

Week 14 0.07 ± 0.01 0.07 ± 0.01 0.09 ± 0.00 0.08 ± 0.01 0.08 ± 0.01 0.08 ± 0.01

Monocytes (103/μL)

Day 3 0.25 ± 0.02 0.23 ± 0.02 0.24 ± 0.03 0.22 ± 0.02 0.25 ± 0.02 0.22 ± 0.02

Day 21 0.22 ± 0.02 0.25 ± 0.01 0.24 ± 0.02 0.20 ± 0.01 0.27 ± 0.02 0.25 ± 0.03

Week 14 0.14 ± 0.01 0.16 ± 0.01 0.17 ± 0.01 0.15 ± 0.01 0.18 ± 0.02 0.13 ± 0.02

Basophils (103/μL)

Day 3 0.061 ± 0.007 0.057 ± 0.014 0.058 ± 0.008 0.051 ± 0.006 0.044 ± 0.005 0.050 ± 0.004

Day 21 0.054 ± 0.005 0.072 ± 0.007 0.071 ± 0.008 0.074 ± 0.009 0.067 ± 0.008 0.065 ± 0.006

Week 14 0.055 ± 0.011 0.046 ± 0.003 0.058 ± 0.007 0.061 ± 0.008 0.072 ± 0.007 0.071 ± 0.011

Eosinophils (103/μL)

Day 3 0.04 ± 0.00 0.04 ± 0.01 0.04 ± 0.01 0.04 ± 0.00 0.03 ± 0.00 0.04 ± 0.00

Day 21 0.05 ± 0.01 0.06 ± 0.01 0.06 ± 0.01 0.05 ± 0.01 0.04 ± 0.00 0.05 ± 0.00

Week 14 0.05 ± 0.00 0.06 ± 0.01 0.05 ± 0.00 0.05 ± 0.00 0.07 ± 0.00* 0.05 ± 0.00

Chemistry Data

Urea nitrogen (mg/dL)

Day 3 13.8 ± 0.6 14.4 ± 0.6 14.8 ± 0.6 16.7 ± 1.1 16.0 ± 0.6 15.4 ± 0.5

Day 21 17.5 ± 0.5 16.7 ± 0.6 17.2 ± 0.9 15.6 ± 0.5 16.7 ± 0.3 16.9 ± 0.3

Week 14 13.5 ± 0.5 15.2 ± 0.6 14.6 ± 0.5 13.1 ± 0.4 14.1 ± 0.8 13.5 ± 0.4

Creatinine (mg/dL)

Day 3 0.50 ± 0.00 0.49 ± 0.01 0.49 ± 0.01 0.48 ± 0.01 0.50 ± 0.02 0.46 ± 0.02

Day 21 0.50 ± 0.02 0.51 ± 0.01 0.51 ± 0.01 0.49 ± 0.01 0.50 ± 0.00 0.51 ± 0.01

Week 14 0.62 ± 0.01 0.61 ± 0.01 0.59 ± 0.02 0.60 ± 0.02 0.62 ± 0.01 0.58 ± 0.03

Total Protein (g/dL)

Day 3 5.6 ± 0.1 5.6 ± 0.1 5.7 ± 0.1 6.0 ± 0.2 5.8 ± 0.1 5.7 ± 0.1

Day 21 6.1 ± 0.1 6.0 ± 0.1 6.2 ± 0.1 6.0 ± 0.1 6.1 ± 0.1 6.0 ± 0.1

Week 14 6.5 ± 0.1 6.7 ± 0.1 6.6 ± 0.1 6.6 ± 0.1 6.8 ± 0.1 6.6 ± 0.1

Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft
NOT FOR DISTRIBUTION OR ATTRIBUTION

Chromium Picolinate Monohydrate, NTP TR 556 F-7

TABLE F1

Hematology and Clinical Chemistry Data for Rats in the 3-Month Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 80 ppm 240 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Female (continued)

Clinical Chemistry (continued)

n

Day 3 10 10 10 10 10 10

Day 21 10 10 10 9 10 10

Week 14 10 10 10 10 10 10

Albumin (g/dL)

Day 3 4.0 ± 0.0 4.0 ± 0.0 4.1 ± 0.1 4.3 ± 0.1 4.1 ± 0.1 4.0 ± 0.0

Day 21 4.2 ± 0.0 4.2 ± 0.0 4.3 ± 0.1 4.1 ± 0.1 4.2 ± 0.0 4.2 ± 0.0

Week 14 4.6 ± 0.1 4.7 ± 0.1 4.6 ± 0.1 4.6 ± 0.1 4.7 ± 0.0 4.6 ± 0.1

Alanine aminotransferase (IU/L)

Day 3 53 ± 1 49 ± 1 51 ± 2 47 ± 1** 48 ± 1** 48 ± 2*

Day 21 36 ± 1 31 ± 1 30 ± 0** 34 ± 1 33 ± 1 33 ± 1

Week 14 74 ± 10 82 ± 10 81 ± 13 60 ± 8 72 ± 12 65 ± 7

Alkaline phosphate (IU/L)

Day 3 591 ± 16 578 ± 12 596 ± 14 589 ± 13 574 ± 7 582 ± 9

Day 21 356 ± 5 378 ± 7 369 ± 8 399 ± 5** 372 ± 9 375 ± 9

Week 14 160 ± 5 172 ± 7 171 ± 5 164 ± 4 163 ± 6 155 ± 4

Creatine kinase (IU/L)

Day 3 468 ± 57 340 ± 31 348 ± 28 484 ± 57 413 ± 45 434 ± 50

Day 21 405 ± 40 380 ± 50 314 ± 36 323 ± 28 319 ± 30 378 ± 46

Week 14 188 ± 18 160 ± 21 183 ± 20 174 ± 29 222 ± 44 227 ± 28

Sorbitol dehydrogenase (IU/L)

Day 3 18 ± 0 19 ± 1 20 ± 1 20 ± 1 19 ± 1 18 ± 1

Day 21 16 ± 1 16 ± 1 19 ± 1 17 ± 1 19 ± 1 17 ± 1

Week 14 25 ± 2 25 ± 2 26 ± 3 25 ± 2 25 ± 2 24 ± 1

Bile acids (μmol/L)

Day 3 22.9 ± 1.4 22.1 ± 2.9 17.9 ± 1.6 21.7 ± 1.7 18.7 ± 1.7 19.3 ± 2.1

Day 21 19.0 ± 2.3 20.5 ± 1.8 14.3 ± 0.6 19.7 ± 1.7 18.4 ± 1.9 17.5 ± 1.8

Week 14 33.4 ± 3.4 34.3 ± 5.0 38.6 ± 2.6 35.1 ± 4.1 30.1 ± 2.9 36.2 ± 3.4

* Significantly different (P#0.05) from the control group by Dunn’s or Shirley’s test

** P#0.01 a Mean ± standard error. Statistical tests were performed on unrounded data. b n=9

Board Draft 
F-8 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft

TABLE F2

Hematology Data for Mice in the 3-Month Feed Study of Chromium Picolinate Monohydratea

0 ppm 80 ppm 240 ppm 2,000 ppm 10,000 ppm 50,000 ppm

n 10 10 10 10 10 10

Male

Hematocrit (auto) (%) 47.6 ± 0.5 46.9 ± 1.0 47.2 ± 0.8 46.5 ± 0.9 46.8 ± 0.8 48.1 ± 0.6

Hematocrit (spun) (%) 47.3 ± 0.7 47.0 ± 0.8 46.5 ± 0.6 46.2 ± 0.9 46.9 ± 0.9 47.7 ± 0.5

Hemoglobin (g/dL) 16.0 ± 0.1 15.8 ± 0.3 15.9 ± 0.2 15.6 ± 0.3 15.7 ± 0.3 16.1 ± 0.2

Erythrocytes (106/μL) 10.38 ± 0.10 10.17 ± 0.21 10.27 ± 0.15 10.09 ± 0.19 10.18 ± 0.16 10.44 ± 0.14

Reticulocytes (106/μL) 2.96 ± 0.09 2.97 ± 0.04 3.12 ± 0.12 2.97 ± 0.07 2.98 ± 0.08 2.97 ± 0.12

Reticulocytes (%) 2.85 ± 0.09 2.93 ± 0.06 3.04 ± 0.15 2.96 ± 0.10 2.91 ± 0.07 2.82 ± 0.11

Nucleated erythrocytes

/100 leukocytes 0.10 ± 0.10 0.00 ± 0.00 0.00 ± 0.00 0.10 ± 0.10 0.00 ± 0.00 0.10 ± 0.10

Mean cell volume (fL) 45.9 ± 0.1 46.1 ± 0.1 46.0 ± 0.3 46.1 ± 0.2 46.0 ± 0.2 46.0 ± 0.1

Mean cell 

hemoglobin (pg) 15.4 ± 0.0 15.5 ± 0.1 15.5 ± 0.1 15.4 ± 0.0 15.4 ± 0.1 15.4 ± 0.1

Mean cell hemoglobin

concentration (g/dL) 33.5 ± 0.1 33.6 ± 0.2 33.6 ± 0.2 33.4 ± 0.2 33.5 ± 0.2 33.5 ± 0.1

Platelets (103/μL) 740.9 ± 41.6 783.7 ± 30.8 804.4 ± 51.1 827.9 ± 36.1 741.8 ± 38.7 739.9 ± 70.0

Leukocytes (103/μL) 6.42 ± 0.53 5.75 ± 0.43 5.99 ± 0.64 6.52 ± 0.42 6.46 ± 0.63 5.75 ± 0.28

Segmented 

neutrophils (103/μL) 0.90 ± 0.15 0.77 ± 0.08 0.71 ± 0.08 1.06 ± 0.15 1.13 ± 0.33 0.88 ± 0.04

Lymphocytes (103/μL) 5.11 ± 0.40 4.64 ± 0.35 4.97 ± 0.57 5.13 ± 0.30 5.01 ± 0.31 4.55 ± 0.24

Activated 

lymphocytes (103/μL) 0.03 ± 0.00 0.02 ± 0.00 0.03 ± 0.01 0.02 ± 0.00 0.03 ± 0.00 0.02 ± 0.00

Monocytes (103/μL) 0.07 ± 0.01 0.06 ± 0.01 0.06 ± 0.01 0.06 ± 0.01 0.06 ± 0.01 0.06 ± 0.01

Basophils (103/μL) 0.028 ± 0.002 0.020 ± 0.003 0.026 ± 0.004 0.023 ± 0.003 0.024 ± 0.003 0.023 ± 0.004

Eosinophils (103/μL) 0.29 ± 0.02 0.23 ± 0.05 0.20 ± 0.03* 0.22 ± 0.03 0.22 ± 0.02 0.22 ± 0.02

Female

Hematocrit (auto) (%) 47.7 ± 0.5 46.0 ± 0.7 47.8 ± 0.8 48.2 ± 0.5 47.0 ± 1.1 47.2 ± 0.5

Hematocrit (spun) (%) 47.8 ± 0.4 46.4 ± 0.7 47.8 ± 0.6 49.1 ± 0.5 47.1 ± 1.0 47.9 ± 0.6

Hemoglobin (g/dL) 16.3 ± 0.2 15.6 ± 0.3 16.2 ± 0.3 16.5 ± 0.2 16.0 ± 0.4 16.1 ± 0.2

Erythrocytes (106/μL) 10.45 ± 0.11 10.02 ± 0.16 10.39 ± 0.17 10.48 ± 0.11 10.19 ± 0.27 10.32 ± 0.12

Reticulocytes (106/μL) 3.03 ± 0.19 3.46 ± 0.20 3.17 ± 0.10 3.14 ± 0.16 3.60 ± 0.32 2.82 ± 0.22

Reticulocytes (%) 2.90 ± 0.18 3.46 ± 0.19 3.06 ± 0.10 3.01 ± 0.14 3.58 ± 0.38 2.74 ± 0.18

Nucleated erythrocytes

/100 leukocytes 0.20 ± 0.13 0.30 ± 0.15 0.00 ± 0.00 0.00 ± 0.00 0.30 ± 0.15 0.00 ± 0.00

Mean cell volume (fL) 45.7 ± 0.1 45.9 ± 0.1 46.0 ± 0.1 46.0 ± 0.1 46.2 ± 0.3 45.7 ± 0.1

Mean cell 

hemoglobin (pg) 15.6 ± 0.0 15.6 ± 0.1 15.6 ± 0.0 15.7 ± 0.0 15.8 ± 0.1 15.6 ± 0.0

Mean cell hemoglobin

concentration (g/dL) 34.1 ± 0.1 33.9 ± 0.2 33.9 ± 0.1 34.1 ± 0.1 34.1 ± 0.2 34.2 ± 0.1

Platelets (103/μL) 672.8 ± 62.7 721.5 ± 58.0 679.8 ± 60.6 558.5 ± 44.4 702.8 ± 58.0 664.2 ± 44.5

Leukocytes (103/μL) 5.85 ± 0.60 6.11 ± 0.40 6.18 ± 0.29 6.26 ± 0.32 6.03 ± 0.55 5.52 ± 0.46

Segmented 

neutrophils (103/μL) 0.68 ± 0.06 0.78 ± 0.06 0.68 ± 0.07 0.82 ± 0.10 0.88 ± 0.14 0.67 ± 0.09

Lymphocytes (103/μL) 4.87 ± 0.52 5.02 ± 0.34 5.15 ± 0.26 5.10 ± 0.22 4.85 ± 0.48 4.60 ± 0.39

Activated 

lymphocytes (103/μL) 0.03 ± 0.01 0.03 ± 0.00 0.03 ± 0.00 0.03 ± 0.00 0.03 ± 0.01 0.02 ± 0.00

Monocytes (103/μL) 0.05 ± 0.01 0.06 ± 0.01 0.07 ± 0.01 0.06 ± 0.01 0.06 ± 0.01 0.06 ± 0.01

Basophils (103/μL) 0.021 ± 0.002 0.023 ± 0.003 0.024 ± 0.003 0.025 ± 0.002 0.027 ± 0.003 0.020 ± 0.003

Eosinophils (103/μL) 0.17 ± 0.01 0.21 ± 0.04 0.22 ± 0.03 0.22 ± 0.04 0.19 ± 0.03 0.15 ± 0.01

* Significantly different (P#0.05) from the control group by Dunn’s test a Mean ± standard error. Statistical tests were performed on unrounded data.
NOT FOR DISTRIBUTION OR ATTRIBUTION

G-1

Board Draft 

APPENDIX G

ORGAN WEIGHTS

AND ORGAN-WEIGHT-TO-BODY-WEIGHT RATIOS

TABLE G1 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats

in the 3-Month Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . G-2

TABLE G2 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice

in the 3-Month Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . G-3
Board Draft 

G-2 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION

TABLE G1

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 3-Month Feed Study 

of Chromium Picolinate Monohydratea

0 ppm 80 ppm 240 ppm 2,000 ppm 10,000 ppm 50,000 ppm

n 10 10 10 10 10 10

Male

Necropsy body wt 326 ± 8 328 ± 2 323 ± 4 329 ± 6 319 ± 5 316 ± 9

Heart

Absolute 0.851 ± 0.018 0.848 ± 0.016 0.811 ± 0.012 0.832 ± 0.018 0.825 ± 0.018 0.784 ± 0.026

Relative 2.616 ± 0.029 2.587 ± 0.045 2.511 ± 0.033 2.527 ± 0.033 2.587 ± 0.059 2.481 ± 0.023*

R. Kidney

Absolute 0.933 ± 0.028 0.971 ± 0.018 0.932 ± 0.017 0.950 ± 0.022 0.939 ± 0.019 0.947 ± 0.029

Relative 2.867 ± 0.066 2.963 ± 0.057 2.885 ± 0.044 2.884 ± 0.032 2.941 ± 0.028 3.001 ± 0.039

Liver

Absolute 10.363 ± 0.276 10.541 ± 0.242 10.451 ± 0.232 10.533 ± 0.277 9.850 ± 0.242 9.780 ± 0.291

Relative 31.807 ± 0.263 32.167 ± 0.746 32.321 ± 0.449 31.976 ± 0.568 30.831 ± 0.443 31.030 ± 0.605

Lung

Absolute 1.693 ± 0.105 1.864 ± 0.093 1.654 ± 0.083 1.748 ± 0.064 1.706 ± 0.106 1.661 ± 0.085

Relative 5.193 ± 0.297 5.685 ± 0.276 5.117 ± 0.249 5.323 ± 0.210 5.374 ± 0.380 5.255 ± 0.215

R. Testis

Absolute 1.397 ± 0.032 1.361 ± 0.022 1.388 ± 0.018 1.336 ± 0.035 1.333 ± 0.029 1.357 ± 0.036

Relative 4.294 ± 0.067 4.153 ± 0.057 4.298 ± 0.061 4.060 ± 0.100 4.176 ± 0.072 4.316 ± 0.115

Thymus

Absolute 0.251 ± 0.010 0.227 ± 0.008 0.236 ± 0.010 0.225 ± 0.011 0.242 ± 0.008 0.219 ± 0.011

Relative 0.771 ± 0.025 0.693 ± 0.022 0.729 ± 0.031 0.685 ± 0.030 0.758 ± 0.023 0.690 ± 0.027

Females

Necropsy body wt 190 ± 4 189 ± 4 189 ± 3 190 ± 4 189 ± 4 190 ± 3

Heart

Absolute 0.547 ± 0.013 0.552 ± 0.010 0.548 ± 0.010 0.563 ± 0.016 0.552 ± 0.010 0.562 ± 0.014

Relative 2.889 ± 0.056 2.930 ± 0.034 2.911 ± 0.049 2.962 ± 0.063 2.931 ± 0.028 2.962 ± 0.059

R. Kidney

Absolute 0.571 ± 0.009 0.627 ± 0.019* 0.608 ± 0.012* 0.634 ± 0.013** 0.644 ± 0.018** 0.630 ± 0.016**

Relative 3.019 ± 0.047 3.323 ± 0.063** 3.227 ± 0.045** 3.340 ± 0.058** 3.414 ± 0.046** 3.316 ± 0.050**

Liver

Absolute 5.285 ± 0.120 5.783 ± 0.165 5.653 ± 0.138 5.910 ± 0.164* 5.756 ± 0.175 5.615 ± 0.114

Relative 27.898 ± 0.333 30.653 ± 0.523** 29.985 ± 0.509* 31.078 ± 0.520** 30.501 ± 0.482** 29.585 ± 0.389

Lung

Absolute 1.077 ± 0.060 1.003 ± 0.039 1.012 ± 0.029 1.045 ± 0.050 1.049 ± 0.042 1.035 ± 0.039

Relative 5.666 ± 0.243 5.307 ± 0.131 5.376 ± 0.156 5.486 ± 0.209 5.573 ± 0.210 5.446 ± 0.164

Thymus

Absolute 0.208 ± 0.008 0.204 ± 0.009 0.192 ± 0.012 0.202 ± 0.007 0.213 ± 0.008 0.205 ± 0.012

Relative 1.099 ± 0.028 1.084 ± 0.048 1.021 ± 0.065 1.059 ± 0.030 1.129 ± 0.038 1.076 ± 0.049

* Significantly different (P#0.05) from the control group by Williams’ or Dunnett’s test

** (P#0.01) a Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ 

weight/g body weight (mean ± standard error).
Chromium Picolinate Monohydrate, NTP TR 556 G-3

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TABLE G2

Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 3-Month Feed Study 

of Chromium Picolinate Monohydratea

0 ppm 80 ppm 240 ppm 2,000 ppm 10,000 ppm 50,000 ppm

n 10 10 10 10 10 10

Male

Necropsy body wt 30.0 ± 0.5 30.6 ± 1.2 31.4 ± 0.7 30.0 ± 0.6 29.3 ± 0.5 29.6 ± 0.6

Heart

Absolute 0.132 ± 0.004 0.128 ± 0.009 0.133 ± 0.004 0.132 ± 0.004 0.131 ± 0.004 0.128 ± 0.003

Relative 4.420 ± 0.168 4.243 ± 0.386 4.248 ± 0.115 4.400 ± 0.089 4.482 ± 0.142 4.351 ± 0.161

R. Kidney

Absolute 0.256 ± 0.007 0.252 ± 0.012 0.250 ± 0.007 0.239 ± 0.009 0.240 ± 0.008 0.246 ± 0.008

Relative 8.561 ± 0.253 8.221 ± 0.197 7.975 ± 0.171 7.977 ± 0.292 8.203 ± 0.232 8.332 ± 0.292

Liver

Absolute 1.169 ± 0.041 1.183 ± 0.048 1.235 ± 0.039 1.220 ± 0.055 1.150 ± 0.044 1.170 ± 0.056

Relative 39.054 ± 1.342 38.695 ± 0.991 39.503 ± 1.335 40.750 ± 1.889 39.249 ± 1.208 39.384 ± 1.190

Lung

Absolute 0.221 ± 0.013 0.224 ± 0.012 0.224 ± 0.010 0.221 ± 0.011 0.220 ± 0.009 0.226 ± 0.014

Relative 7.413 ± 0.482 7.402 ± 0.478 7.172 ± 0.357 7.392 ± 0.399 7.520 ± 0.280 7.699 ± 0.550

R. Testis

Absolute 0.120 ± 0.002 0.113 ± 0.003 0.116 ± 0.003 0.116 ± 0.002 0.115 ± 0.002 0.117 ± 0.002

Relative 4.024 ± 0.082 3.735 ± 0.140 3.708 ± 0.115 3.897 ± 0.121 3.938 ± 0.097 3.975 ± 0.098

Thymus

Absolute 0.033 ± 0.001 0.038 ± 0.002* 0.036 ± 0.002 0.033 ± 0.001 0.033 ± 0.001 0.034 ± 0.002

Relative 1.089 ± 0.036 1.243 ± 0.053 1.156 ± 0.059 1.098 ± 0.040 1.135 ± 0.037 1.157 ± 0.036

Females

Necropsy body wt 24.5 ± 0.5 24.4 ± 0.3 25.3 ± 0.3 24.8 ± 0.4 24.3 ± 0.5 24.8 ± 0.5

Heart

Absolute 0.113 ± 0.002 0.112 ± 0.003 0.119 ± 0.003 0.114 ± 0.002 0.112 ± 0.004 0.107 ± 0.003

Relative 4.634 ± 0.118 4.585 ± 0.121 4.717 ± 0.129 4.593 ± 0.077 4.613 ± 0.124 4.333 ± 0.127

R. Kidney

Absolute 0.162 ± 0.004 0.163 ± 0.005 0.159 ± 0.004 0.157 ± 0.005 0.153 ± 0.003 0.148 ± 0.006

Relative 6.633 ± 0.174 6.678 ± 0.214 6.308 ± 0.177 6.326 ± 0.169 6.318 ± 0.130 5.994 ± 0.242

Liver

Absolute 0.922 ± 0.024 0.921 ± 0.023 1.015 ± 0.021 0.953 ± 0.019 0.903 ± 0.044 0.926 ± 0.031

Relative 37.688 ± 0.564 37.683 ± 0.711 40.260 ± 0.998 38.440 ± 0.866 37.104 ± 1.350 37.353 ± 0.681

Lung

Absolute 0.197 ± 0.010 0.186 ± 0.016 0.219 ± 0.014 0.222 ± 0.012 0.211 ± 0.014 0.212 ± 0.013

Relative 8.082 ± 0.425 7.595 ± 0.614 8.650 ± 0.502 8.936 ± 0.436 8.692 ± 0.536 8.600 ± 0.543

Thymus

Absolute 0.047 ± 0.001 0.048 ± 0.003 0.042 ± 0.001 0.043 ± 0.002 0.040 ± 0.003 0.043 ± 0.002

Relative 1.909 ± 0.061 1.942 ± 0.101 1.647 ± 0.059 1.741 ± 0.049 1.657 ± 0.096 1.755 ± 0.099

* Significantly different (P#0.05) from the control group by Dunnett’s test a Organ weights (absolute weights) and body weights are given in grams; organ-weight-to-body-weight ratios (relative weights) are given as mg organ 

weight/g body weight (mean ± standard error).
Board Draft 

G-4 Chromium Picolinate Monohydrate, NTP TR 556 

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H-1

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APPENDIX H

REPRODUCTIVE TISSUE EVALUATIONS

AND ESTROUS CYCLE CHARACTERIZATION

TABLE H1 Summary of Reproductive Tissue Evaluations for Male Rats

in the 3-Month Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . H-2

TABLE H2 Estrous Cycle Characterization for Female Rats

in the 3-Month Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . H-2

TABLE H3 Summary of Reproductive Tissue Evaluations for Male Mice

in the 3-Month Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . H-3

TABLE H4 Estrous Cycle Characterization for Female Mice

in the 3-Month Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . H-3
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TABLE H1

Summary of Reproductive Tissue Evaluations for Male Rats in the 3-Month Feed Study

of Chromium Picolinate Monohydratea

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

n 10 10 10 10

Weights (g)

Necropsy body wt 326 ± 8 329 ± 6 319 ± 5 316 ± 9

L. Cauda epididymis 0.2265 ± 0.0427 0.1862 ± 0.0045 0.1823 ± 0.0062 0.1781 ± 0.0073

L. Epididymis 0.4582 ± 0.0088 0.4404 ± 0.0109 0.4440 ± 0.0129 0.4494 ± 0.0157

L. Testis 1.5000 ± 0.0340 1.4757 ± 0.0216 1.4756 ± 0.0272 1.4635 ± 0.0337

Spermatid measurements

Spermatid heads (107/g testis) 109.47 ± 4.91 111.75 ± 3.94 100.41 ± 3.45 120.74 ± 7.93

Spermatid heads (107/testis) 147.13 ± 6.02 151.50 ± 7.63 133.25 ± 4.47 158.63 ± 11.27

Epididymal spermatozoal measurements

Sperm motility (%) 72.34 ± 2.11 71.22 ± 1.33 70.86 ± 0.82 70.09 ± 0.91

Sperm (107

/g cauda epididymis) 625.59 ± 62.68 576.71 ± 33.76 570.01 ± 32.04 595.47 ± 26.03

Sperm (107

/cauda epididymis) 120.41 ± 4.68 106.90 ± 5.78 102.78 ± 5.08 106.03 ± 6.29

a Data are presented as mean ± standard error. Differences from the control group are not significant by Dunnett’s test (body and tissue 

weights) or Dunn’s test (spermatid and epididymal spermatozoal measurements).

TABLE H2

Estrous Cycle Characterization for Female Rats in the 3-Month Feed Study

of Chromium Picolinate Monohydratea

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Number weighed at necropsy 10 10 10 10

Necropsy body wt (g) 189 ± 4 190 ± 4 188 ± 4 190 ± 3

Proportion of regular cycling femalesb 6/8 7/10 8/9 10/10

Estrous cycle length (days) 4.94 ± 0.47c 4.75 ± 0.21 4.44 ± 0.15d 5.00 ± 0.31

Estrous stages (% of cycle)

Diestrus 51.7 38.3 38.3 44.2

Proestrus 6.7 12.5 14.2 7.5

Estrus 25.8 33.3 30.0 30.0

Metestrus 15.8 15.8 17.5 18.3

a Necropsy body weights and estrous cycle length data are presented as mean ± standard error. Differences from the control group are not 

significant by Dunnett’s test (body weight), Dunn’s test (estrous cycle length), or Fisher’s exact test (proportion of regular cycling females). 

By multivariate analysis of variance, exposed females do not differ significantly from the control females in the relative length of time spent 

in the estrous stages. b Number of females with a regular cycle/number of females cycling c Estrous cycle was longer than 12 days or unclear in 2 of 10 animals. d Estrous cycle was longer than 12 days or unclear in 1 of 10 animals.
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TABLE H3

Summary of Reproductive Tissue Evaluations for Male Mice in the 3-Month Feed Study

of Chromium Picolinate Monohydratea

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

n 10 10 10 10

Weights (g)

Necropsy body wt 30.0 ± 0.5 30.0 ± 0.6 28.9 ± 0.6 29.6 ± 0.6

L. Cauda epididymis 0.0241 ± 0.0019 0.0247 ± 0.0011 0.0241 ± 0.0011 0.0239 ± 0.0013

L. Epididymis 0.0531 ± 0.0027 0.0519 ± 0.0020 0.0538 ± 0.0017 0.0550 ± 0.0020

L. Testis 0.1230 ± 0.0023 0.1233 ± 0.0015 0.1175 ± 0.0019 0.1208 ± 0.0015

Spermatid measurements

Spermatid heads (107

/g testis) 204.15 ± 6.18 190.85 ± 13.69 214.29 ± 14.32 221.64 ± 8.15

Spermatid heads (107

/testis) 21.13 ± 0.59 18.54 ± 1.29 20.84 ± 1.08 21.81 ± 0.94

Epididymal spermatozoal measurements

Sperm motility (%) 65.61 ± 2.83 66.63 ± 0.92 70.04 ± 1.03 66.68 ± 1.73

Sperm heads (107

/g cauda epididymis) 802.99 ± 56.27 814.71 ± 52.23 850.90 ± 57.47 824.25 ± 47.63

Sperm heads (107/cauda epididymis) 18.81 ± 1.28 19.72 ± 0.74 20.11 ± 1.17 19.36 ± 0.87

a Data are presented as mean ± standard error. Differences from the control group are not significant by Dunnett’s test (body and tissue 

weights) or Dunn’s test (spermatid and epididymal spermatozoal measurements).

TABLE H4

Estrous Cycle Characterization for Female Mice in the 3-Month Feed Study 

of Chromium Picolinate Monohydratea

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Number weighed at necropsy 10 10 10 10

Necropsy body wt (g) 24.5 ± 0.4 24.8 ± 0.4 24.3 ± 0.5 24.8 ± 0.5

Proportion of regular cycling femalesb 5/10 7/10 3/10 4/10

Estrous cycle length (days) 3.87 ± 0.24 4.19 ± 0.24 5.10 ± 0.51* 4.00 ± 0.46

Estrous stages (% of cycle)

Diestrus 41.7 41.7 44.2 43.3

Proestrus 0.0 0.0 0.0 0.0

Estrus 45.0 41.7 44.2 45.0

Metestrus 13.3 16.7 11.7 11.7

* Significantly different (P#0.05) from the control group by Shirley’s test a Necropsy body weights and estrous cycle length data are presented as mean ± standard error. Differences from the control group are not 

significant by Dunnett’s test (body weight) or Fisher’s exact test (proportion of regular cycling females). By multivariate analysis of 

variance, exposed females do not differ significantly from the control females in the relative length of time spent in the estrous stages. b Number of females with a regular cycle/number of females cycling
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I-1

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APPENDIX I

CHEMICAL CHARACTERIZATION

AND DOSE FORMULATION STUDIES

PROCUREMENT AND CHARACTERIZATION OF CHROMIUM PICOLINATE MONOHYDRATE . . . . . . . . . . . . . . . . . . I-2

PREPARATION AND ANALYSIS OF DOSE FORMULATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I-3

FIGURE I1 Infrared Absorption Spectrum of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . I-4

FIGURE I2 Proton Nuclear Magnetic Resonance Spectrum of Chromium Picolinate Monohydrate . . I-5

FIGURE I3 X-Ray Diffraction Spectrum of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . I-6

TABLE I1 High-Performance Liquid Chromatography Systems Used 

in the Feed Studies of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . I-7

TABLE I2 Preparation and Storage of Dose Formulations in the Feed Studies 

of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I-8

TABLE I3 Results of Analyses of Dose Formulations Administered to Rats and Mice

in the 3-Month Feed Studies of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . I-9

TABLE I4 Results of Analyses of Dose Formulations Administered to Rats and Mice

in the 2-Year Feed Studies of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . I-11
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CHEMICAL CHARACTERIZATION

AND DOSE FORMULATION STUDIES

PROCUREMENT AND CHARACTERIZATION

OF CHROMIUM PICOLINATE MONOHYDRATE

Chromium picolinate monohydrate was obtained from TCI America (Portland, OR) in one lot (OGJ01) and from

Sigma-Aldrich (St. Louis, MO) in one lot (CHESS0204DFCI). Lot OGJ01 was used in the 3-month studies; the

unused remainder of lot OGJ01 was combined with lot CHESS0204DFCI by the analytical chemistry laboratory at

Battelle Toxicology Northwest (Richland, WA) and assigned lot number 672002, which was used in the 2-year

studies. Identity, purity, and stability analyses were performed by the analytical chemistry laboratory, the study

laboratory at Southern Research Institute (Birmingham, AL), PIXE Analytical Laboratories (Tallahassee, FL),

Element Analysis Corporation (Lexington, KY), Galbraith Laboratories, Inc. (Knoxville, TN), and Oneida

Research Services, Inc. (Whitesboro, NY). Reports on analyses performed in support of the chromium picolinate

monohydrate studies are on file at the National Institute of Environmental Health Sciences.

Lots OGJ01 and 672002 of the chemical, a reddish-purple crystalline powder, were identified as chromium

picolinate monohydrate by the analytical chemistry laboratory using infrared (IR) spectroscopy, X-ray diffraction

(XRD), proton nuclear magnetic resonance (NMR) spectroscopy, and electrospray ionization-mass spectrometry

(EI-MS); the identity of these lots was also confirmed by the study laboratory using IR spectroscopy. All spectra

were consistent with literature spectra (Green et al., 1984; Stearns and Armstrong, 1992; Broadhurst et al., 1997),

spectra of reference samples from each lot, and the structure of chromium picolinate monohydrate. Representative

IR, NMR, and XRD spectra are presented in Figures I1, I2, and I3, respectively. 

The moisture contents of lots OGJ01 and 672002 were determined by Galbraith Laboratories, Inc., using Karl

Fischer titration; weight loss on drying was determined by the analytical chemistry laboratory for lot 672002.

Elemental analyses for carbon, hydrogen, and nitrogen were performed by Oneida Research Services, Inc.

(lot OGJ01) and Galbraith Laboratories, Inc. (lot 672002). Proton-induced X-ray emission (PIXE) spectroscopy

was conducted by PIXE Analytical Laboratories (lot OGJ01) and Element Analysis Corporation (lot 672002).

PIXE analyses for 72 elements in proton-irradiated samples of the test chemical were performed on a noncommercial Van de Graff accelerator (lot OGJ01) or a General Ionex (Bellaire, MI) tandem accelerator

(lot 672002). Inductively coupled plasma-atomic emission spectroscopy (ICP-AES) was used by the analytical

chemistry laboratory to determine the total chromium contents of lots OGJ01 and 672002; the system utilized an

Applied Research Laboratories spectrometer (Thermo Electron Corp., Waltham, MA) with coolant, plasma, and

carrier argon pressures of 24, 25, and 29 or 30 psig, respectively, a sample flow rate of approximately

2.4 mL/minute, and analysis of chromium at 284.325 nm. High-performance liquid chromatography (HPLC) was

used by the analytical chemistry laboratory to determine the purity of the test chemical using diode-array detection

(HPLC-DAD) by system A (lot OGJ01), ultraviolet-visible detection (HPLC-UV/Vis) by system B (lot 672002),

UV detection (HPLC-UV) by system C (lots OGJ01 and 672002), and ICP-mass spectrometric detection

(HPLC-ICP-MS) by system D (lots OGJ01 and 672002) (Table I1). The study laboratory determined the purities

of lots OGJ01 and 672002 using HPLC-UV by system E.

For lot OGJ01, the results of Karl Fischer titration for water content, elemental analyses for carbon, hydrogen, and

nitrogen, and ICP-AES analysis for total chromium were all consistent with the theoretical values for chromium

picolinate monohydrate. PIXE analysis indicated the absence of significant metallic impurities and a total

chromium content of 117% of the theoretical value; the high value was considered a result of experimental artifacts

involving high vacuum and/or thermal degradation of the test chemical. HPLC-DAD by system A revealed a

major component at 96%, one impurity at 2.1%, and five additional impurities at less than 1%, but greater than or

equal to 0.1% each. HPLC-UV by system C followed by HPLC-ICP-MS indicated that the maximum
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concentrations of free (uncomplexed) Cr(III) or Cr(VI) were less than 0.025%. The overall purity of lot OGJ01

was determined to be greater than 96%.

For lot 672002, Karl Fischer titration and weight loss on drying assays indicated the presence of approximately

1 mole of water in the test chemical complex. Results of elemental analyses for carbon, hydrogen, and nitrogen

and of ICP-AES analysis for total chromium content were consistent with the theoretical values for chromium

picolinate monohydrate. PIXE analysis indicated a chromium content consistent with the theoretical value and

absence of significant metallic impurities. HPLC-UV/Vis indicated one major peak with an area percent purity of

approximately 95%. HPLC-UV by system C coupled with HPLC-ICP-MS indicated that the maximum

concentrations of free Cr III or Cr VI were less than 0.025%. The overall purity of lot 672002 was determined to

be greater than 95%.

In an attempt to identify the impurities indicated by HPLC-DAD in lot OGJ01, the analytical chemistry laboratory

made preparations of chromium:picolinate complexes according to procedures reported in the literature (Stearns

and Armstrong, 1992; Evans and Pouchnik, 1993). The complexes were prepared from chromium (III) chloride

hexahydrate and picolinic acid obtained from Sigma-Aldrich and were analyzed using HPLC-DAD by system F

and HPLC-EI-MS by system G. Results were inconclusive due to insufficient assay sensitivity and the lack of

authentic reference standards. However, these analyses provided evidence that the impurities in the test chemical

were probably chromium:picolinate complexes, although the exact structures and ratios were uncertain. 

Stability studies of lot OGJ01 of the bulk chemical were performed by the analytical chemistry laboratory using

ICP-AES by the same method as that used for purity analyses and HPLC-UV by a system similar to system A with

detection at 265 nm. These studies indicated that chromium picolinate monohydrate was stable as a bulk chemical

for at least 2 weeks when stored in sealed amber glass containers at temperatures up to 60° C. To ensure stability,

the bulk chemical was stored at room temperature (~25° C), protected from light, in sealed plastic buckets.

Periodic reanalyses of the bulk chemical were performed during the 3-month and 2-year studies by the study

laboratory using HPLC-UV by system E, and no degradation of the bulk chemical was detected. 

PREPARATION AND ANALYSIS OF DOSE FORMULATIONS

The dose formulations were prepared by mixing chromium picolinate monohydrate with feed (Table I2).

Formulations were stored in sealed double-thick plastic bags, protected from light at 5° C for up to 42 days.

Homogeneity studies of 82 and 50,000 ppm dose formulations of lot OGJ01 and stability studies of the 82 ppm

dose formulation of lot OGJ01 were performed by the analytical chemistry laboratory using ICP-AES as described

for the purity assays. Additional homogeneity studies of 80 and 50,000 ppm dose formulations of this lot were

performed by the study laboratory using HPLC-UV by system E (Table I1). Homogeneity was confirmed, and

stability was confirmed for at least 42 days for dose formulations stored in double-thick sealed plastic bags,

protected from light at room temperature and for at least 8 days under simulated animal room conditions; to ensure

stability, storage at 5° C was recommended. 

Periodic analyses of the dose formulations of chromium picolinate monohydrate were conducted by the study

laboratory using HPLC-UV by system E. During the 3-month studies, the dose formulations were analyzed at the

beginning, midpoint, and end of the studies; all 35 dose formulations analyzed for rats and mice were within 10%

of the target concentrations (Table I3). Animal room samples of these dose formulations were also analyzed; all 10

animal room samples for rats and eight of 10 for mice were within 10% of the target concentrations. During the

2-year studies, the dose formulations were analyzed approximately every 12 weeks (Table I4). Of the dose

formulations analyzed, all 167 for rats and all 99 for mice were within 10% of the target concentrations; all 12

animal room samples for rats and eight of 15 for mice were within 10% of the target concentrations. 
I-4 Chromium Picolinate Monohydrate, NTP TR 556 

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FIGURE I1

Infrared Absorption Spectrum of Chromium Picolinate Monohydrate
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Chromium Picolinate Monohydrate, NTP TR 556 I-5

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FIGURE I2

Proton Nuclear Magnetic Resonance Spectrum of Chromium Picolinate Monohydrate
I-6 Chromium Picolinate Monohydrate, NTP TR 556 

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FIGURE I3

X-Ray Diffraction Spectrum of Chromium Picolinate Monohydrate
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TABLE I1 

High-Performance Liquid Chromatography Systems Used in the Feed Studies 

of Chromium Picolinate Monohydratea

Detection System Column Solvent System

System A

Ultraviolet/visible photodiode array

(scanning from 190 to 600 nm) with

monitoring at 265 nm

System B

Ultraviolet (265 nm)/visible (509 nm) light

System C

Ultraviolet (265 nm) light

System D

Inductively coupled plasmamass spectrometry

System E

Ultraviolet (265 nm) light

System F

Ultraviolet/visible photodiode array

(scanning from 190 to 600 nm) with

monitoring at 265 and 509 nm

System G

Electrospray ionization-mass spectrometry

Supelco Discovery® C18, 150 mm × 4.6 mm,

5 μm (Supelco, Bellefonte, PA)

Supelco Discovery® C18, 150 mm × 4.6 mm,

5 μm (Supelco)

Supelco Discovery® C18, 150 mm × 4.6 mm,

5 μm (Supelco) 

Dionex IonPac® AS7, 250 mm × 4.4 mm,

10 μm (Dionex, Sunnyvale, CA) 

Supelco Discovery® C18, 150 mm × 4.6 mm,

5 μm (Supelco)

Supelco Discovery® C18, 150 mm × 4.6 mm,

5 μm (Supelco)

Supelco Discovery® C18, 150 mm × 4.6 mm,

5 μm (Supelco)

A) 50 mM potassium dihydrogen phosphate

and B) methanol; linear gradient from

98% A:2% B to 70% A:30% B in 8 minutes,

held for 2 minutes, then linear gradient to

98% A:2% B in 0.01 minutes, held for

5 minutes; flow rate 1 mL/minute 

A) 50 mM ammonium acetate and

B) methanol; linear gradient from 100% A to

100% B in 8 minutes, held for 4 minutes,

then linear gradient to 100% A in

0.1 minutes, held for 10 minutes; flow rate

1 mL/minute 

A) 50 mM potassium dihydrogen phosphate

and B) methanol; linear gradient from

100% A to 70% A:30% B in 8 minutes, held

for 2 minutes, then linear gradient to 100% A

in 0.01 minute, held for 5 minutes; flow rate

1 mL/minute 

35 mM ammonium sulfate (adjusted to

pH 9.2 with ammonium hydroxide),

isocratic; flow rate 1 mL/minute

50 mM potassium dihydrogen

phosphate:methanol (70:30), isocratic; flow

rate 0.5 mL/minute 

A) 50 mM ammonium acetate with

0.1% acetic acid and B) methanol; linear

gradient from 100% A to 90% A:10% B in

8 minutes, held for 4 minutes, then linear

gradient to 100% B in 0.01 minute, held for

4 minutes, then linear gradient to 100% A in

0.01 minute, held for 4 minutes; flow rate

1 mL/minute 

A) 50 mM ammonium acetate with

0.1% acetic acid and B) methanol; linear

gradient from 100% A to 90% A:10% B in

8 minutes, held for 4 minutes, then linear

gradient to 100% B in 0.01 minute, then

linear gradient to 100% A in 4 minutes, held

for 4 minutes; flow rate 1 mL/minute 

a The high-performance liquid chromatographs were manufactured by Hewlett-Packard Company (Palo Alto, CA) (systems A, C, D, E, F,

and G) or Agilent (Palo Alto, CA) (system B). The mass spectrometers were manufactured by Hewlett-Packard Company (system D) or

Finnigan, Inc. (San Jose, CA) (system G).
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TABLE I2 

Preparation and Storage of Dose Formulations in the Feed Studies of Chromium Picolinate Monohydrate

3-Month Studies 2-Year Studies

Preparation

A premix of feed and chromium picolinate monohydrate was

prepared, then layered into the remaining feed and blended in a

Patterson-Kelly twin-shell blender for 30 minutes using an intensifier

bar. The dose formulations were prepared four times. 

Chemical Lot Number

OGJ01

Maximum Storage Time

42 days

Storage Conditions

Stored in sealed double-thick plastic bags, protected from light at

5° C.

Study Laboratory

Southern Research Institute (Birmingham, AL)

Same as 3-month studies until June 16, 2003, and later, when the

dose formulations were mixed for only 15 minutes with the intensifier

bar on. The dose formulations were prepared approximately monthly.

672002

42 days

Stored in sealed double-thick plastic bags, protected from light at

5° C.

Southern Research Institute (Birmingham, AL)
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TABLE I3 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 3-Month Feed Studies

of Chromium Picolinate Monohydrate

Target Determined Difference

Date Prepared Date Analyzed Concentration Concentrationa from Target

(ppm) (ppm) (%)

Rats and Mice

October 5, 2001 October 8-9, 2001 80 76.4 –5

80 79.8 0

80 79.2 –1

240 226 –6

240 236 –2

240 228 –5

2,000 1,959 –2

2,000 1,983 –1

2,000 1,993 0

10,000 9,899 –1

10,000 10,027 0

10,000 10,024 0

50,000 50,477 +1

50,000 48,980 –2

50,000 49,528 –1

November 7, 2001 November 8-9, 2001 80 84.9 +6

80 83.9 +5

80 86.7 +8

240 236 –2

240 240 0

240 233 –3

2,000 1,988 –1

2,000 2,010 +1

2,000 1,993 0

10,000 9,933 –1

10,000 9,725 –3

10,000 9,788 –2

50,000 49,168 –2

50,000 49,050 –2

50,000 49,127 –2

January 2, 2002 January 3-4, 2002 80 84.9 +6

240 234 –3

2,000 1,975 –1

10,000 9,906 –1

50,000 50,601 +1

Animal Room Samples

Rats

November 7, 2001 December 17-18, 2001 80 73.3 –8

240 225 –6

2,000 1,943 –3

10,000 9,446 –6

50,000 50,650 +1

January 2, 2002 January 22-23, 2002 80 73.2 –9

240 223 –7

2,000 1,986 –1

10,000 9,539 –5

50,000 47,983 –4
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TABLE I3 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 3-Month Feed Studies

of Chromium Picolinate Monohydrate

Target Determined Difference

Date Prepared Date Analyzed Concentration Concentration from Target

(ppm) (ppm) (%)

Animal Room Samples (continued)

Mice

November 7, 2001 December 17-18, 2001 80 77.7 –3

240 220 –8

2,000 1,983 –1

10,000 8,749 –13

50,000 44,579 –11

January 2, 2002 January 22-23, 2002 80 72.9 –9

240 223 –7

2,000 1,990 –1

10,000 9,328 –7

50,000 46,916 –6

a Results of duplicate analyses
NOT FOR DISTRIBUTION OR ATTRIBUTION

Chromium Picolinate Monohydrate, NTP TR 556 I-11

Board Draft 

TABLE I4 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies

of Chromium Picolinate Monohydrate

Target Determined Difference

Date Prepared Date Analyzed Concentration Concentrationa from Target

(ppm) (ppm) (%)

Rats 

July 30-31, 2002 August 1-2, 2002 2,000 1,876 –6

2,000 1,916 –4

2,000 1,927 –4

2,000 1,900 –5

2,000 1,951 –2

10,000 9,594 –4

10,000 9,738 –3

10,000 9,617 –4

10,000 9,619 –4

10,000 9,634 –4

50,000 45,532 –9

50,000 46,787 –6

50,000 47,633 –5

50,000 48,569 –3

50,000 47,618 –5

50,000 48,443 –3

August 28-29, 2002b 2,000 1,920 –4

10,000 9,498 –5

50,000 46,033 –8

October 8-9, 2002 October 9-11, 2002 2,000 2,128 +6

2,000 2,102 +5

2,000 2,113 +6

2,000 2,126 +6

2,000 2,131 +7

10,000 9,288 –7

10,000 9,693 –3

10,000 9,444 –6

10,000 9,495 –5

10,000 9,418 –6

10000 9,742 –3

50,000 48,737 –3

50,000 47,964 –4

50,000 48,685 –3

50,000 48,782 –2

50,000 49,746 –1

50,000 47,923 –4

December 17-18, 2002 February 4-5, 2003c 2,000 1,950 –3

2,000 1,950 –3

2,000 1,935 –3

2,000 1,916 –4

2,000 1,937 –3

2,000 1,933 –3

10,000 9,503 –5

10,000 9,696 –3

10,000 9,806 –2

10,000 9,534 –5

10,000 9,887 –1

10,000 9,650 –4

50,000 47,842 –4

50,000 49,627 –1

50,000 48,422 –3

50,000 49,463 –1

50,000 48,614 –3
I-12 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft

TABLE I4 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies

of Chromium Picolinate Monohydrate

Target Determined Difference

Date Prepared Date Analyzed Concentration Concentration from Target

(ppm) (ppm) (%)

Rats (continued)

December 30-31, 2002 February 4-5, 2003 2,000 1,881 –6

2,000 1,918 –4

February 25-26, 2003 February 27-28, 2003 2,000 1,885 –6

2,000 1,903 –5

2,000 1,894 –5

2,000 1,847 –8

10,000 9,781 –2

10,000 9,735 –3

10,000 9,677 –3

10,000 9,878 –1

50,000 49,518 –1

50,000 49,805 0

50,000 48,428 –3

50,000 47,364 –5

March 25-26, 2003b 2,000 1,941 –3

10,000 9,826 –2

50,000 51,000 +2

May 5-6, 2003 May 8-9, 2003 2,000 1,984 –1

2,000 1,934 –3

2,000 1,997 0

2,000 1,979 –1

2,000 2,009 0

10,000 9,670 –3

10,000 9,191 –8

10,000 9,345 –7

10,000 9,878 –1

10,000 10,032 0

50,000 49,675 –1

50,000 51,526 +3

50,000 49,656 –1

50,000 51,185 +2

50,000 51,334 +3

June 16-17, 2003 June 17-18, 2003 2,000 1,935 –3

2,000 1,834 –8

50,000 47,562 –5

50,000 47,944 –4

July 14, 2003 July 17-18, 2003 2,000 1,970 –2

2,000 1,881 –6

2,000 1,917 –4

2,000 1,938 –3

10,000 9,609 –4

10,000 9,461 –5

10,000 9,717 –3

10,000 9,905 –1

50,000 50,232 0

50,000 49,768 0

50,000 49,996 0

50,000 49,938 0
NOT FOR DISTRIBUTION OR ATTRIBUTION

Chromium Picolinate Monohydrate, NTP TR 556 I-13

Board Draft 

TABLE I4 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies

of Chromium Picolinate Monohydrate

Target Determined Difference

Date Prepared Date Analyzed Concentration Concentration from Target

(ppm) (ppm) (%)

Rats (continued)

July 18, 2003 July 21, 2003 2,000 1,939d –3

October 6, 2003 October 7-8, 2003 2,000 2,016 +1

2,000 1,984 –1

2,000 2,028 +1

2,000 2,024 +1

10,000 9,931 –1

10,000 9,854 –1

10,000 9,887 –1

10,000 9,930 –1

50,000 50,385 +1

50,000 50,129 0

50,000 50,222 0

50,000 51,759 +4

November 3-4, 2003b 2,000 1,807 –10

10,000 9,627 –4

50,000 47,787 –4

December 1, 2003 December 2-3, 2003 2,000 1,998 0

2,000 1,989 –1

2,000 1,948 –3

2,000 1,997 0

2,000 1,964 –2

2,000 1,963 –2

10,000 9,916 –1

10,000 9,660 –3

10,000 9,704 –3

10,000 9,897 –1

10,000 9,872 –1

50,000 50,001 0

50,000 49,036 –2

50,000 47,796 –4

50,000 47,978 –4

50,000 46,649 –7

February 9, 2004 February 10-11, 2004 2,000 1,920 –4

2,000 1,912 –4

2,000 1,934 –3

2,000 1,867 –7

2,000 1,915 –4

2,000 1,931 –3

10,000 9,848 –2

10,000 9,876 –1

10,000 9,933 –1

10,000 9,791 –2

10,000 9,853 –1

10,000 10,010 0

50,000 49,467 –1

50,000 49,013 –2

50,000 49,402 –1

50,000 49,894 0

50,000 50,317 +1

50,000 49,699 –1
I-14 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft

TABLE I4 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies

of Chromium Picolinate Monohydrate

Target Determined Difference

Date Prepared Date Analyzed Concentration Concentration from Target

(ppm) (ppm) (%)

Rats (continued)

April 19, 2004 April 20-21, 2004 2,000 1,924 –4

2,000 1,962 –2

2,000 1,976 –1

2,000 1,952 –2

2,000 1,942 –3

10,000 9,489 –5

10,000 9,610 –4

10,000 9,564 –4

10,000 9,369 –6

50,000 46,940 –6

50,000 47,582 –5

50,000 47,221 –6

50,000 48,254 –3

May 17-18, 2004b 2,000 1,950 –3

10,000 9,617 –4

50,000 46,405 –7

June 28, 2004 June 30-July 1, 2004 2,000 1,896 –5

2,000 1,894 –5

2,000 1,921 –4

2,000 1,925 –4

10,000 9,996 0

10,000 10,250 +3

10,000 10,143 +1

10,000 10,040 0

50,000 51,365 +3

50,000 50,314 +1

50,000 50,600 +1

50,000 49,784 0

Mice

July 18, 2002 July 19-21, 2002 2,000 1,914 –4

2,000 1,972 –1

10,000 9,827 –2

10,000 9,828 –2

50,000 49,250 –2

50,000 49,261 –1

August 12-13, 2002b 2,000 1,733 –13

10,000 7,340 –27

50,000 36,359 –27
NOT FOR DISTRIBUTION OR ATTRIBUTION

Chromium Picolinate Monohydrate, NTP TR 556 I-15

Board Draft 

TABLE I4 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies

of Chromium Picolinate Monohydrate

Target Determined Difference

Date Prepared Date Analyzed Concentration Concentration from Target

(ppm) (ppm) (%)

Mice (continued)

July 30-31, 2002 August 1-2, 2002 2,000 1,897 –5

2,000 1,900 –5

10,000 9,682 –3

10,000 9,738 –3

50,000 47,633 –5

50,000 47,618 –5

50,000 47,083 –6

August 28-29, 2002b 2,000 1,803 –10

10,000 7,783 –22

50,000 39,189 –22

October 8-9, 2002 October 9-11, 2002 2,000 2,102 +5

2,000 2,126 +6

2,000 2,111 +6

10,000 9,444 –6

10,000 9,495 –5

50,000 48,782 –2

50,000 49,746 –1

December 17-18, 2002 February 4-5, 2003 2,000 1,950 –3

2,000 1,935 –3

2,000 1,916 –4

2,000 1,937 –3

10,000 9,503 –5

10,000 9,534 –5

10,000 9,887 –1

50,000 47,842 –4

50,000 48,614 –3

February 25-26, 2003 February 27-28, 2003 2,000 1,870 –7

2,000 1,893 –5

10,000 9,896 –1

10,000 9,839 –2

50,000 49,973 0

50,000 49,879 0

March 25-26, 2003b 2,000 1,919 –4

10,000 9,902 –1

50,000 50,205 0

May 5-6, 2003 May 8-9, 2003 2,000 1,984 –1

2,000 1,984 –1

10,000 9,191 –8

10,000 9,879 –1

50,000 48,987 –2

50,000 49,675 –1

50,000 51,334 +3
I-16 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft

TABLE I4 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies

of Chromium Picolinate Monohydrate

Target Determined Difference

Date Prepared Date Analyzed Concentration Concentration from Target

(ppm) (ppm) (%)

Mice (continued)

June 16-17, 2003 June 17-18, 2003 2,000 1,935 –3

2,000 1,834 –8

50,000 47,562 –5

50,000 47,944 –4

July 14, 2003 July 17-18, 2003 2,000 1,881 –6

2,000 1,976 –1

2,000 1,917 –4

10,000 9,646 –4

10,000 9,461 –5

10,000 9,799 –2

50,000 48,863 –2

50,000 49,621 –1

July 18, 2003 July 21, 2003 2,000 1,939d –3

October 6, 2003 October 7-8, 2003 2,000 1,985 –1

2,000 1,984 –1

2,000 1,990 –1

10,000 9,806 –2

10,000 9,931 –1

10,000 9,887 –1

50,000 49,718 –1

50,000 50,427 +1

50,000 50,129 0

November 3-4, 2003b 2,000 1,806 –10

10,000 9,564 –4

50,000 46,790 –6

December 1, 2003 December 2-3, 2003 2,000 1,989 –1

2,000 1,948 –3

2,000 1,997 0

10,000 9,916 –1

10,000 9,825 –2

50,000 50,001 0

50,000 49,338 –1

February 9, 2004 February 10-11, 2004 2,000 1,867 –7

2,000 1,915 –4

2,000 1,931 –3

10,000 9,876 –1

10,000 9,933 –1

50,000 49,467 –1

50,000 49,013 –2

50,000 50,317 +1
NOT FOR DISTRIBUTION OR ATTRIBUTION

Chromium Picolinate Monohydrate, NTP TR 556 I-17

Board Draft 

TABLE I4 

Results of Analyses of Dose Formulations Administered to Rats and Mice in the 2-Year Feed Studies

of Chromium Picolinate Monohydrate

Target Determined Difference

Date Prepared Date Analyzed Concentration Concentration from Target

(ppm) (ppm) (%)

Mice (continued)

April 19, 2004 April 20-21, 2004 2,000 1,922 –4

2,000 1,952 –2

2,000 1,942 –3

10,000 9,509 –5

10,000 9,631 –4

50,000 46,940 –6

50,000 46,816 –6

50,000 47,021 –6

50,000 47,221 –6

May 17-18, 2004b 2,000 1,972 –1

10,000 8,895 –11

50,000 48,280 –3

June 28, 2004 June 30-July 1, 2004 2,000 1,921 –4

2,000 1,925 –4

2,000 1,937 –3

2,000 1,942 –3

10,000 10,246 +2

10,000 10,143 +1

10,000 10,040 0

10,000 10,016 0

50,000 49,974 0

50,000 50,884 +2

50,000 50,600 +1

a Results of duplicate analyses b Animal room samples c Results of reanalyses: analyses performed in December, 2002 (data not shown) were erratic due to low temperature in the laboratory that 

was believed to have caused saturation of the solvent; extraction solvent volume was increased and formulations were reanalyzed. d Results of remix
I-18 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft
NOT FOR DISTRIBUTION OR ATTRIBUTION

J-1

Board Draft 

APPENDIX J

FEED AND COMPOUND CONSUMPTION

IN THE 2-YEAR FEED STUDIES

OF CHROMIUM PICOLINATE MONOHYDRATE

TABLE J1 Feed and Compound Consumption by Male Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J-2

TABLE J2 Feed and Compound Consumption by Female Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J-3

TABLE J3 Feed and Compound Consumption by Male Mice in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J-4

TABLE J4 Feed and Compound Consumption by Female Mice in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . J-5
J-2 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft

TABLE J1

Feed and Compound Consumption by Male Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Feed Body Feed Body Doseb Feed Body Dose Feed Body Dose/

(g/day)a Weight (g/day) Weight (mg/kg) (g/day) Weight (mg/kg) (g/day) Weight (mg/kg)

Week (g) (g) (g) (g)

1 16.4 114 15.5 114 272 15.6 113 1,375 15.8 113 6,975

2 14.9 143 14.7 141 209 15.1 139 1,084 15.3 138 5,540

3 17.1 170 16.1 166 194 16.6 166 1,001 16.9 162 5,206

4 17.3 199 16.2 192 168 16.9 193 878 17.9 188 4,773

5 16.3 208 16.1 206 156 16.5 205 807 16.6 198 4,202

6 17.9 235 17.9 232 154 18.3 232 790 19.5 226 4,306

7 17.5 251 17.5 246 142 19.0 248 767 19.4 244 3,978

8 16.4 269 16.5 263 125 17.9 267 672 18.4 263 3,503

9 18.3 271 18.5 269 138 18.3 276 663 19.1 271 3,522

10 16.8 286 16.6 286 116 17.2 292 588 17.2 287 3,000

11 16.8 301 16.5 298 111 17.5 301 582 18.1 296 3,061

12 17.6 311 17.2 309 111 17.2 312 551 18.9 307 3,079

13 15.8 318 15.8 315 100 18.3 320 572 17.4 315 2,762

17 17.4 350 17.1 344 100 18.5 351 527 18.5 346 2,672

21 18.0 367 18.9 364 104 19.3 370 522 19.6 366 2,679

25 17.4 385 16.8 380 89 17.0 387 440 19.3 379 2,544

29 18.0 400 17.3 392 88 18.4 386 477 20.8 392 2,652

33 17.5 414 17.4 406 86 17.9 413 434 19.0 409 2,325

37 18.0 423 17.6 415 85 18.5 424 437 19.2 419 2,289

41 17.8 435 18.3 428 86 18.3 434 421 19.6 431 2,276

45 17.0 439 16.6 433 77 17.0 439 387 18.0 437 2,062

49 17.2 443 16.0 435 74 17.4 445 391 18.4 443 2,078

53 18.2 448 18.8 443 85 18.3 448 409 19.2 443 2,170

58 18.0 451 17.3 443 78 18.0 449 401 18.7 446 2,096

61 16.9 447 16.4 438 75 17.8 444 401 18.1 438 2,065

65 17.9 453 18.2 445 82 18.7 449 417 20.0 444 2,253

69 16.6 450 15.8 444 71 17.0 451 377 18.1 448 2,022

73 17.6 451 16.5 443 74 16.2 451 359 13.3 448 1,485

77 16.4 451 17.1 445 77 17.8 452 394 19.0 448 2,120

82 16.9 449 17.4 442 79 17.7 448 395 18.1 448 2,021

85 16.6 445 14.8 435 68 17.2 450 382 16.5 444 1,858

89 16.9 444 17.2 437 79 17.8 445 400 18.5 445 2,077

93 15.7 443 16.2 439 74 16.5 451 366 17.0 447 1,902

97 16.5 443 14.9 436 68 15.5 443 350 16.2 441 1,837

101 17.1 439 14.7 438 67 14.2 435 326 15.0 426 1,761

Mean for weeks

1-13 16.9 237 16.5 234 154 17.3 236 795 17.7 231 4,147

14-52 17.6 406 17.3 400 88 18.0 405 448 19.2 402 2,397

53-101 17.0 447 16.6 441 75 17.1 447 383 17.5 444 1,974

a Grams of feed consumed per animal per day b Milligrams of chromium picolinate monohydrate consumed per kilogram body weight per day
NOT FOR DISTRIBUTION OR ATTRIBUTION

Chromium Picolinate Monohydrate, NTP TR 556 J-3

Board Draft 

TABLE J2

Feed and Compound Consumption by Female Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Feed Body Feed Body Doseb Feed Body Dose Feed Body Dose/

(g/day)a Weight (g/day) Weight (mg/kg) (g/day) Weight (mg/kg) (g/day) Weight (mg/kg)

Week (g) (g) (g) (g)

1 12.6 95 12.0 95 252 11.9 95 1,250 12.4 96 6,490

2 11.4 116 11.2 115 195 11.4 113 1,006 11.7 115 5,067

3 12.2 133 12.7 132 192 12.8 130 981 12.6 132 4,774

4 11.2 142 11.5 142 162 11.9 141 844 13.2 141 4,683

5 12.4 147 11.0 147 150 12.1 146 827 12.5 148 4,221

6 11.1 153 12.9 156 165 11.8 153 773 12.4 156 3,986

7 11.0 161 11.1 162 137 11.6 160 727 12.0 162 3,709

8 10.1 165 10.5 166 127 10.9 163 669 11.3 165 3,416

9 12.1 169 11.6 170 136 11.1 166 671 11.2 169 3,320

10 10.0 173 9.9 175 113 10.3 170 606 10.4 173 3,005

11 9.4 175 9.8 179 110 10.4 174 597 11.6 177 3,270

12 10.1 177 10.2 181 113 10.3 177 583 11.1 180 3,080

13 10.0 181 10.0 184 109 10.1 179 565 10.2 182 2,802

17 10.2 191 10.2 193 106 10.6 189 561 10.9 192 2,840

21 10.6 198 11.3 203 112 11.6 198 585 11.7 201 2,914

25 10.5 204 10.7 207 103 11.3 203 557 11.5 206 2,797

29 10.6 210 10.7 213 100 9.5 205 463 11.9 212 2,809

33 12.1 218 11.7 224 104 11.0 217 507 11.8 221 2,671

37 11.2 226 11.6 230 101 11.7 223 525 11.9 226 2,629

41 10.9 234 12.0 241 100 11.9 232 514 12.0 236 2,548

45 10.4 237 11.2 247 91 11.1 236 471 11.6 240 2,412

49 10.8 243 11.2 253 89 11.2 241 464 11.9 246 2,416

53 12.6 252 12.6 260 97 12.7 250 509 13.2 254 2,602

58 12.1 260 12.3 270 91 12.6 259 487 12.8 263 2,438

61 12.1 267 11.7 275 85 11.8 262 450 12.3 267 2,307

65 12.8 277 14.0 284 99 14.4 271 532 14.8 275 2,695

69 12.5 284 12.7 292 87 12.5 279 449 13.6 283 2,400

73 12.6 294 12.7 303 84 11.0 289 381 11.2 289 1,941

77 12.4 296 13.2 306 86 13.7 291 471 13.8 295 2,341

82 11.8 301 13.1 315 83 13.3 299 444 13.9 303 2,298

85 12.6 307 12.1 315 77 12.7 305 416 13.0 306 2,127

89 12.9 315 13.0 322 81 12.9 308 419 13.7 310 2,210

93 10.9 309 12.1 317 76 11.9 307 388 11.6 307 1,888

97 13.1 312 11.2 316 71 11.6 309 376 12.7 313 2,028

101 12.6 311 13.0 318 82 12.5 311 402 13.4 315 2,126

Mean for weeks

1-13 11.0 153 11.1 154 151 11.3 151 777 11.7 154 3,986

14-52 10.8 218 11.2 223 101 11.1 216 516 11.7 220 2,671

53-101 12.4 291 12.6 299 85 12.6 288 440 13.1 291 2,262

a Grams of feed consumed per animal per day b Milligrams of chromium picolinate monohydrate consumed per kilogram body weight per day
J-4

TABLE J3

Feed and Compound Consumption by Male Mice in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Feed Body Feed Body Doseb Feed Body Dose Feed Body Dose/

(g/day)a Weight (g/day) Weight (mg/kg) (g/day) Weight (mg/kg) (g/day) Weight (mg/kg)

Week (g) (g) (g) (g)

1 5.2 20.3 4.9 20.5 478 4.7 20.4 2,302 5.0 20.5 12,173

2 4.4 21.5 4.3 21.4 402 4.4 21.2 2,071 4.7 21.8 10,765

3 5.4 22.8 5.6 22.9 488 5.2 22.7 2,294 5.5 22.9 12,017

4 5.3 23.5 5.3 23.5 452 4.9 23.1 2,118 5.2 23.6 11,027

5 5.4 23.7 5.3 24.2 437 5.2 23.7 2,193 5.2 24.5 10,633

6 5.2 25.0 5.2 25.3 411 5.1 25.1 2,036 5.3 25.4 10,416

7 5.1 26.2 5.7 25.9 441 5.2 25.7 2,023 5.0 25.8 9,692

8 5.3 26.2 5.2 26.9 387 4.9 26.1 1,875 5.2 26.6 9,786

9 4.9 27.3 5.5 27.5 400 5.4 26.9 2,005 5.2 27.5 9,443

10 5.2 27.4 4.9 27.0 363 5.0 26.9 1,858 5.0 27.3 9,154

11 5.4 28.7 5.5 28.9 380 5.7 28.5 1,999 5.5 29.0 9,476

12 5.3 29.6 5.2 29.8 349 5.3 29.2 1,816 5.3 29.7 8,908

13 5.0 30.5 5.4 31.0 348 5.3 30.4 1,744 5.4 31.2 8,657

17 4.9 33.4 4.8 34.1 282 4.8 33.0 1,453 5.0 33.6 7,431

21 4.7 35.1 4.7 36.1 260 4.9 35.0 1,402 5.1 36.0 7,083

25 4.8 37.3 5.0 38.4 260 5.0 37.1 1,347 5.3 38.0 6,965

29 4.7 39.5 4.9 40.8 240 4.9 38.8 1,263 4.9 40.1 6,110

33 4.9 42.4 5.1 43.3 236 5.1 40.8 1,251 5.4 42.0 6,426

37 4.9 45.2 5.0 46.0 218 5.1 44.0 1,160 5.3 44.8 5,912

41 4.8 46.1 5.0 47.1 212 4.8 45.3 1,060 5.1 45.9 5,558

45 5.4 48.8 5.2 48.9 213 5.2 47.2 1,101 5.3 48.1 5,509

49 5.1 49.4 5.3 49.7 213 5.3 48.4 1,096 5.4 49.0 5,513

53 5.2 49.3 5.7 49.4 231 5.6 48.2 1,163 5.3 48.5 5,458

58 5.3 49.5 5.1 49.5 206 5.5 48.7 1,129 5.7 49.6 5,751

61 5.3 49.6 4.3 47.3 182 4.3 46.7 922 4.3 47.7 4,507

65 4.9 47.2 5.0 46.5 215 5.0 45.5 1,100 5.3 46.4 5,714

69 5.5 49.1 5.8 47.7 243 5.5 47.1 1,168 5.7 48.1 5,922

73 5.7 49.5 5.7 49.3 231 5.7 47.8 1,192 5.8 49.3 5,882

77 5.6 49.7 5.7 49.2 232 5.7 48.3 1,181 5.9 49.8 5,928

81 5.6 48.8 5.7 49.1 232 5.8 47.4 1,224 6.1 48.9 6,235

85 5.5 49.2 5.8 49.2 236 5.8 47.7 1,215 5.9 48.5 6,087

89 5.9 50.0 6.1 49.7 246 6.0 47.7 1,259 6.2 48.7 6,367

93 5.7 48.6 5.9 48.2 245 5.8 46.2 1,254 5.8 47.0 6,175

97 5.7 48.1 5.6 47.0 239 5.6 46.1 1,216 5.8 46.1 6,286

101 5.0 45.8 5.0 44.3 226 4.9 44.5 1,100 5.3 43.4 6,109

Mean for weeks

1-13 5.2 25.6 5.2 25.8 410 5.1 25.4 2,026 5.2 25.8 10,165

14-52 4.9 41.9 5.0 42.7 237 5.0 41.1 1,237 5.2 41.9 6,279

53-101 5.5 48.8 5.5 48.2 228 5.5 47.1 1,163 5.6 47.8 5,879

a Grams of feed consumed per animal per day b Milligrams of chromium picolinate monohydrate consumed per kilogram body weight per day

Chromium Picolinate Monohydrate, NTP TR 556 

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Chromium Picolinate Monohydrate, NTP TR 556 J-5

TABLE J4

Feed and Compound Consumption by Female Mice in the 2-Year Feed Study 

of Chromium Picolinate Monohydrate

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

Feed Body Feed Body Doseb Feed Body Dose Feed Body Dose/

(g/day)a Weight (g/day) Weight (mg/kg) (g/day) Weight (mg/kg) (g/day) Weight (mg/kg)

Week (g) (g) (g) (g)

1 3.2 16.6 3.0 16.7 359 3.2 16.6 1,930 3.0 16.5 9,065

2 2.4 17.4 2.0 17.0 235 2.3 16.9 1,361 2.2 17.2 6,390

3 3.6 18.2 3.6 18.4 390 3.5 18.3 1,910 3.5 18.1 9,672

4 3.6 19.5 3.6 19.4 372 3.8 19.1 1,986 3.7 19.2 9,631

5 3.8 19.5 3.8 20.1 379 3.7 20.3 1,826 3.8 20.1 9,442

6 2.7 21.2 3.4 21.2 321 3.6 20.8 1,730 3.6 21.0 8,575

7 3.7 22.1 3.7 21.3 348 3.5 22.1 1,582 3.7 21.6 8,581

8 3.9 22.8 3.7 22.4 330 3.9 22.9 1,701 3.8 22.8 8,339

9 3.8 23.2 3.9 23.0 340 3.8 22.2 1,708 3.9 22.8 8,544

10 3.9 24.3 3.7 23.9 309 3.9 23.1 1,689 3.9 23.9 8,160

11 4.2 24.9 4.1 24.3 337 4.3 24.2 1,774 4.2 24.3 8,653

12 3.9 24.0 3.8 24.1 315 3.8 23.8 1,599 3.9 23.7 8,243

13 4.2 25.7 4.0 25.7 312 4.3 25.7 1,676 4.3 25.6 8,400

17 4.2 29.2 4.2 28.4 296 4.3 28.0 1,537 4.2 28.6 7,340

21 3.8 30.7 3.8 29.8 255 3.8 30.3 1,256 3.9 30.0 6,491

25 4.3 33.1 4.4 32.0 275 4.5 31.4 1,434 4.8 32.1 7,486

29 3.8 36.0 3.9 34.8 224 3.9 33.8 1,156 3.9 34.3 5,680

33 4.4 37.4 4.2 34.3 245 4.3 33.9 1,269 4.4 35.1 6,264

37 4.2 39.3 4.3 37.1 232 4.2 36.7 1,144 4.2 37.3 5,631

41 4.3 40.2 4.6 37.8 244 4.6 38.4 1,196 4.9 38.1 6,432

45 4.3 42.2 4.3 39.6 217 4.4 39.7 1,109 4.3 39.7 5,422

49 4.1 42.8 4.3 39.5 218 4.4 40.5 1,086 4.4 39.8 5,523

53 4.2 42.6 4.1 39.8 206 4.1 40.7 1,008 3.7 38.3 4,837

58 4.5 45.2 4.7 43.2 218 4.8 43.3 1,108 4.9 42.6 5,753

61 4.5 45.1 3.9 42.2 185 3.9 42.5 918 4.3 42.1 5,105

65 4.4 43.3 4.4 41.2 214 4.2 41.3 1,017 4.5 41.1 5,468

69 4.1 44.2 4.3 40.6 212 4.2 42.2 994 4.4 41.7 5,278

73 5.0 46.2 4.8 42.7 225 4.9 44.8 1,094 5.0 43.9 5,688

77 4.8 48.8 4.8 44.7 215 4.8 46.6 1,030 4.9 46.0 5,324

81 4.9 49.9 5.0 45.9 218 4.9 47.6 1,029 5.2 46.8 5,561

85 5.1 49.8 4.8 46.7 206 5.1 48.3 1,057 5.2 47.3 5,498

89 4.9 50.3 4.8 46.5 206 4.8 48.8 983 5.3 48.2 5,496

93 4.5 49.3 4.6 47.0 196 4.9 48.6 1,008 5.0 46.7 5,357

97 4.7 48.4 4.7 46.9 201 4.7 48.4 970 5.1 47.6 5,356

101 4.4 46.4 4.5 45.4 198 4.6 46.0 1,001 5.0 45.7 5,473

Mean for weeks

1-13 3.6 21.5 3.6 21.3 334 3.7 21.2 1,729 3.7 21.3 8,592

14-52 4.2 36.8 4.2 34.8 245 4.3 34.7 1,243 4.3 35.0 6,252

53-101 4.6 46.9 4.6 44.1 208 4.6 45.3 1,017 4.8 44.5 5,400

a Grams of feed consumed per animal per day b Milligrams of chromium picolinate monohydrate consumed per kilogram body weight per day

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J-6 Chromium Picolinate Monohydrate, NTP TR 556 

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K-1

Board Draft 

APPENDIX K

INGREDIENTS, NUTRIENT COMPOSITION,

AND CONTAMINANT LEVELS

IN NTP-2000 RAT AND MOUSE RATION

TABLE K1 Ingredients of NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . K-2

TABLE K2 Vitamins and Minerals in NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . K-2

TABLE K3 Nutrient Composition of NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . K-3

TABLE K4 Contaminant Levels in NTP-2000 Rat and Mouse Ration . . . . . . . . . . . . . . . . . . . . . . . . . . . K-4
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TABLE K1

Ingredients of NTP-2000 Rat and Mouse Ration

Ingredients Percent by Weight

Ground hard winter wheat 22.26

Ground #2 yellow shelled corn 22.18

Wheat middlings 15.0

Oat hulls 8.5

Alfalfa meal (dehydrated, 17% protein) 7.5

Purified cellulose 5.5

Soybean meal (49% protein) 5.0

Fish meal (60% protein) 4.0

Corn oil (without preservatives) 3.0

Soy oil (without preservatives) 3.0

Dried brewer’s yeast 1.0

Calcium carbonate (USP) 0.9

Vitamin premixa 0.5

Mineral premixb 0.5

Calcium phosphate, dibasic (USP) 0.4

Sodium chloride 0.3

Choline chloride (70% choline) 0.26

Methionine 0.2

a Wheat middlings as carrier b Calcium carbonate as carrier

TABLE K2

Vitamins and Minerals in NTP-2000 Rat and Mouse Rationa

Amount Source

Vitamins

A 4,000 IU Stabilized vitamin A palmitate or acetate

D 1,000 IU D-activated animal sterol

K 1.0 mg Menadione sodium bisulfite complex

"-Tocopheryl acetate 100 IU

Niacin 23 mg

Folic acid 1.1 mg

d-Pantothenic acid 10 mg d-Calcium pantothenate

Riboflavin 3.3 mg

Thiamine 4 mg Thiamine mononitrate

B12 52 μg

Pyridoxine 6.3 mg Pyridoxine hydrochloride

Biotin 0.2 mg d-Biotin

Minerals

Magnesium 514 mg Magnesium oxide

Iron 35 mg Iron sulfate

Zinc 12 mg Zinc oxide

Manganese 10 mg Manganese oxide

Copper 2.0 mg Copper sulfate

Iodine 0.2 mg Calcium iodate

Chromium 0.2 mg Chromium acetate

a Per kg of finished product
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Chromium Picolinate Monohydrate, NTP TR 556 K-3

TABLE K3

Nutrient Composition of NTP-2000 Rat and Mouse Ration

Mean ± Standard

Nutrient Deviation Range Number of Samples

Protein (% by weight) 15.2 ± 0.48 14.4 – 16.2 24

Crude fat (% by weight) 8.1 ± 0.33 7.5 – 9.0 24

Crude fiber (% by weight) 9.1 ± 0.60 8.1 – 10.6 24

Ash (% by weight) 5.0 ± 0.30 4.5 – 5.6 24

Amino Acids (% of total diet)

Arginine 0.750 ± 0.048 0.670 – 0.850 15

Cystine 0.225 ± 0.025 0.150 – 0.250 15

Glycine 0.701 ± 0.039 0.620 – 0.750 15

Histidine 0.365 ± 0.090 0.310 – 0.680 15

Isoleucine 0.533 ± 0.038 0.430 – 0.590 15

Leucine 1.077 ± 0.059 0.960 – 1.150 15

Lysine 0.703 ± 0.125 0.310 – 0.830 15

Methionine 0.402 ± 0.049 0.260 – 0.460 15

Phenylalanine 0.615 ± 0.035 0.540 – 0.660 15

Threonine 0.492 ± 0.040 0.430 – 0.590 15

Tryptophan 0.135 ± 0.018 0.110 – 0.160 15

Tyrosine 0.378 ± 0.048 0.280 – 0.460 15

Valine 0.658 ± 0.043 0.550 – 0.710 15

Essential Fatty Acids (% of total diet)

Linoleic 3.90 ± 0.256 3.49 – 4.54 15

Linolenic 0.30 ± 0.035 0.21 – 0.35 15

Vitamins

Vitamin A (IU/kg) 6,092 ± 317 3,210 – 17,600 24

Vitamin D (IU/kg) 1,000a

"-Tocopherol (ppm) 84.2 ± 16.60 52.0 – 110.0 15

Thiamine (ppm)b 10.2 ± 5.86 6.4 – 30.6 24

Riboflavin (ppm) 6.8 ± 2.11 4.20 – 11.20 15

Niacin (ppm) 79.0 ± 10.50 66.4 – 98.2 15

Pantothenic acid (ppm) 23.9 ± 3.73 17.4 – 29.8 15

Pyridoxine (ppm)b 9.21 ± 2.20 6.4 – 13.7 15

Folic acid (ppm) 1.75 ± 0.54 1.20 – 3.27 15

Biotin (ppm) 0.332 ± 0.12 0.225 – 0.704 15

Vitamin B12 (ppb) 60.5 ± 46.5 18.3 – 174.0 15

Choline (ppm)b 3,064 ± 270 2,700 – 3,790 15

Minerals

Calcium (%) 0.986 ± 0.047 1.080 – 0.909 24

Phosphorus (%) 0.604 ± 0.037 0.539 – 0.721 24

Potassium (%) 0.665 ± 0.023 0.626 – 0.694 15

Chloride (%) 0.376 ± 0.041 0.300 – 0.474 15

Sodium (%) 0.191 ± 0.017 0.160 – 0.222 15

Magnesium (%) 0.201 ± 0.009 0.185 – 0.217 15

Sulfur (%) 0.170 ± 0.029 0.116 – 0.209 15

Iron (ppm) 182 ± 46.7 135 – 311 15

Manganese (ppm) 54.1 ± 7.89 42.1 – 73.1 15

Zinc (ppm) 55.0 ± 9.55 43.3 – 78.5 15

Copper (ppm) 6.65 ± 1.790 3.21 – 10.50 15

Iodine (ppm) 0.512 ± 0.221 0.233 – 0.972 15

Chromium (ppm) 0.604 ± 0.253 0.330 – 1.380 14

Cobalt (ppm) 0.25 ± 0.074 0.20 – 0.47 14

a From formulation b As hydrochloride (thiamine and pyridoxine) or chloride (choline)

Board Draft 
K-4

TABLE K4

Contaminant Levels in NTP-2000 Rat and Mouse Rationa

Mean ± Standard

Deviationb Range Number of Samples

Contaminants

Arsenic (ppm) 0.38 ± 0.156 0.14 – 0.50 24

Cadmium (ppm) 0.66 ± 0.023 0.39 – 0.11 24

Lead (ppm) 0.07 ± 0.019 0.05 – 0.11 24

Mercury (ppm) <0.02 24

Selenium (ppm) 0.21 ± 0.057 0.14 – 0.40 24

Aflatoxins (ppb) <5.00 24

Nitrate nitrogen (ppm)c 16.2 ± 4.99 10.0 – 28.7 24

Nitrite nitrogen (ppm)c <0.88 24

BHA (ppm)d <1.0 24

BHT (ppm)d <1.0 24

Aerobic plate count (CFU/g) 12 ± 8 10 – 50 24

Coliform (MPN/g) 3.0 ± 0.0 3.0 – 3.0 24

Escherichia coli (MPN/g) <10 24

Salmonella (MPN/g) Negative 24

Total nitrosoamines (ppb)e 4.8 ± 2.55 2.4 – 12.0 24

N-Nitrosodimethylamine (ppb)e 3.2 ± 2.26 1.2 – 9.3 24

N-Nitrosopyrrolidine (ppb)e 1.6 ± 0.52 0.9 – 2.8 24

Pesticides (ppm)

"-BHC <0.01 24

$-BHC <0.02 24

(-BHC <0.01 24

*-BHC <0.01 24

Heptachlor <0.01 24

Aldrin <0.01 24

Heptachlor epoxide <0.01 24

DDE <0.01 24

DDD <0.01 24

DDT <0.01 24

HCB <0.01 24

Mirex <0.01 24

Methoxychlor <0.05 24

Dieldrin <0.01 24

Endrin <0.01 24

Telodrin <0.01 24

Chlordane <0.05 24

Toxaphene <0.10 24

Estimated PCBs <0.20 24

Ronnel <0.01 24

Ethion <0.02 24

Trithion <0.05 24

Diazinon <0.10 24

Methyl chlorpyrifos 0.081 ± 0.072 0.020 – 0.280 24

Methyl parathion <0.02 24

Ethyl parathion <0.02 24

Malathion 0.227 ± 0.372 0.021 – 1.640 24

Endosulfan I <0.01 24

Endosulfan II <0.01 24

Endosulfan sulfate <0.03 24

a All samples were irradiated. CFU=colony-forming units; MPN=most probable number; BHC=hexachlorocyclohexane or 

benzene hexachloride b For values less than the limit of detection, the detection limit is given as the mean. c Sources of contamination: alfalfa, grains, and fish meal d Sources of contamination: soy oil and fish meal e All values were corrected for percent recovery.

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APPENDIX L

SENTINEL ANIMAL PROGRAM

METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L-2

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . L-3

L-1

Board Draft 
L-2

SENTINEL ANIMAL PROGRAM

METHODS

Rodents used in the Carcinogenesis Program of the National Toxicology Program are produced in optimally clean

facilities to eliminate potential pathogens that may affect study results. The Sentinel Animal Program is part of the

periodic monitoring of animal health that occurs during the toxicologic evaluation of chemical compounds. Under

this program, the disease state of the rodents is monitored via serology on sera from extra (sentinel) animals in the

study rooms. These animals and the study animals are subject to identical environmental conditions. The sentinel

animals come from the same production source and weanling groups as the animals used for the studies of

chemical compounds.

Serum samples were collected from five male and five female control rats (core study) and mice at the end of the

3-month studies and from five male and five female sentinel rats and mice at 6, 12, and 18 months and five male

and five female rats and mice from the 50,000 ppm groups at the end of the 2-year studies. Blood from each

animal was collected and allowed to clot, and the serum was separated. The samples were processed appropriately

and sent to BioReliance Corporation (Rockville, MD) for determination of antibody titers. Fecal samples were

taken from sentinel mice at 18 months in the 2-year study for PCR analysis. The laboratory methods and agents

for which testing was performed and the times at which samples were collected during the studies are tabulated

below.

Method and Test Time of Analysis

RATS

3-Month Study

ELISA

PVM (pneumonia virus of mice) Study termination

RCV/SDA 

(rat coronavirus/sialodacryoadenitis virus) Study termination

Sendai Study termination

Immunofluorescence Assay

Parvovirus Study termination

2-Year Study

ELISA

Mycoplasma arthritidis Study termination

Mycoplasma pulmonis Study termination

PVM 6, 12, and 18 months, study termination

RCV/SDA 6, 12, and 18 months, study termination

Sendai 6, 12, and 18 months, study termination

Immunofluorescence Assay

Parvovirus 6, 12, and 18 months, study termination

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Method and Test Time of Analysis

MICE

3-Month Study

ELISA

Mouse adenoma virus-FL Study termination

Ectromelia virus Study termination

EDIM (epizootic diarrhea of infant mice) Study termination

GDVII (mouse encephalomyelitis virus) Study termination

LCM (lymphocytic choriomeningitis virus) Study termination

MHV (mouse hepatitis virus) Study termination

PVM Study termination

Reovirus 3 Study termination

Sendai Study termination

Immunofluorescence Assay

Parvovirus Study termination

2-Year Study

ELISA

Ectromelia virus 6, 12, and 18 months, study termination

EDIM 6, 12, and 18 months, study termination

GDVII 6, 12, and 18 months, study termination

LCM 6, 12, and 18 months, study termination

Mouse adenoma virus-FL 6, 12, and 18 months, study termination

MHV 6, 12, and 18 months, study termination

M. arthritidis Study termination

M. pulmonis Study termination

PVM 6, 12, and 18 months, study termination

Reovirus 3 6, 12, and 18 months, study termination

Sendai 6, 12, and 18 months, study termination

MMV VP2 (mice minute virus VP2) Study termination

MPV VP2 (mouse parvovirus VP2) Study termination

Immunofluorescence Assay

EDIM 12 and 18 months

GDVII Study termination

MCMV (mouse cytomegalovirus) Study termination

MHV 18 months

Parvovirus 6, 12, and 18 months

Reovirus 3 6 months

Polymerase chain reaction (PCR)

Helicobacter species 18 months

RESULTS

All test results were negative.

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M-1

APPENDIX M

CHROMIUM TISSUE DISTRIBUTION STUDY

Board Draft 

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M-2

TABLE M1 Tissue Concentrations and Excreta Content of Chromium in Male Rats

in the 2-Year Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . M-4

TABLE M2 Tissue Concentrations and Excreta Content of Chromium in Female Mice

in the 2-Year Feed Study of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . . . . . . M-5
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CHROMIUM TISSUE DISTRIBUTION STUDY

MATERIALS AND METHODS

Groups of 30 special study male rats and female mice were randomly assigned to the tissue distribution study at

the beginning of the 2-year studies and treated identically to the core study groups. On days 4, 11, and 180, up to

10 animals per exposure group were removed from treatment and placed in individual metabolism cages to allow

separate collection of urine and feces. The animals had access to drinking water and undosed feed ad libitum.

Two collections of urine and feces were made to include the intervals from 0 to 24 and 24 to 48 hours; measured

values were combined to yield the reported 48-hour values. Urine and feces collections were frozen at –20° C and

shipped to the analytical laboratory (RTI International, Research Triangle Park, NC).

At the end of 48 hours, the animals were anesthetized with CO2/O2 and blood was taken from the retroorbital sinus

into heparinized centrifuge tubes. The blood was separated into cells and plasma. While the animals were still

under CO2/O2 anesthesia, the abdominal wall was opened and the aorta was severed. The entire liver, both

kidneys, and the stomach (separated into glandular and forestomach parts) were removed, weighed, and frozen at

–20° C and shipped frozen to the analytical lab. Stainless steel was avoided during the tissue collection; only

plastic, TeflonTM, ceramic, or tungsten carbide instruments were used.

The tissue samples were stored in a freezer. Prior to homogenization, the samples were allowed to thaw at room

temperature in a Class 100 environment. Tissue samples were added to tared glass scintillation vials, and the

whole wet tissue mass was recorded. A minimal amount of deionized water was added to each sample, and the

total mass (tissue plus deionized water) was recorded. Each tissue sample was homogenized using a Polytron

tissue homogenizer (Brinkmann Instruments, Westbury, NY) and returned to frozen storage.

Prior to aliquot transfer, the tissue homogenate was removed from frozen storage and allowed to thaw at room

temperature in a Class 100 environment. Each tissue homogenate was mixed well to ensure complete

homogeneity. An aliquot of homogenate containing approximately 0.500 g of tissue was transferred to a tared

50 mL centrifuge tube, and the mass was recorded. Approximately 5% of the rat kidney samples were prepared

with duplicate aliquots, and masses were recorded. The actual mass for each sample was calculated by applying

the percent tissue in the homogenate to the measured mass. Homogenization was not necessary if the tissue mass

was less than 0.500 g, but duplicate aliquots for these samples could not be prepared.

Five mL of concentrated nitric acid was added to each centrifuge tube containing tissue. The tubes were capped

with microwavable caps and allowed to stand overnight at room temperature. The following morning, 2 mL of

deionized water was added to each centrifuge tube. The tubes were arranged in a microwave tray, the thermowell

and temperature sensors were inserted into one of the sample tubes to monitor the digestion, and the samples were

subjected to a four-stage microwave digestion program that ramped the power from 60% to 100% with hold times

of 5 to 20 minutes. Samples achieved a maximum temperature of 110° C, which was held for 10 minutes.

After the digestion was complete, the samples were allowed to cool to room temperature. To each tube, 0.500 mL

of hydrogen peroxide and 0.125 mL of hydrofluoric acid were added as was more deionized water, if necessary, to

avoid charring. The samples were subjected to the same microwave program a second time and then allowed to

cool. The content of each tube was transferred to a 25 mL volumetric flask, and 0.250 mL of a previously

prepared 1.00 μg/mL internal standard solution was added. The internal standard solution was prepared by

transferring 100 μL aliquots of NIST-traceable yttrium, bismuth, indium, and scandium stock solutions

(1,000 μg/mL) to a 100 mL volumetric flask, adding 1 mL of concentrated nitric acid, and diluting to volume with

deionized water. The tubes were rinsed with deionized water, and the rinses were added to the flasks. Each flask

was brought to volume with deionized water and mixed well. Samples were transferred to clean plastic storage

bottles and stored in a refrigerator until the day of analysis.
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The solvent standards, digested matrix standards, and experimental samples were analyzed by inductively coupled

plasma-mass spectrometry using a Thermo X7 instrument (ThermoElectron Corp., Winsford, Cheshire, U.K.) or a

Plasma Quad XR Instrument (VG Elemental Ltd., Winsford, Cheshire, U.K.) with a concentric nebulizer and a

Peltier impact-bead spray chamber cooled to 5° C. A calibration curve was constructed at the beginning of each

analysis, and the performance of the calibration was evaluated prior to sample analysis. A successful calibration

was indicated by an acceptable correlation coefficient (r > 0.99) and residual percent read-backs within 20% of the

lowest standard and within 10% of all other solvent standards. A valid calibration included a minimum of six

calibration standards and a calibration blank.

Analysis data were considered valid if they were bracketed by valid quality control (QC) sets. A maximum of

10 samples (including method blanks and controls) were bracketed by a QC set, which consisted of the calibration

blank and two midlevel calibration standards. A QC set passed when the measured concentration for one midlevel

calibration standard was within 10% of its nominal value. This selected midlevel calibration standard was then

required to pass consistently throughout the analysis. If the selected midlevel calibration standard failed, it was

necessary to reanalyze the bracketed samples. Samples with responses greater than the calibration range were

diluted with diluent solution to get a response within the range. The diluent solution was prepared by adding 1 mL

of the internal standard solution and 20 mL of concentrated nitric acid to a 100 mL plastic volumetric flask and

diluting to volume with deionized water.
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TABLE M1

Tissue Concentrations and Excreta Content of Chromium in Male Rats in the 2-Year Feed Study 

of Chromium Picolinate Monohydratea

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

n 3333

Erythrocytes (μg/g)

Day 6 0.0343 ± 0.0100 0.0446 ± 0.0023 0.0600 ± 0.0057 0.0392 ± 0.0109

Day 13 0.0405 ± 0.0114 0.0517 ± 0.0089 0.0725 ± 0.0095 0.0676 ± 0.0058

Day 182 0.0462 ± 0.0028 0.0616 ± 0.0104 0.0956 ± 0.0078* 0.1154 ± 0.0064**

Plasma (μg/g)b

Day 6 0.0609 ± 0.0068 0.0919 ± 0.0048** 0.1050 ± 0.0044** 0.1259 ± 0.0064**

Day 13 0.0782 ± 0.0057 0.0940 ± 0.0030* 0.1141 ± 0.0061** 0.1351 ± 0.0060**

Day 182 0.0796 ± 0.0034 0.1118 ± 0.0100** 0.1638 ± 0.0042** 0.1760 ± 0.0087**

Liver (μg/g)

Day 6 0.0610 ± 0.0072 0.1423 ± 0.0056* 0.2151 ± 0.0038** 0.2357 ± 0.0164**

Day 13 0.0779 ± 0.0093 0.2333 ± 0.0116* 0.3384 ± 0.0181** 0.3954 ± 0.0305**

Day 182 0.1110 ± 0.0120 0.5130 ± 0.0328* 1.1857 ± 0.0622** 1.2843 ± 0.0171**

Kidney (μg/g)

Day 6 0.1247 ± 0.0172 0.3757 ± 0.0228* 0.4946 ± 0.0210** 0.5778 ± 0.0285**

Day 13 0.1019 ± 0.0098 0.7173 ± 0.0336* 1.2823 ± 0.0900** 1.2500 ± 0.0896*

Day 182 0.1534 ± 0.0080 2.8677 ± 0.0159* 6.0673 ± 0.3618** 6.7137 ± 0.0489**

Glandular stomach (μg/g)

Day 6 0.0721 ± 0.0005 0.1671 ± 0.0639 0.1061 ± 0.0101 0.1604 ± 0.0463

Day 13 0.4378 ± 0.3547 0.1450 ± 0.0298 0.1679 ± 0.0288 0.2299 ± 0.0745

Day 182 1.0366 ± 0.8837 0.6991 ± 0.1218 1.4057 ± 0.6062 0.6560 ± 0.1255

Forestomach (μg/g)

Day 6 0.0709 ± 0.0068 0.1222 ± 0.0092* 0.1991 ± 0.0385* 0.1701 ± 0.0202*

Day 13 0.1183 ± 0.0133 0.1354 ± 0.0222 0.2400 ± 0.1112 0.1390 ± 0.0109

Day 182 0.0863 ± 0.0155 0.1592 ± 0.0100* 0.3615 ± 0.0589** 0.3479 ± 0.0332*

Feces (μg)c

Day 6 15.5 ± 5.4 981.5 ± 133.5* 2,670.5 ± 761.1** 32,055.2 ± 3,383.3**

Day 13 10.5 ± 0.3 1,974.1 ± 97.1* 8,619.4 ± 839.5** 34,786.4 ± 10,954.5**

Day 182 23.3 ± 9.3 2,182.5 ± 308.7* 11,451.2 ± 1,417.2** 59,338.5 ± 6,272.3**

Urine (μg)c

Day 6 0.313 ± 0.056 4.755 ± 1.183* 10.323 ± 1.727* 23.549 ± 4.426**

Day 13 0.539 ± 0.068 6.728 ± 0.427* 14.653 ± 2.218** 31.366 ± 5.451**

Day 182 0.430 ± 0.101 8.169 ± 1.195* 23.106 ± 1.102** 30.026 ± 9.869**

* Significantly different (P#0.05) from the control group by Shirley’s test

** P#0.01 a Mean ± standard error. Statistical tests were performed on unrounded data. Measured values less than the experimental limit of quantitation 

(ELOQ) were included in the analysis as 1⁄2 ELOQ. b n=6 for all plasma values c Cumulative chromium content for 48 hours ending on the day indicated.
NOT FOR DISTRIBUTION OR ATTRIBUTION

Chromium Picolinate Monohydrate, NTP TR 556 M-5

Board Draft 

TABLE M2

Tissue Concentrations and Excreta Content of Chromium in Female Mice in the 2-Year Feed Study 

of Chromium Picolinate Monohydratea

0 ppm 2,000 ppm 10,000 ppm 50,000 ppm

n 3333

Erythrocytes (μg/g)

Day 6 0.0688 ± 0.0171 0.0543 ± 0.0110 0.0749 ± 0.0182 0.0813 ± 0.0073

Day 13 0.0672 ± 0.0110 0.0609 ± 0.0052 0.1635 ± 0.0065 0.7116 ± 0.5998

Day 182 0.0361 ± 0.0040 0.0773 ± 0.0084* 0.1198 ± 0.0132** 0.1292 ± 0.0204*

Plasma (μg/g)b

Day 6 0.0703 ± 0.0080 0.1283 ± 0.0097** 0.1389 ± 0.0145** 0.1749 ± 0.0354**

Day 13 0.0792 ± 0.0024 0.1130 ± 0.0147* 0.1404 ± 0.0293* 0.1960 ± 0.0453**

Day 182 0.0644 ± 0.0055 0.1162 ± 0.0066** 0.1262 ± 0.0081** 0.2762 ± 0.0606**

Liver (μg/g)

Day 6 0.1732 ± 0.0093 0.3566 ± 0.0026* 0.5619 ± 0.0885** 0.4904 ± 0.0075*

Day 13 0.1860 ± 0.0107 0.5705 ± 0.0273* 0.8671 ± 0.0591** 1.0055 ± 0.1068**

Day 182 0.2118 ± 0.0421 1.3467 ± 0.0552* 2.6553 ± 0.3195** 2.8937 ± 0.4221**

Kidney (μg/g)

Day 6 0.0970 ± 0.0092 0.2165 ± 0.0220* 0.3253 ± 0.0309** 0.3242 ± 0.0543*

Day 13 0.0813 ± 0.0021 0.3533 ± 0.0129* 0.7171 ± 0.1738** 0.6933 ± 0.1200*

Day 182 0.0799 ± 0.0013 0.7907 ± 0.0309* 1.0035 ± 0.0357** 1.1981 ± 0.1849**

Glandular stomach (μg/g)

Day 6 0.3810 ± 0.1045 0.7671 ± 0.2943 0.4174 ± 0.1225 0.9349 ± 0.3279

Day 13 0.4913 ± 0.1635 0.4520 ± 0.1541 6.4730 ± 3.3601* 1.3614 ± 0.5943

Day 182 0.2594 ± 0.0951 1.9187 ± 0.5852* 1.6635 ± 0.5503 1.2947 ± 0.0846

Forestomach (μg/g)

Day 6 0.2618 ± 0.0558 1.0015 ± 0.3925 0.3211 ± 0.0420 0.6222 ± 0.2099

Day 13 0.3582 ± 0.0214 0.3746 ± 0.2022 17.1577 ± 14.2041c 1.3203 ± 0.8859

Day 182 0.2941 ± 0.1099 1.4537 ± 0.3915* 0.7424 ± 0.0737 1.1491 ± 0.2961

Feces (μg)d

Day 6 5.0 ± 2.5 129.2 ± 22.9* 632.5 ± 149.1** 2,831.8 ± 522.2**

Day 13 4.0 ± 0.9 156.6 ± 8.1* 586.9 ± 81.9** 3,360.1 ± 77.3**

Day 182 2.7 ± 1.2 129.9 ± 37.5* 1,022.8 ± 230.0** 5,562.8 ± 258.9**

* Significantly different (P#0.05) from the control group by Dunn’s or Shirley’s test

** P#0.01 a Mean ± standard error. Statistical tests were performed on unrounded data. Urine data are not presented due to inadequate sample sizes. 

Measured values less than the experimental limit of quantitation (ELOQ) were included in the analysis as 1⁄2 ELOQ. b n=6 for all plasma values c Measured chromium concentrations for the three samples were 5.692, 45.4 and 0.4007 μg/g forestomach. Because reanalysis of the first two 

samples resulted in similar chromium concentrations (measured at 5.518 and 44.05 μg/g forestomach) and there were three samples analyzed 

from the group, outliers could not be excluded. These data were excluded from significance testing. d Cumulative chromium content for 48 hours ending on the day indicated.
M-6 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft
NOT FOR DISTRIBUTION OR ATTRIBUTION

N-1

Board Draft 

APPENDIX N

ABSORPTION, DISTRIBUTION, 

METABOLISM, AND EXCRETION STUDIES

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-2

MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-2

RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-5

DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-7

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-7

TABLE N1 Excretion of Radioactivity and Total Chromium by Male Rats 

After a Single Gavage Dose of 15.3 mg/kg [14C]-Chromium Picolinate 

in Propylene Glycol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-8

TABLE N2 Urinary Excretion of Chromium Picolinate and Metabolites by Male Rats

After a Single Gavage Dose of 15.3 mg/kg [14C]-Chromium Picolinate 

in Propylene Glycol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-8

TABLE N3 Distribution of Radioactivity in Tissues of Male Rats at 52 Hours 

After a Single Gavage Dose of 15.3 mg/kg [14C]-Chromium Picolinate 

in Propylene Glycol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-9

TABLE N4 Excretion of Radioactivity and Total Chromium by Male Rats

After a Single Gavage Dose of 17.4 mg/kg [14C]-Chromium Picolinate 

as an Aqueous Slurry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-9

TABLE N5 Distribution of Radioactivity in Tissues of Male Rats 48 Hours 

After a Single Gavage Dose of 17.4 mg/kg [14C]-Chromium Picolinate 

as an Aqueous Slurry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-10

TABLE N6 Excretion of Radioactivity and Total Chromium by Male Mice

After a Single Gavage Dose of 20.5 mg/kg [14C]-Chromium Picolinate 

as an Aqueous Slurry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-10

TABLE N7 Urinary Excretion of Chromium Picolinate and Metabolites by Male Mice

After a Single Gavage Dose of 20.5 mg/kg [14C]-Chromium Picolinate 

as an Aqueous Slurry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-11

TABLE N8 Excretion of Radioactivity and Total Chromium by Male Mice

After a Single Gavage Dose of 19.0 mg/kg [14C]-Chromium Picolinate 

in Propylene Glycol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-11

TABLE N9 Distribution of Radioactivity in Tissues of Male Mice 48 Hours

After a Single Gavage Dose of 19.0 mg/kg [14C]-Chromium Picolinate 

in Propylene Glycol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-12

TABLE N10 Urinary Excretion of Chromium Picolinate and Metabolites by Male Mice

After a Single Gavage Dose of 19.0 mg/kg [14C]-Chromium Picolinate 

in Propylene Glycol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-12
N-2 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft

ABSORPTION, DISTRIBUTION, METABOLISM, 

AND EXCRETION STUDIES

INTRODUCTION

Chromium picolinate monohydrate (chromium picolinate, chromium trispicolinate, chromium tripicolinate) is a

synthetic compound that has widespread use as a nutritional supplement. 

Chromium is an essential element to life, being required for proper glucose metabolism and potentiation of the

action of insulin. Only 2% to 3% of dietary chromium is absorbed systemically (Anderson et al., 1993; Gargas

et al., 1994). Because of this low systemic bioavailability of chromium from dietary sources, forms of chromium

having higher bioavailability were sought. Chromium picolinate is claimed to be such a compound although

Gargas et al. (1994) reported that the extent of absorption of chromium picolinate by adult volunteers was

2.8% ± 1.1%. 

Anderson et al. (1997) fed chromium picolinate to rats for 20 weeks at concentrations of 5, 25, 50, or 100 μg

chromium/g feed. At the end of the treatment period, concentration of chromium in the liver was proportional to

the concentration of chromium in the feed; rats fed 5 or 100 μg chromium/g feed had liver concentrations of

chromium of 26 and 542 ng chromium/g dry weight, respectively. Chromium concentration in kidney was 4 to

5 times that measured in liver (Anderson et al., 1993), possibly because absorbed chromium is excreted almost

entirely in urine. 

In another study, Anderson et al. (1996) incorporated chromium picolinate, chromium nicotinate, or complexes of

Cr III and mixtures of several amino acids into rat chow at concentrations of 5 μg Cr/g feed. Groups of weanling

rats were then fed these diets for 3 weeks. Concentrations of chromium in the liver following exposure to diets

containing no added chromium, chromium picolinate, or chromium nicotinate were 4, 50, and 13 ng/g dry weight

respectively, whereas kidney concentrations of chromium were 23, 368, and 166 ng/g dry weight, respectively. In

heart and leg muscle, chromium concentrations above those of controls were found in animals treated with

chromium picolinate, but not with chromium nicotinate. It thus appears that exposure to chromium picolinate and

chromium nicotinate leads to somewhat different patterns of chromium concentrations in tissues. 

The purposes of these studies are to determine the absorption of chromium picolinate into the systemic circulation,

and the fate of both the chromium and the picolinate parts of chromium picolinate. 

MATERIALS AND METHODS

Test Chemicals

Nonradiolabeled Chromium Picolinate

Nonradiolabeled chromium picolinate was obtained from Spectrum Quality Products, Inc. (Gardena, CA) in one lot

(NG0569). Aliquots were examined by high-performance liquid chromatography (HPLC) using HPLC system 1

(see below), mass spectrometry (MS), and nuclear magnetic resonance spectroscopy. In addition, the Cr VI and

total chromium contents were determined. Samples of a chromium picolinate standard were dissolved in distilled

water to produce solutions with chromium picolinate concentrations of 0.0909 mg/mL and 0.0709 mg/mL,

respectively. For Cr VI determinations, samples were diluted with deionized water and analyzed by ion

chromatography with postcolumn colorimetric detection. In this mode, the eluting Cr VI ion is reacted with

diphenylcarbazide and the absorbance of the resulting complex is measured at 530 nm. For measurement of total

chromium, the samples were diluted with dilute nitric acid and assayed by graphite furnace atomic absorption

(GFAA) directly.
NOT FOR DISTRIBUTION OR ATTRIBUTION

Chromium Picolinate Monohydrate, NTP TR 556 N-3

Board Draft 

[

14C]-Chromium Picolinate

Radiolabeled chromium picolinate, labeled with 14C at multiple sites in the aromatic rings, was synthesized by

Wizard Laboratories (West Sacramento, CA) at a specific activity of 52.0 mCi/mmole of chromium picolinate

(17.3 mCi/mmole of each of the three picolinate ligands). The [14C]-chromium picolinate was supplied as a neat

solid and its radiochemical purity was determined by HPLC. Following injection of [14C]-chromium picolinate,

the column effluent was collected in fractions. The radioactivity eluting in each fraction was measured by liquid

scintillation spectrometry (LSS). The identity of the [14C]-chromium picolinate was tentatively confirmed by

comparison of its retention time in HPLC system 1 with that of the chromium picolinate standard. 

High-Performance Liquid Chromatography

HPLC analysis was performed on a system consisting of two Waters 6000A pumps, a Waters 715 ULTRA WISP

(Waters, Milford, MA), an ABI 785A Programmable Absorbance Detector (ABI, Forest City, CA) set at 264 nm,

and an IN/US Systems $-Ram Radioactivity Detector (IN/US Systems, Fairfield, NJ). Mobile phase solvents were

combined by volume and all mobile phase gradients were linear. 

A Waters [μBondapakTM column (300 mm × 3.9 mm)] was used for HPLC system 1. For HPLC system 1, the

mobile phase was isocratic for 8 minutes at 20:80 methanol:water and then changed over 2 minutes to 80:20

methanol:water and held for 2 minutes. The flow rate was 2 mL/minute. A Phenomenex Luna® (Phenomenex,

Torrance, CA) C18(2) column (150 mm × 4.6 mm) was used for HPLC systems 2 and 3. For HPLC system 2, the

mobile phase was isocratic at 10:90 0.01% trifluoroacetic acid in methanol:0.01% aqueous trifluoroacetic acid for

2 minutes and then changed over 8 minutes to a 30:70 ratio of these solvents where it was held for 2 minutes. The

flow rate was 2 mL/minute. For HPLC system 3, the mobile phase was isocratic at 10:90 0.01% trifluoroacetic acid

in methanol:0.01% aqueous trifluoroacetic acid for 2 minutes and then changed over 15 minutes to a 40:60 ratio of

these solvents. The flow rate was 1 mL/minute. A Phenomenex Luna® (Anasys Technologies, Lake Forest, CA)

phenyl-hexyl column (150 mm × 4.6 mm) was used for HPLC system 4. For HPLC system 4, the mobile phase

was isocratic for 8 minutes at 10:90 methanol:water and then changed over 2 minutes to 80:20 methanol:water and

held for 2 minutes. The flow rate was 2 mL/minute. For HPLC system 5, the mobile phase was isocratic at 10:90

0.01% trifluoroacetic acid in methanol:0.01% aqueous trifluoroacetic acid for 2 minutes and then changed over

8 minutes to a 50:50 ratio of these solvents and held there for 2 minutes. The flow rate was 2 mL/minute. For

system 7, the mobile phase was isocratic at 25:75 0.01% trifluoroacetic acid in methanol:0.01% aqueous

trifluoroacetic acid and the flow rate was 0.5 mL/minute.

Mass Spectrometry

Electrospray mass spectra (including MS/MS and MS3 spectra) of nonlabeled chromium picolinate, urinary

metabolite Ur1, and the glycine conjugate of picolinic acid (N-picolinoylglycine), and were obtained on a Finnigan

LCQ (Finnigan Thermo Electron, San Jose, CA) mass spectrometer. Solutions containing the analytes of interest

were introduced by direct infusion at a rate of 3.0 μL/minute or by HPLC.

Animals

Adult male Fischer 344/N rats and B6C3F1 mice were purchased from Charles River Laboratories, Inc. (Raleigh,

NC). The animals were quarantined at least 1 week before they were used in a study. The animals were identified

by individual ear tags and they were given Certified Purina Rodent Chow #5002 and tap water ad libitum. All

animals were stored in standard polycarbonate cages until they were used in an experiment. Chromium picolinate

was formulated in water (pH, .3) or propylene glycol at a dose volume of 5 mL/kg for both formulations. Oral

doses were administered from a syringe fitted with a 16 gauge ball-tipped gavage needle. Following dosing, the

animals were housed in individual glass metabolism chambers that provided for separate collection of urine, feces,

and breath (volatile organics and CO2

). 
N-4 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft

Preparation of Aqueous Oral Dose Formulations of [14C]-Chromium Picolinate

A known amount of [14C]-chromium picolinate was dissolved in water. Weighed aliquots of the solution were

transferred to silylated glass vials to prepare individual doses for each animal in the studies. The contents of the

vials were frozen and lyophilized to dryness. Immediately prior to dosing, 0.5 mL of water was added to each dose

vial and after mixing on a vortex mixer to form a chromium picolinate/water slurry, the slurry was given by

gavage.

Single 15.3 mg/kg Oral Dose of [14C]-Chromium Picolinate in Propylene Glycol to Rats

A dose formulation of [14C]-chromium picolinate dissolved in propylene glycol at a concentration of 2.84 mg

(33.6 μCi) chromium picolinate per gram of dose preparation was prepared. The dose preparation was analyzed for

radiochemical purity using HPLC system 4. Single oral doses were given by gavage to four male rats (71 days old,

219 to 233 grams). Urine and feces were collected until excretion was essentially complete (52 hours) and

analyzed for total radiolabel by LSS and chromium by the method described below. Exhaled organic volatiles and

CO2 were collected for 24 and 48 hours, respectively. The animals were sacrificed at 52 hours postdosing. The

following tissues were collected: adipose (two sites), bladder, blood, brain, heart, kidney, liver, lung, muscle (two

sites), skin (ear), spleen, testis, stomach (with contents), small intestine (with contents), large intestine (with

contents), and cecum (with contents). The collected tissues were stored at approximately –20° C until analyzed for

total radiolabel by tissue solubilization followed by LSS. Aliquots of the 8- and 24-hour urine collections were

analyzed for chromium picolinate and metabolites by HPLC/LSS using HPLC system 2.

Single 17.4 mg/kg Oral Dose of [14C]-Chromium Picolinate to Rats as an Aqueous Slurry

A slurry of [14C]-chromium picolinate was prepared in water at an activity of 9.93 μCi per mg chromium

picolinate. Following mixing on a vortex mixer to form a chromium picolinate water slurry, doses were given by

gavage to four male rats (58 days old, 206 to 217 grams). Excreta and tissues were collected, stored and analyzed

as described above.

Single 20.5 mg/kg Oral Dose of [14C]-Chromium Picolinate to Mice as an Aqueous Slurry

A slurry of [14C]-chromium picolinate was prepared in water at an activity of 30.4 μCi per mg chromium

picolinate. Following mixing on a vortex mixer to form a chromium picolinate water slurry, doses were given by

gavage to four male mice (67 days old, 20.0 to 22.7 grams). Urine and feces were collected at 6, 12, 24, and

48 hours and analyzed for total radiolabel by LSS. The remaining urine and feces were stored at approximately

–20° C for later analysis for chromium picolinate, metabolites, and chromium. The animals were sacrificed at

48 hours postdosing. The following tissues were collected: muscle, adipose, liver, and blood and stored at

approximately –20° C for later analysis. Aliquots of the 6-, 12-, and 24-hour urine collections were analyzed for

chromium picolinate and metabolites by HPLC/LSS using HPLC system 3.

Single 19.0 mg/kg Oral Dose of [14C]-Chromium Picolinate in Propylene Glycol to Mice

A dose formulation of [14C]-chromium picolinate dissolved in propylene glycol at a concentration of 1.58 mg

(39.1 μCi) chromium picolinate per gram of dose preparation was prepared. Single oral doses were given by

gavage to four male mice (68 days old, 20.7 to 22.6 grams). Tissues and excreta were collected, stored, and

analyzed as described above. An additional timepoint at 48 hours was included in the urine metabolite analysis. 

Analysis of Biological Samples for Carbon-14 Content 

Samples were assayed for carbon-14 either directly (after dissolution in a scintillation cocktail) or following

solubilization in 2 N ethanolic sodium hydroxide or Soluene®

-350 (PerkinElmer, Inc., Boston, MA). Samples that

were too dark were bleached and neutralized (perchloric acid/hydrogen peroxide) prior to analysis by LSS. Ultima

GoldTM scintillation cocktail (PerkinElmer, Inc.) was used in all determinations of radiochemical content.
NOT FOR DISTRIBUTION OR ATTRIBUTION

Chromium Picolinate Monohydrate, NTP TR 556 N-5

Samples of urine and feces excreted during the acclimation period were collected immediately prior to dosing for

determination of background carbon-14 levels. Duplicate aliquots of urine and cage rinse were weighed into

scintillation vials containing approximately 15 mL of scintillation cocktail for 14C analysis. Fecal samples were

homogenized after adding an approximately equal mass of water. The total homogenate weight was recorded.

Triplicate homogenate aliquots were weighed into scintillation vials containing 2 mL of Soluene®-350. After

dissolution, scintillation cocktail was added. Duplicate aliquots of the trapping solution collections were weighed

into scintillation vials containing scintillation cocktail. Tissue and blood samples were added to Soluene®

-350 and

agitated using a reciprocating shaker until they were fully dissolved. Liver, lung, blood, and kidney samples were

then neutralized with 125 μL of perchloric acid and decolorized with 300 μL of hydrogen peroxide. Scintillation

cocktail was added to all tissue vials. Stomach (with contents), cecum (with contents), small and large intestines

(with contents), and the residual carcass placed in 2 N ethanolic sodium hydroxide were also agitated until

dissolved. Aliquots were weighed into scintillation vials containing approximately 15 mL of scintillation cocktail. 

Analysis of Urine, Feces, and Tissues for Total Chromium

Aliquots of urine and feces from animals receiving [14C]-chromium picolinate were analyzed for total chromium.

The fecal samples were prepared for analysis as described above. The resulting solutions were analyzed for

chromium using a PerkinElmer Optima 4300 DV inductively coupled plasma-atomic emission spectrometer. 

Isolation and Identification of the Urinary Metabolite 

Urinary metabolite Ur1, the single major radioactive peak found in the urine of rats receiving [14C]-chromium

picolinate orally, was isolated from urine by multiple HPLC injections (system 5). The fractions containing the

metabolite were collected, combined, and concentrated. The isolated metabolite was analyzed by HPLC/MS/MS

using the conditions of HPLC system 7 on a Finnigan LCQ mass spectrometer. The isolated metabolite was also

diluted with nitric acid and assayed directly for total chromium by GFAA. The glycine conjugate of picolinic acid

(N-picolinoylglycine) was synthesized as described by Reddy et al. (1990) from picolinic acid and glycine ethyl

ester using dicyclohexylcarbodiimide as the coupling agent followed by hydrolysis of the resulting ethyl ester. The

synthetic product and isolated Ur1 were co-chromatographed using HPLC systems 5 and 6. The synthetic

N-picolinoylglycine was also analyzed by HPLC/MS/MS using the same conditions as those for the isolated

urinary metabolite.

RESULTS

15.3 mg/kg Oral Dose of [14C]-Chromium Picolinate in Propylene Glycol to Rats

Excretion of radioactivity in rats following administration of [14C]-chromium picolinate is shown in Table N1. The

rats dosed orally with [14C]-chromium picolinate dissolved in propylene glycol excreted an average of 53% of the

14C dose in urine, 39% in feces, and 1.5% as CO2 in breath in 24 hours. No measurable dose was collected as

volatiles in breath. An additional 8% of the 14C dose was excreted during the collections at 48 and 52 hours. The

results of the assay of total chromium excreted in urine and feces collected from the animals are also shown in

Table N1. An average of 1.25% ± 0.24% and 97.5% ± 7.4% of the chromium dose was excreted in urine and

feces, respectively, in 48 hours. The results of the HPLC assay of the 8- and 24-hour urine collections are shown

in Table N2. An average of 49.0% ± 1.2% the 14C dose received was excreted in 24 hours as N-picolinoylglycine.

An average of 1.9% ± 0.4% and 1.1% ± 0.7% of the 14C dose received was excreted in 24 hours as picolinic acid

and chromium picolinate, respectively. The results of the assay of radioactivity in all tissues collected at 52 hours

postdosing from the four rats are shown in Table N3. Less than 1% of the 14C dose was found in these tissues. 

17.4 mg/kg Oral Dose of [14C]-Chromium Picolinate to Rats as an Aqueous Slurry

Excretion of radioactivity in rats sacrificed at 48 hours following administration of [14C]-chromium picolinate is

shown in Table N4. The rats dosed orally with [14C]-chromium picolinate water slurry excreted an average of 41%

Board Draft 
N-6

of the 14C dose in urine, 47% in feces, and 1.4% as CO2 in breath in 24 hours. An additional 4% of the 14C dose

was excreted during the collections at 48 hours. Excretion of total chromium from rats sacrificed at 48 hours

following administration of chromium picolinate is also shown in Table N4. An average of 1.53% ± 0.51% and

97.6% ± 7.4% of the chromium dose was excreted in urine and feces, respectively, in 48 hours. The results of the

assay of radioactivity in all tissues collected at 48 hours postdosing from four rats are shown in Table N5. Less

than 1% of the 14C dose administered was found in the non-gastrointestinal tract tissues at 48 hours; the

gastrointestinal tract tissues and contents contained 0.15% of the administered radioactivity.

Single 20.5 mg/kg Oral Dose of [14C]-Chromium Picolinate to Mice as an Aqueous Slurry

Excretion of radioactivity in mice sacrificed at 48 hours following administration of [14C]-chromium picolinate is

shown in Table N6. The mice excreted an average of 26% of the 14C dose in urine and 59% in feces in 48 hours.

Less than 0.1% of the 14C dose was excreted as CO2 in breath in 48 hours, and an average of 0.2% of the 14C dose

was recovered in the digested carcasses (data not shown). The results of the assay of total chromium excreted in

urine and feces collected from these animals are also shown in Table N6. An average of 1.1% ± 0.7% and

105% ± 7.8% of the chromium dose was excreted in urine and feces, respectively, in 48 hours. The results of the

HPLC assay of the 6-, 12-, and 24-hour urine collections are shown in Table N7. An average of 22.3% ± 4.2% of

the 14C dose was excreted in 24 hours as N-picolinoylglycine, the urinary metabolite isolated and identified from

rat urine. An average of 0.29% ± 0.04% and 0.51% ± 0.20% of the 14C dose was excreted in 24 hours as picolinic

acid and chromium picolinate, respectively. Also present in mouse urine was an additional metabolite, MUr1, that

accounted for an average of 0.16% ± 0.12% of the 14C dose.

Single 19.0 mg/kg Oral Dose of [14C]-Chromium Picolinate in Propylene Glycol to Mice

Excretion of radioactivity in mice sacrificed at 48 hours following administration of [14C]-chromium picolinate is

shown in Table N8. The mice excreted an average of 42.0% ± 7.8% of the 14C dose in urine and 55.1% ± 5.6% in

feces in 48 hours. The results of the assay of total chromium excreted in urine and feces are also shown in

Table N8. An average of 3.9% ± 1.1% and 91.3% ± 12.7% of the chromium dose was excreted in urine and feces,

respectively, in 48 hours. The results of the assay of radioactivity in tissues, gastrointestinal tract and contents, and

residual carcass collected at 48 hours postdosing are shown in Table N9. A total of less than 1% of the 14C dose

was found in the tissues of these animals at 48 hours postdosing. The results of the HPLC assay of the 6-, 12-,

24-, and 48-hour urine collections are shown in Table N10. An average of 31.8% ± 7.6% of the 14C dose was

excreted in 48 hours as N-picolinoylglycine, the urinary metabolite isolated and identified from rat urine. An

average of 1.3% ± 0.8% and 2.8% ± 1.0% of the 14C dose was excreted in 48 hours as picolinic acid and chromium

picolinate, respectively. Also present in mouse urine was an additional metabolite, MUr1, that accounted for an

average of 0.25% ± 0.11% of the 14C dose.

Isolation and Identification of the Urinary Metabolite

Only a single radiolabeled metabolite (Ur1) was excreted in rat urine following an oral dose of [14C]-chromium

picolinate. This metabolite was isolated and purified by HPLC. No apparent decomposition was observed during

the purification process. Analysis of the isolated and purified metabolite for total chromium showed that Ur1 does

not contain chromium. A sample of Ur1 that would have contained 75 μg-Eq/L of chromium based on the

chromium/14C ratio in chromium picolinate was found to contain less than 10 μg/L chromium (the limit of

quantitation of the method).

The isolated urinary metabolite Ur1 and synthetic N-picolinoylglycine co-eluted when analyzed on two different

HPLC columns. HPLC/MS spectra of N-picolinoylglycine and Ur1 each contained a base peak at m/z 181

(NPG + H)+. HPLC/MS/MS experiments showed the m/z peak at 181 in both spectra fragmented to produce major

peaks at m/z 163 (loss of OH) and m/z 135 (loss of CHO2

).

Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft
NOT FOR DISTRIBUTION OR ATTRIBUTION

Chromium Picolinate Monohydrate, NTP TR 556 N-7

DISCUSSION

The poor bioavailability of chromium III, even though it is an essential mineral, is well known. The poor

bioavailability is little improved when chromium is complexed with picolinic acid or similar complexing agents.

Chromium picolinate has low water solubility. The rate of dissolution of the solid complex could be slow enough

to affect bioavailability. One of the goals of these studies was to establish the effect of formulation on

bioavailability. Propylene glycol was found to be a good solvent for the complex allowing comparison of

bioavailability of a homogenous formulation to an aqueous slurry. Comparing total chromium excreted in urine

following dosing with either the solution or slurry reveals little difference in rats. However, formulation may have

an effect on bioavailability in mice, as less of the dose was excreted in urine when the formulation was an aqueous

slurry (1.1%) than when the formulation was a solution (3.9%).

A second question these studies sought to answer was the form of chromium in circulation. The analysis of urine

revealed that about 1% of the administered chromium picolinate is excreted unchanged in the urine of rats. In

mice, 0.5% of the administered dose is excreted unchanged when the formulation is an aqueous slurry, and 2.5%

when the formulation is a solution. Analysis of blood 1 hour following a 17.4 mg/kg oral dose indicated a

concentration of 31 ng chromium picolinate/g of blood (data not shown). At this same time the total radioactivity

in blood was 290 ng-Eq of chromium picolinate, so no more than 10% of the chromium in the blood was

associated with picolinate.

Picolinic acid represents about 83% of the mass of chromium picolinate. The fate of this part of the complex has

received little attention. Excretion of picolinate-derived radioactivity in rats is about equally divided between urine

and feces. Excretion of chromium, however, is nearly 100% in feces. This implies that much of the picolinate is

absorbed without the chromium attached. The major urinary metabolite is a glycine conjugate of picolinic acid.

The excretion pattern is similar in mice. The ratio of excretion in urine versus feces may be affected more by

formulation in mice. Picolinic acid is cleared rapidly in both rats and mice with elimination being primarily by

conjugation with glycine. There is little evidence for accumulation of picolinate-derived material in tissues. Less

than 1% of the administered radioactivity remains in tissues after 48 hours. 

REFERENCES

Anderson, R.A., Colton, T., Doull, J., Marks, J.G., Smith, R.G., Bruce, G.M., Finley, B.L., and Paustenbach, D.J.

(1993). Designing a biological monitoring program to assess community exposure to chromium: Conclusions of

an expert panel. J. Toxicol. Environ. Health 40, 555-583.

Anderson, R.A., Bryden, N.A., Polansky, M.M., and Gautschi, K. (1996). Dietary chromium effects on tissue

chromium concentrations and chromium absorption in rats. J. Trace Elem. Exp. Med. 9, 11-25.

Anderson, R.A., Bryden, N.A., and Polansky, M.M. (1997). Lack of toxicity of chromium chloride and chromium

picolinate in rats. J. Am. Coll. Nutr. 16, 273-279.

Gargas, M.L., Norton, R.L., Paustenbach, D.J., and Finley, B.L. (1994). Urinary excretion of chromium by

humans following ingestion of chromium picolinate. Implications for biomonitoring. Drug Metab. Dispos. 22,

522-529.

Reddy, K.V., Jin, S.-J., Arora, P.K., Sfeir, D.S., Maloney, S.C.F., Urbach, F.L., and Sayre, L.M. (1990).

Copper-mediated oxidative C-terminal N-dealkylation of peptide-derived ligands. A possible model for enzymatic

generation of desglycine peptide amides. J. Am. Chem. Soc. 112, 2332-2340. 

Board Draft 
N-8 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft

TABLE N1

Excretion of Radioactivity and Total Chromium by Male Rats After a Single Gavage Dose 

of 15.3 mg/kg [14C]-Chromium Picolinate in Propylene Glycola

Time (hours) Urine Feces CO2 Total

Radioactivity

8 22.8 ± 2.1 —b 0.1 ± 0.1 22.9 ± 2.1

24 53.4 ± 0.9 39 ± 4.9 1.5 ± 0.5 94.2 ± 6.1

48 56.3 ± 0.9 43.6 ± 3.4 2.2 ± 0.5 102 ± 3.8

52 56.4 ± 0.9 43.6 ± 3.4 2.2 ± 0.5 102 ± 3.8

Total Chromium

8 0.97 ± 0.26 — NA 0.97 ± 0.26

24 1.18 ± 0.24 82.5 ± 12.2 NA 83.7 ± 12.1

48 1.25 ± 0.24 97.5 ± 7.4 NA 98.8 ± 7.3

a Values are presented as cumulative percent of dose (mean ± standard deviation) for four rats at each timepoint. NA=not applicable. b Not collected

TABLE N2

Urinary Excretion of Chromium Picolinate and Metabolites by Male Rats After a Single Gavage Dose 

of 15.3 mg/kg [14C]-Chromium Picolinate in Propylene Glycola

Percent of Dose Co-eluting with:

Time Interval Chromium Picolinate Picolinic Acid N-Picolinoylglycine

(hours)

0-8 1.06 ± 0.66 1.07 ± 0.35 20.0 ± 2.0

8-24 0.08 ± 0.06 0.822 ± 0.053 28.2 ± 2.8

0-24 1.12 ± 0.67 1.90 ± 0.37 49.0 ± 1.2

a Values are presented as mean ± standard deviation for four rats at each time interval.
NOT FOR DISTRIBUTION OR ATTRIBUTION

Chromium Picolinate Monohydrate, NTP TR 556 N-9

Board Draft 

TABLE N3

Distribution of Radioactivity in Tissues of Male Rats 52 Hours After a Single Gavage Dose 

of 15.3 mg/kg [14C]-Chromium Picolinate in Propylene Glycola

Tissue ng-Eqb Chromium Picolinate Tissue/Blood Ratio Percent Dose in

per gram Tissue Total Tissue

Adipose 149 ± 52 2.70 ± 1.28 0.067 ± 0.022

Bladder 142 ± 44 2.54 ± 1.1 0.0005 ± 0.0003

Blood 62.5 ± 23.2 Unity 0.021 ± 0.0079

Brain 35.0 ± 5.0 0.596 ± 0.140 0.002 ± 0.000

Heart 71.8 ± 17 1.22 ± 0.35 0.001 ± 0.000

Kidney 281 ± 29 4.82 ± 0.77 0.014 ± 0.002

Liver 437 ± 163 7.01 ± 0.77 0.117 ± 0.047

Lung 71.8 ± 41.3 1.09 ± 0.23 0.002 ± 0.001

Muscle 33.1 ± 11.2 0.542 ± 0.129 0.103 ± 0.035

Skin 211 ± 45 3.75 ± 1.40 0.232 ± 0.052

Spleen 98.1 ± 21.5 1.71 ± 0.57 0.001 ± 0.000

Testis 37.4 ± 7.0 0.631 ± 0.132 0.003 ± 0.000

Total non-gastrointestinal tract 0.459 ± 0.099

Total gastrointestinal tract 0.177 ± 0.040

Total in tissues 0.726 ± 0.100

a Values are mean ± standard deviation for four rats. b Eq=Equivalents

TABLE N4

Excretion of Radioactivity and Total Chromium by Male Rats After a Single Gavage Dose 

of 17.4 mg/kg [14C]-Chromium Picolinate as an Aqueous Slurrya

Time (hours) Urine Feces CO2 Total

Radioactivity

4 3.6 ± 0.1 0.00 ± 0.00 —b 3.6 ± 2.1

8 15.5 ± 3.8 5.3 ± 10.6 0.1 ± 0.0 20.9 ± 11.1

12 26.6 ± 3.1 8.9 ± 8.3 0.1 ± 0.1 35.6 ± 7.8

24 40.9 ± 3.6 46.6 ± 6.8 1.4 ± 0.7 88.9 ± 4.7

48 43.4 ± 3.5 47.8 ± 6.9 1.7 ± 0.9 92.9 ± 4.4

Total Chromium

2 0.16 ± 0.14 — NA 0.18 ± 0.14

4 0.34 ± 0.04 0.0 ± 0.0 NA 0.34 ± 0.04

8 0.66 ± 0.05 7.64 ± 15.3 NA 8.30 ± 15.3

12 0.84 ± 0.05 13.7 ± 11.5 NA 14.6 ± 11.5

24 1.16 ± 0.34 92.9 ± 8.9 NA 94.1 ± 8.8

48 1.53 ± 0.51 97.6 ± 7.4 NA 99.2 ± 7.2

a Values are presented as cumulative percent of dose (mean ± standard) deviation for four rats at each timepoint. NA=not applicable. b Not collected
N-10 Chromium Picolinate Monohydrate, NTP TR 556 

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft

TABLE N5

Distribution of Radioactivity in Tissues of Male Rats 48 Hours After a Single Gavage Dose 

of 17.4 mg/kg [14C]-Chromium Picolinate as an Aqueous Slurrya

Tissue ng-Eqb Chromium Picolinate Tissue/Blood Ratio Percent Dose in

per gram Tissue Total Tissue

Adipose 248 ± 125 3.54 ± 0.74 0.094 ± 0.048

Bladder 114 ± 50 1.65 ± 0.17 0.000 ± 0.000

Blood 66.9 ± 24.7 Unity 0.019 ± 0.007

Brain 25.9 ± 11.1 0.377 ± 0.048 0.001 ± 0.001

Heart 66.9 ± 28.0 0.977 ± 0.192 0.001 ± 0.001

Kidney 235 ± 76 3.57 ± 0.26 0.010 ± 0.004

Liver 263 ± 89 4.00 ± 0.61 0.049 ± 0.018

Lung 125 ± 55 1.81 ± 0.317 0.003 ± 0.001

Muscle 40.3 ± 21.7 0.569 ± 0.155 0.105 ± 0.059

Skin 169 ± 64 2.56 ± 0.29 0.156 ± 0.060

Spleen 133 ± 62 1.89 ± 0.46 0.002 ± 0.001

Testis 23.8 ± 11.8 0.350 ± 0.138 0.002 ± 0.001

Total non-gastrointestinal tract 0.44 ± 0.19

Total gastrointestinal tract 0.15 ± 0.07

Total in tissues 0.59 ± 0.21

a Values are mean ± standard deviation for four rats. b Eq=Equivalents

TABLE N6

Excretion of Radioactivity and Total Chromium by Male Mice After a Single Gavage Dose 

of 20.5 mg/kg [14C]-Chromium Picolinate as an Aqueous Slurrya

Time (hours) Urine Feces Total

Radioactivity

6 14.6 ± 8.9 37.8 ± 25.7 52.4 ± 33.9

12 18.1 ± 8.4 55.0 ± 6.0 73.0 ± 12.4

24 23.8 ± 4.4 57.5 ± 4.9 81.3 ± 5.6

48 25.5 ± 3.0 59.4 ± 5.0 85.0 ± 3.1

Total Chromium

6 0.8 ± 0.7 64.5 ± 43.1 65.2 ± 43.2

12 0.8 ± 0.6 98.2 ± 5.2 99.0 ± 5.4

24 0.9 ± 0.5 105 ± 7.8 106 ± 7.5

48 1.1 ± 0.7 105 ± 7.8 106 ± 7.4

a Values are presented as cumulative percent of dose (mean ± standard deviation) for four mice at each timepoint.
NOT FOR DISTRIBUTION OR ATTRIBUTION

Chromium Picolinate Monohydrate, NTP TR 556 N-11

Board Draft 

TABLE N7

Urinary Excretion of Chromium Picolinate and Metabolites by Male Mice After a Single Gavage Dose 

of 20.5 mg/kg [14C]-Chromium Picolinate as an Aqueous Slurrya

Percent of Dose Co-eluting with:

Time Interval Chromium Picolinate Picolinic Acid Mouse N-Picolinoylglycine

(hours) Unknown 1

0-6 0.40 ± 0.25 0.18 ± 0.11 0.09 ± 0.14 13.6 ± 8.3

6-12 0.038 ± 0.025 0.038 ± 0.022 0.015 ± 0.03 3.28 ± 1.34

12-24 0.078 ± 0.033 0.07 ± 0.04 0.062 ± 0.067 5.38 ± 3.98

0-24 0.51 ± 0.20 0.29 ± 0.04 0.163 ± 0.124 22.3 ± 4.16

a Values are presented as mean ± standard deviation for four mice at each time interval. 

TABLE N8

Excretion of Radioactivity and Total Chromium by Male Mice After a Single Gavage Dose 

of 19.0 mg/kg [14C]-Chromium Picolinate in Propylene Glycola

Time (hours) Urine Feces Total

Radioactivity

6 18.0 ± 6.2 19.1 ± 7.8 37.1 ± 10.1

12 24.8 ± 7.0 49.8 ± 5.4 74.6 ± 5.6

24 30.7 ± 8.2 53.0 ± 4.8 83.7 ± 4.4

48 42.0 ± 7.8 55.1 ± 5.6 97.1 ± 3.7

Total Chromium

6 2.3 ± 0.8 28.6 ± 9.7 30.9 ± 9.1

12 2.5 ± 0.7 80.3 ± 10.9 82.8 ± 11.3

24 2.8 ± 0.5 87.8 ± 12.5 90.6 ± 12.5

48 3.9 ± 1.1 91.3 ± 12.7 95.2 ± 13.7

a Values are presented as cumulative percent of dose (mean ± standard deviation) for four mice at each timepoint.
N-12 Chromium Picolinate Monohydrate, NTP TR 556 

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TABLE N9

Distribution of Radioactivity in Tissues of Male Mice 48 Hours After a Single Gavage Dose 

of 19.0 mg/kg [14C]-Chromium Picolinate in Propylene Glycola

Tissue ng-Eqb Chromium Picolinate Tissue/Blood Ratio Percent Dose in

per gram Tissue Total Tissue

Blood 4.8 ± 0.8 Unity 0.0019 ± 0.0003

Liver 63 ± 22 13.2 ± 4.3 0.014 ± 0.005

Muscle 7.5 ± 4.6 1.8 ± 0.9 0.018 ± 0.012

Adipose 7.9 ± 3.5 1.6 ± 0.5 0.0041 ± 0.0016

Total gastrointestinal tract 

(with contents) 0.047 ± 0.016

Carcass 0.432 ± 0.203

a Values are mean ± standard deviation for four mice. b Eq=Equivalents

TABLE N10

Urinary Excretion of Chromium Picolinate and Metabolites by Male Mice After a Single Gavage Dose 

of 19.0 mg/kg [14C]-Chromium Picolinate in Propylene Glycola

Percent of Dose Co-eluting with:

Time Interval Chromium Picolinate Picolinic Acid Mouse N-Picolinoylglycine

(hours) Unknown 1

0-6 2.31 ± 0.96 0.34 ± 0.12 0.12 ± 0.07 14.6 ± 5.05

6-12 0.22 ± 0.03 0.10 ± 0.011 0.056 ± 0.38 8.07 ± 2.45

12-24 0.12 ± 0.053 0.085 ± 0.04 0.032 ± 0.02 5.5 ± 3.48

24-48 0.162 ± 0.111 0.77 ± 0.91 0.052 ± 0.02 5.68 ± 1.37

0-24 2.54 ± 0.95 0.51 ± 0.10 0.19 ± 0.11 24.7 ± 9.0

0-48 2.75 ± 0.97 1.28 ± 0.83 0.248 ± 0.108 31.8 ± 7.63

a Values are presented as mean ± standard deviation for four mice at each time interval.