## 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

### NOT FOR DISTRIBUTION OR ATTRIBUTION Chromium Picolinate Monohydrate, NTP TR 556 5

Board Draft 

### 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

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft

### 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: C 18 

### H 12 

### CrN3 

### O 6 

# @ H2 

### O 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 H 2 

## O 

# @

### 8 Chromium Picolinate Monohydrate, NTP TR 556

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft 

# 3-M ONTH 

# S TUDY IN 

# R ATS 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-M ONTH 

# S TUDY 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 

# S TUDY IN 

# R ATS 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.

### NOT FOR DISTRIBUTION OR ATTRIBUTION Chromium Picolinate Monohydrate, NTP TR 556 9

Board Draft 

# 2-YEAR 

# S TUDY 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 

# T OXICOLOGY 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


# C ONCLUSIONS 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

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft

### NOT FOR DISTRIBUTION OR ATTRIBUTION 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

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft 

## 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 limitedset 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 studi es 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 increaseof 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 cl ear 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 differentlevels of evidence categories, the following convention has been adopted to convey completely the study results. In a study with clear evidence o f carcinogenic activity at some tissue sites, other responses that alone might be deemed some evidence are indicated as “were also related” tochemical 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 impo ssible 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 le sion 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.

### NOT FOR DISTRIBUTION OR ATTRIBUTION Chromium Picolinate Monohydrate, NTP TR 556 13

Board Draft 

## 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

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft 

## 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.

### NOT FOR DISTRIBUTION OR ATTRIBUTION 15

Board Draft 

# C HEMICAL AND 

# P HYSICAL 

# P ROPERTIES 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: C 18 

### H 12 

### CrN3 

### O 6 

# @ H2 

### O 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 H 2 

## O 

# @

### 16 Chromium Picolinate Monohydrate, NTP TR 556

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft 

### 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).



# P RODUCTION

# , U SE 

# , AND 

# HUMAN 

# E XPOSURE Cr III is the predominant form of chromium in nature, occurring in ores such as ferrochromite (FeCr2 

### O 4

### ). 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

# , D ISTRIBUTION

# , METABOLISM

# , AND 

# E XCRETION 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

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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 (HOCrO 3 –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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# T OXICITY 

## 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 

# D EVELOPMENTAL 

# T OXICITY 

## 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

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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.



# C ARCINOGENICITY 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 

# T OXICITY 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/ 

### p un

### ) 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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# S TUDY 

# R ATIONALE 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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# P ROCUREMENT AND 

# C HARACTERIZATION OF 

# C HROMIUM 

# P ICOLINATE 

# M ONOHYDRATE 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.



# P REPARATION AND 

# ANALYSIS OF 

# D OSE 

# F ORMULATIONS 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-M ONTH 

# S TUDIES 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 andhomogenizing 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 

# S TUDIES 

## 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,

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### T ABLE 

### 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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### T ABLE 

### 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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# S TATISTICAL 

# 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 fromthe 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. 

### T ABLE 

### 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.



# Q UALITY 

# A SSURANCE 

# 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 

# T OXICOLOGY 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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# R

ATS 

# 3-M ONTH 

# S TUDY 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

T ABLE 

### 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 Consumption c 

### (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.

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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 

# S TUDY 

## 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.

T ABLE 

### 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 35 d 28 Percent probability of survival at end of studya 74 72 70 56 Mean survival (days)b 705 683 705 683 Survival analysis c 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 36 e 35 36 40 e 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

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### F IGURE 

### 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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### T ABLE 

### 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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### T ABLE 

### 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

### 52 Chromium Picolinate Monohydrate, NTP TR 556

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### F IGURE 

### 2 Growth Curves for Male and Female Rats Exposed to Chromium Picolinate Monohydrate in Feed for 2 Years

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Board Draft 

## 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 theducts 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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### T ABLE 

### 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 rate d 1/50 (2%) 1/50 (2%) 7/50 (14%) 4/50 (8%) Adjusted rate e 2.2% 2.3% 14.9% 9.3% Terminal rate f 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-M ONTH 

# S TUDY 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 Consumption c 

### (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. Chromium Picolinate Monohydrate, NTP TR 556

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### NOT FOR DISTRIBUTION OR ATTRIBUTION Chromium Picolinate Monohydrate, NTP TR 556 57

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### T ABLE 

### 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 45 d Percent probability of survival at end of studya 92 86 76 90 Mean survival (days)b 715 701 704 705 Survival analysis c 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 

# S TUDY 

## 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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### F IGURE 

### 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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### T ABLE 

### 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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### T ABLE 

### 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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### F IGURE 

### 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.

### 64 Chromium Picolinate Monohydrate, NTP TR 556

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# GENETIC 

# T OXICOLOGY 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).

### NOT FOR DISTRIBUTION OR ATTRIBUTION Chromium Picolinate Monohydrate, NTP TR 556 65


# ABSORPTION

# , D ISTRIBUTION

# , METABOLISM

# , AND 

# E XCRETION Absorption, distribution, metabolism, and excretion studies were conducted using [ 14 

### C]-chromium picolinate monohydrate in rats and mice; the [ 14 

### C]-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 14 

### C, 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 14 

### C. 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 14 

### C, 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 14 

### C. 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 [ 14 

### C]-picolinate-derived radioactivity was excreted about equally in feces and urine, implying that most of picolinate was absorbed without the chromium.



# C HROMIUM 

# T ISSUE 

# D ISTRIBUTION 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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### 66


### 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 Chromium Picolinate Monohydrate, NTP TR 556

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### F IGURE 

### 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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### 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

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### 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 Chromium Picolinate Monohydrate, NTP TR 556

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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 [ 14 

### C]-chromium picolinate monohydrate were conducted to aid in the design and interpretation of the present studies (Appendix N); the 14 

### C-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. Chromium Picolinate Monohydrate, NTP TR 556

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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 ofCr 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.



# C ONCLUSIONS 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. Department of Health and Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry. Alexander J., and Aaseth, J. (1995). Uptake of chromate in human red blood cells and isolated rat liver cells: The 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. 10 , 179-183. 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. 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. Andersson, M.A., Petersson Grawé, K.V., Karlsson, O.M., Abramsson-Zetterberg, L.A., and Hellman, B.E. (2007). Evaluation of the potential genotoxicity of chromium picolinate in mammalian cells in vivo and in vitro

. Food Chem. Toxicol. 45 , 1097-1106. Ashby, J., and Tennant, R.W. (1991). Definitive relationships among chemical structure, carcinogenicity and mutagenicity for 301 chemicals tested by the U.S. NTP. Mutat. Res. 257 , 229-306. Bailer, A.J., and Portier, C.J. (1988). Effects of treatment-induced mortality and tumor-induced mortality on tests for carcinogenicity in small samples. Biometrics 44 , 417-431. Bailey, M.M., Boohaker, J.G., Sawyer, R.D., Behling, J.E., Rasco, J.F., Jernigan, J.J., Hood, R.D., and Vincent, J.B. (2006). Exposure of pregnant mice to chromium picolinate results in skeletal defects in their offspring. Birth Defects Res. 77 , 244-249. Barnhart, J. (1997). Chromium, chemistry and implications for environmental fate and toxicity. J. Soil Contam. 6 , 561-568. Bataineh, H., al-Hamood, M.H., Elbetieha, A., and Bani Hani, I. (1997). Effect of long-term ingestion of chromium compounds on aggression, sex behavior and fertility in adult male rat. Drug Chem. Toxicol. 20 , 133-149. 

# REFERENCES

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### Bieler, G.S., and Williams, R.L. (1993). Ratio estimates, the delta method, and quantal response tests for increased carcinogenicity. Biometrics 49 , 793-801. Boorman, G.A., Montgomery, C.A., Jr., Eustis, S.L., Wolfe, M.J., McConnell, E.E., and Hardisty, J.F. (1985). Quality assurance in pathology for rodent carcinogenicity studies. In Handbook of Carcinogen Testing (H.A. Milman and E.K. Weisburger, Eds.), pp. 345-357. Noyes Publications, Park Ridge, NJ. Bragt, P.C., and van Dura, E.A. (1983). Toxicokinetics of hexavalent chromium in the rat after intratracheal administration of chromates of different solubilities. Ann. Occup. Hyg. 27 , 315-322. Bridgewater, L.C., Manning, F.C.R., Woo, E.S., and Patierno, S.R. (1994). DNA polymerase arrest by adducted trivalent chromium. Mol. Carcinog. 9 , 122-133. Broadhurst, L.C., Schmidt, W.F., Reeves, J.B., III, Polansky, M.M., Gautschi, K., and Anderson, R.A. (1997). Characterization and structure by NMR and FTIR spectroscopy, and molecular modeling of chromium (III) picolinate and nicotinate complexes utilized for nutritional supplementation. J. Inorg. Biochem

. 66 , 119-130. Campbell, W.W., Joseph, L.J., Anderson, R.A., Davey, S.L., Hinton, J., and Evans, W.J. (2002). Effects of resistive training and chromium picolinate on body composition and skeletal muscle size in older women. Int. J. Sport Nutr. Exerc. Metab. 12 , 125-135. Cerulli, J., Grabe, D.W., Gauthier, I., Malone, M., and McGoldrick, M.D. (1998). Chromium picolinate toxicity. Ann. Pharmacother. 32 , 428-431. Clodfelder, B.J., Emamaullee, J., Hepburn, D.D.D., Chakov, N.E., Nettles, H.S., and Vincent, J.B. (2001). The trail of chromium(III) in vivo from the blood to the urine: The roles of transferrin and chromodulin. J. Biol. Inorg. Chem. 6 , 608-617. Code of Federal Regulations (CFR) 21 , Part 58. Cohen, M.D., Kargacin, B., Klein, C.B., and Costa, M. (1993). Mechanisms of chromium carcinogenicity and toxicity. Crit. Rev. Toxicol. 23 , 255-281. Copeland-Haines, D., and Eustis, S.L. (1990). Specialized sebaceous glands. In Pathology of the Fischer Rat. Reference and Atlas (G.A. Boorman, S.L. Eustis, M.R. Elwell, C.A. Montgomery, Jr., and W.F. MacKenzie, Eds.), pp. 279–293. Academic Press, Inc., San Diego. Coryell, V.H., and Stearns, D.M. (2006). Molecular analysis of hprt mutations induced by chromium picolinate in CHO AA8 cells. Mutat. Res. 610 , 114-123. Costa, M. (1997). Toxicity and carcinogenicity of Cr(VI) in animal models and humans. Crit. Rev. Toxicol. 27 , 431-442. Costa, M., and Klein, C.B. (2006). Toxicity and carcinogenicity of chromium compounds in humans. Crit. Rev. Toxicol. 36 , 155-163. Cox, D.R. (1972). Regression models and life-tables. J. R. Stat. Soc. B34 , 187-220. Crawford, B.D. (1985). Perspectives on the somatic mutation model of carcinogenesis. In Advances in Modern Environmental Toxicology. Mechanisms and Toxicity of Chemical Carcinogens and Mutagens (M.A. Mehlman, W.G. Flamm, and R.J. Lorentzen, Eds.), pp. 13-59. Princeton Scientific Publishing Co., Inc., Princeton, NJ.

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### De Flora, S., Bagnasco, M., Serra, D., and Zanacchi, P. (1990). Genotoxicity of chromium compounds. A review. Mutat. Res. 238 , 99-172. Derelanko, M.J., Rinehart, W.E., Hilaski, R.J., Thompson, R.B., and Löser, E. (1999). Thirteen-week subchronic rat inhalation toxicity study with a recovery phase of trivalent chromium compounds, chromic oxide, and basic chromium sulfate. Toxicol. Sci. 52 , 278-288. Dixon, W.J., and Massey, F.J., Jr. (1957). Introduction to Statistical Analysis, 2nd ed., pp. 276-278, 412. McGraw-Hill Book Company, Inc., New York. Donaldson, R.M., Jr., and Barreras, R.F. (1966). Intestinal absorption of trace quantities of chromium. J. Lab. Clin. Med. 68 , 484-493. Dunn, O.J. (1964). Multiple comparisons using rank sums. Technometrics 6 , 241-252. Dunnett, C.W. (1955). A multiple comparison procedure for comparing several treatments with a control. J. Am. Stat. Assoc. 50 , 1096-1121. Elbetieha, A., and Al-Hamood, M.H. (1997). Long-term exposure of male and female mice to trivalent and hexavalent chromium compounds: Effect on fertility. Toxicology 116 , 39-47. Ernst, E. (1990). Testicular toxicity following short-term exposure to tri- and hexavalent chromium: An experimental study in the rat. Toxicol. Lett. 51 , 269-275. Evans, G.W., and Pouchnik, D.J. (1993). Composition and biological activity of chromium-pyridine carboxylate complexes. J. Inorg. Biochem. 49 , 177-187. Fowler, J.F., Jr. (2000). Systemic contact dermatitis caused by oral chromium picolinate. Cutis 65 , 116. 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. Gart, J.J., Chu, K.C., and Tarone, R.E. (1979). Statistical issues in interpretation of chronic bioassay tests for carcinogenicity. JNCI 62 , 957-974. Girard, D.M., and Sager, D.B. (1987). The use of Markov chains to detect subtle variation in reproductive cycling. Biometrics 43 , 225-234. Gray, S.J., and Sterling, K. (1950). The tagging of reds cells and plasma proteins with radioactive chromium. J. Clin. Invest. 29 , 1604-1613. Green, C.A., Bianchini, R.J., and Legg, J.I. (1984). Characterization of a stable chromium(III)-nicotinic acid complex by deuteron NMR. Inorg. Chem. 23 , 2713-2715. Gudi, R., Slesinski, R.S., Clarke, J.J., and San, R.H. (2005). Chromium picolinate does not produce chromosome damage in CHO cells. Mutat. Res. 587 , 140-146. Hartford, W.H. (1979). Chromium compounds. In Kirk-Othmer Encyclopedia of Chemical Technology , 3rd ed., (M. Grayson, Ed.), Vol. 6, pp. 82-120. John Wiley and Sons, New York.

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### Heddle, J.A., Hite, M., Kirkhart, B., Mavournin, K., MacGregor, J.T., Newell, G.W., and Salamone, M.F. (1983). The induction of micronuclei as a measure of genotoxicity. A report of the U.S. Environmental Protection Agency Gene-Tox Program. Mutat. Res. 123 , 61-118. Hepburn, D.D., and Vincent, J.B. (2002). In vivo distribution of chromium from chromium picolinate in rats and implications for the safety of the dietary supplement. Chem. Res. Toxicol. 15 , 93-100. Hepburn, D.D., and Vincent, J.B. (2003). Tissue and subcellular distribution of chromium picolinate with time after entering the bloodstream. J. Inorg. Biochem. 94 , 86-93. Huszonek, J. (1993). Over-the-counter chromium picolinate. Am. J. Psychiatry 150 , 1560-1561. Integrated Laboratory Systems (ILS) (1990). Micronucleus Data Management and Statistical Analysis Software, Version 1.4. ILS, Inc., P.O. Box 13501, Research Triangle Park, NC 27707. International Agency for Research on Cancer (IARC) (1990). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans: Chromium, Vol. 49, pp. 49-256. IARC, Lyon, France. Institute of Medicine (IOM) (2001). Dietary Reference Intakes for Vitamin A, Vitamin K, Arsenic, Boron, Chromium, Copper, Iodine, Iron, Manganese, Molybdenum, Nickel, Silicon, Vanadium, and Zinc. Food and Nutrition Board, Institute of Medicine. National Academy Press, Washington, DC. Ivankovic, S., and Preussmann, R. (1975). Absence of toxic and carcinogenic effects after administration of high doses of chromic oxide pigment in subacute and long-term experiments in rats. Food Cosmet. Toxicol. 13 , 347-351. Jonckheere, A.R. (1954). A distribution-free k -sample test against ordered alternatives. Biometrika 41 , 133-145. Kaplan, E.L., and Meier, P. (1958). Nonparametric estimation from incomplete observations. J. Am. Stat. Assoc. 53 , 457-481. Kerger, B.D., Finley, B.L., Corbett, G.E., Dodge, D.G., and Paustenbach, D.J. (1997). Ingestion of chromium (VI) in drinking water by human volunteers: Absorption, distribution, and excretion of single and repeated doses. J. Toxicol. Environ. Health 50 , 67-95. Kirpnick-Sobol, Z., Reliene, R., and Schiestl, R.H. (2006). Carcinogenic Cr(VI) and the nutritional supplement Cr(III) induce DNA deletions in yeast and mice. Cancer Res. 66 , 3480-3484. Kurokawa, Y., Matsushima, M., Imazawa, T., Takamura, N., Takahashi, M., and Hayashi, Y. (1985). Promoting effect of metal compounds on rat renal tumorigenesis. J. Am. Coll. Toxicol. 4 , 321-330. Lamson, D.W., and Plaza, S.M. (2002). The safety and efficacy of high-dose chromium. Altern. Med. Rev. 7 , 218-235. Laskin, S., Kuschner, M., and Drew, R.T. (1970). Studies in pulmonary carcinogenesis. In Inhalation Carcinogenesis , U.S. Atomic Energy Commission Symposium Series No. 18, (M.G. Hanna, Jr., P. Nettesheim, J.R. Gilbert, Eds.), pp. 321-351. U.S. Atomic Energy Commission, Division of Technical Information Extension, Oak Ridge, TN. Levy, L.S., and Venitt, S. (1986). Carcinogenicity and mutagenicity of chromium compounds: The association between bronchial metaplasia and neoplasia. Carcinogenesis 7 , 831-835.

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### McConnell, E.E., Solleveld, H.A., Swenberg, J.A., and Boorman, G.A. (1986). Guidelines for combining neoplasms for evaluation of rodent carcinogenesis studies. JNCI 76 , 283-289. MacGregor, J.T., Wehr, C.M., Henika, P.R., and Shelby, M.D. (1990). The in vivo erythrocyte micronucleus test: Measurement at steady state increases assay efficiency and permits integration with toxicity studies. Fundam. Appl. Toxicol. 14 , 513-522. MacKenzie R.D., Byerrum, R.U., Decker, C.F., Hoppert, C.A., and Langham, R.F. (1958). Chronic toxicity studies. II. Hexavalent and trivalent chromium administered in drinking water to rats. AMA Arch. Ind. Health. 18 , 232-234. Mancuso, T.F. (1997). Chromium as an industrial carcinogen: Part II. Chromium in human tissues. Am. J. Ind. Med. 31 , 140-147. Maronpot, R.R., and Boorman, G.A. (1982). Interpretation of rodent hepatocellular proliferative alterations and hepatocellular tumors in chemical safety assessment. Toxicol. Pathol. 10 , 71-80. Martin, W.R., and Fuller, R.E. (1998). Suspected chromium picolinate-induced rhabdomyolysis. Pharmacotherapy 18 , 860-862. Mehler, A.H., and May, E.L. (1956). Studies with carboxyl-labeled 3-hydroxy-anthranilic and picolinic acids in vivo and in vitro. J. Biol. Chem. 223 , 449-455. The Merck Index (1996). 12th ed. (S. Budavari, Ed.), p. 375. Merck and Company, Rahway, NJ. Miller, J.A., and Miller, E.C. (1977). Ultimate chemical carcinogens as reactive mutagenic electrophiles. In Origins of Human Cancer (H.H. Hiatt, J.D. Watson, and J.A. Winsten, Eds.), pp. 605-627. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Mirasol, F. (2000). Chromium picolinate market sees robust growth and high demand. <http://www.icis.com/Articles/Article.aspx?liArticleID=105617&PrinterFriendly=true> Morrison, D.F. (1976). Multivariate Statistical Methods, 2nd ed., pp. 170-179. McGraw-Hill Book Company, New York. National Academy of Sciences (NAS) (1989). Recommended Dietary Allowances, 10th ed. pp. 241-243. National Academy Press, Washington, DC. National Cancer Institute (NCI) (1978). Bioassay of p-Anisidine Hydrochloride for Possible Carcinogenicity (CAS No. 20265-97-8). Technical Report Series No. 116. NIH Publication No. 78-1371. U.S. Department of Health, Education, and Welfare, Public Health Service, National Institutes of Health, Bethesda, MD. National Institutes of Health (NIH) (2007). Dietary Supplement Fact Sheet: Chromium. Office of Dietary Supplements, NIH Clinical Center. <http://ods.od.nih.gov/factsheets/Chromium_pf.asp> National Toxicology Program (NTP) (1989). Toxicology and Carcinogenesis Studies of Nalidixic Acid (CAS No. 389-08-2) in F344/N Rats and B6C3F 1 

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### National Toxicology Program (NTP) (1993). Toxicology and Carcinogenesis Studies of Turmeric Oleoresin (CAS No. 8024-37-1) (Major Component 79%-85% Curcumin, CAS No. 458-37-7) in F344/N Rats and B6C3F 1 

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### Wani, S., Weskamp, C., Marple, J., and Spry, L. (2006). Acute tubular necrosis associated with chromium picolinate—containing dietary supplement. Ann. Pharmacother. 40 , 563-565. Wasser, W.G., Feldman, N.S., and D’Agati, V.D. (1997). Chronic renal failure after ingestion of over-the-counter chromium picolinate. Ann. Intern. Med. 126 , 410. Westbrook, J.H. (1979). Chromium and chromium alloys. In Kirk-Othmer Encyclopedia of Chemical Technology , 3rd ed. (M. Grayson, Ed.), Vol. 6, pp. 54-62. John Wiley and Sons, New York. Whittaker, P., San, R.H., Clarke, J.J., Seifried, H.E., and Dunkel, V.C. (2005). Mutagenicity of chromium picolinate and its components in Salmonella typhimurium and L5178Y mouse lymphoma cells. Food Chem. Toxicol. 43 , 1619-1625. Wiegand, H.J., Ottenwälder, H., and Bolt, H.M. (1988). Recent advances in biological monitoring of hexavalent chromium compounds. Sci. Total Environ. 71 , 309-315. Williams, D.A. (1971). A test for differences between treatment means when several dose levels are compared with a zero dose control. Biometrics 27 , 103-117. Williams, D.A. (1972). The comparison of several dose levels with a zero dose control. Biometrics 28 , 519-531. Williams, D.A. (1986). A note on Shirley’s nonparametric test for comparing several dose levels with a zero-dose control. Biometrics 42 , 183-186. Witt, K.L., Knapton, A., Wehr, C.M., Hook, G.J., Mirsalis, J., Shelby, M.D., and MacGregor, J.T. (2000). Micronucleated erythrocyte frequency in peripheral blood of B6C3F1 mice from short-term, prechronic, and chronic studies of the NTP Carcinogenesis Bioassay Program. Environ. Mol. Mutagen. 36 , 163-194. Young, P.C., Turiansky, G.W., Bonner, M.W., and Benson, P.M. (1999). Acute generalized exanthematous pustulosis induced by chromium picolinate. J. Am. Acad. Dermatol. 41 , 820-823. Zeiger, E., Haseman, J.K., Shelby, M.D., Margolin, B.H., and Tennant, R.W. (1990). Evaluation of four in vitro genetic toxicity tests for predicting rodent carcinogenicity: Confirmation of earlier results with 41 additional chemicals. Environ. Mol. Mutagen. 16 (Suppl. 18), 1-14. Zeiger, E., Anderson, B., Haworth, S., Lawlor, T., and Mortelmans, K. (1992). Salmonella mutagenicity tests: V. Results from the testing of 311 chemicals. Environ. Mol. Mutagen. 19 (Suppl. 21), 2-141. Zhang, J.D., and Li, X.L. (1987). Chromium pollution of soil and water in Jinzhou [in Chinese]. Zhonghua Yu Fang Yi Xue Za Zhi 21 , 262-264. Zhitkovich, A. (2005). Importance of chromium—DNA adducts in mutagenicity and toxicity of chromium (VI). Chem. Res. Toxicol. 18 , 3-9. Zhitkovich, A., Song, Y., Quievryn, G., and Voitkun, V. (2001). Non-oxidative mechanisms are responsible for the induction of mutagenesis by reduction of Cr(VI) with cysteine: Role of ternary DNA adducts in CR(III)-dependent mutagenesis. Biochemistry 40 , 549-560. Zhitkovich, A., Quieveryn, G., Messer, J., and Motylevich, Z. (2002). Reductive activation with cysteine represents a chromium(III)-dependent pathway in the induction of genotoxicity by carcinogenic chromium(VI). Environ. Health Perspect. 110 (Suppl. 5), 729-731.

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### 84 Chromium Picolinate Monohydrate, NTP TR 556

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### NOT FOR DISTRIBUTION OR ATTRIBUTION A-1

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# APPENDIX A SUMMARY OF LESIONS IN MALE RATS IN THE 2-YEAR FEED STUDY OF CHROMIUM PICOLINATE MONOHYDRATE

T ABLE 

### 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

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 A-2

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### T ABLE 

### A1 Summary of the Incidence of Neoplasms in Male Rats in the 2-Year Feed Study of Chromium Picolinate Monohydrate a 

### 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%)

### A-3 Chromium Picolinate Monohydrate, NTP TR 556

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### T ABLE 

### 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 Chromium Picolinate Monohydrate, NTP TR 556 A-4

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### T ABLE 

### 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)

### A-5 Chromium Picolinate Monohydrate, NTP TR 556

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### T ABLE 

### 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 neoplasms c 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 4Total 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 Chromium Picolinate Monohydrate, NTP TR 556 A-6

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### T ABLE 

### 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 rate a 8/50 (16%) 9/49 (18%) 8/50 (16%) 7/50 (14%) Adjusted rateb 17.1% 20.8% 17.4% 16.3% Terminal rate c 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

### A-7 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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 Chromium Picolinate Monohydrate, NTP TR 556 A-8

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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

### A-9 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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 Chromium Picolinate Monohydrate, NTP TR 556 A-10

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### A4 Summary of the Incidence of Nonneoplastic Lesions in Male Rats in the 2-Year Feed Study of Chromium Picolinate Monohydrate a 

### 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

### A-11 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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 Chromium Picolinate Monohydrate, NTP TR 556 A-12

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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)

### A-13 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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 Chromium Picolinate Monohydrate, NTP TR 556 A-14

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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

T ABLE 

### 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

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 B-2

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### B1 Summary of the Incidence of Neoplasms in Female Rats in the 2-Year Feed Study of Chromium Picolinate Monohydrate a 

### 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%)

### B-3 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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 Chromium Picolinate Monohydrate, NTP TR 556 B-4

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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 neoplasms c 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

### B-5 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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 rate a 1/50 (2%) 3/49 (6%) 2/50 (4%) 1/50 (2%) Adjusted rateb 2.2% 6.7% 4.5% 2.1% Terminal rate c 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

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 B-6

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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

### B-7 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 B-8

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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

### B-9 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### B4 Summary of the Incidence of Nonneoplastic Lesions in Female Rats in the 2-Year Feed Study of Chromium Picolinate Monohydrate a 

### 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

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 B-10

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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%)

### B-11 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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%)

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 B-12

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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)

### B-13 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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%)

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 B-14

NOT FOR DISTRIBUTION OR ATTRIBUTION

### NOT FOR DISTRIBUTION OR ATTRIBUTION C-1

Board Draft 

# APPENDIX C SUMMARY OF LESIONS IN MALE MICE IN THE 2-YEAR FEED STUDY OF CHROMIUM PICOLINATE MONOHYDRATE

T ABLE 

### 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

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 C-2

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### C1 Summary of the Incidence of Neoplasms in Male Mice in the 2-Year Feed Study of Chromium Picolinate Monohydrate a 

### 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%)

### C-3 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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%)

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 C-4

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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%)

### C-5 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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 neoplasms c 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

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 C-6

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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 rate a 5/50 (10%) 6/50 (12%) 6/50 (12%) 7/50 (14%) Adjusted rateb 10.4% 12.5% 12.8% 14.6% Terminal rate c 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

### C-7 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 C-8

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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

### C-9 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### C3 Summary of the Incidence of Nonneoplastic Lesions in Male Mice in the 2-Year Feed Study of Chromium Picolinate Monohydrate a 

### 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

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 C-10

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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

### C-11 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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%)

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 C-12

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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%)

### NOT FOR DISTRIBUTION OR ATTRIBUTION D-1

Board Draft 

# APPENDIX D SUMMARY OF LESIONS IN FEMALE MICE IN THE 2-YEAR FEED STUDY OF CHROMIUM PICOLINATE MONOHYDRATE

T ABLE 

### 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

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 D-2

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### D1 Summary of the Incidence of Neoplasms in Female Mice in the 2-Year Feed Study of Chromium Picolinate Monohydrate a 

### 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)

### D-3 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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)

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 D-4

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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)

### D-5 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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 neoplasms c 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

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 D-6

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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 rate a 7/50 (14%) 6/49 (12%) 4/50 (8%) 6/50 (12%) Adjusted rateb 14.8% 12.7% 8.3% 12.9% Terminal rate c 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

### D-7 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 D-8

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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

### D-9 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### D3 Summary of the Incidence of Nonneoplastic Lesions in Female Mice in the 2-Year Feed Study of Chromium Picolinate Monohydrate a 

### 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

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 D-10

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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%)

### D-11 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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%)

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 D-12

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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)

### D-13 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### 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%)

### D-14 Chromium Picolinate Monohydrate, NTP TR 556

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft

### NOT FOR DISTRIBUTION OR ATTRIBUTION E-1

Board Draft 

# APPENDIX E GENETIC TOXICOLOGY

S

ALMONELLA TYPHIMURIUM 

### MUTAGENICITY 

### T EST 

### P ROTOCOL 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-2 M OUSE 

### P ERIPHERAL 

### B LOOD 

### MICRONUCLEUS 

### T EST 

### P ROTOCOL 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-2 R

AT 

### B ONE 

### MARROW 

### MICRONUCLEUS 

### T EST 

### P ROTOCOL 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-3 E VALUATION 

### P ROTOCOL 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-3 RESULTS 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-3 T ABLE 

### E1 Mutagenicity of Chromium Picolinate Monohydrate in Salmonella typhimurium . . . . . . . . E-4 T ABLE 

### E2 Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice Following Administration of Chromium Picolinate Monohydrate in Feed for 3 Months . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E-5 T ABLE 

### E3 Mutagenicity of Chromium Picolinate in Salmonella typhimurium . . . . . . . . . . . . . . . . . . . . E-6 T ABLE 

### E4 Induction of Micronuclei in Bone Marrow Polychromatic Erythrocytes of Male Rats Treated with Chromium Picolinate by Gavage . . . . . . . . . . . . . . . . . . . . . . . . . E-11

### E-2 Chromium Picolinate Monohydrate, NTP TR 556

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft 

# GENETIC TOXICOLOGY S

ALMONELLA TYPHIMURIUM 

# MUTAGENICITY 

# T EST 

# P ROTOCOL 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 thecontents 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.


# M OUSE 

# P ERIPHERAL 

# B LOOD 

# MICRONUCLEUS 

# T EST 

# P ROTOCOL 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.

### NOT FOR DISTRIBUTION OR ATTRIBUTION Chromium Picolinate Monohydrate, NTP TR 556 E-3

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# R

AT 

# B ONE 

# MARROW 

# MICRONUCLEUS 

# T EST 

# P ROTOCOL 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.


# E VALUATION 

# P ROTOCOL 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 typhimurium a


### Revertants/Plateb 

### Dose –S9 +10% rat S9 Strain (

### μ g/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 control c 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 ± 2.0 d 16 ± 1.0 0 ± 0.0 28 ± 4.0 d 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 

### Chromium Picolinate Monohydrate, NTP TR 556

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### NOT FOR DISTRIBUTION OR ATTRIBUTION Chromium Picolinate Monohydrate, NTP TR 556 E-5



### T ABLE 

### E2 Frequency of Micronuclei in Peripheral Blood Erythrocytes of Mice Following Administration of Chromium Picolinate Monohydrate in Feed for 3 Months a 

### Exposure Number of Mice Compound Concentration with Erythrocytes Micronucleated NCEs/ P Value c 

### 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=0.350 e



### 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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### E-6 Chromium Picolinate Monohydrate, NTP TR 556

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### Table E3 Mutagenicity of Chromium Picolinate in Salmonella typhimurium a


### Dose Revertants/Plateb 

### Strain (

### μ g/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 10,000 c 202 ± 6.0 209 ± 18.0 198 ± 10.0 Trial summary Negative Negative Negative Positive control d 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 10,000 c 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 10,000 c 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 10,000 c 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

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### T ABLE 

### E3 Mutagenicity of Chromium Picolinate in Salmonella typhimurium
Revertants/Plate Dose –S9 +hamster S9 Strain (

### μ g/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 ± 3.0 c 137 ± 5.0 c 153 ± 12.0 c 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 10,000 c 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 10,000 c 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

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### T ABLE 

### E3 Mutagenicity of Chromium Picolinate in Salmonella typhimurium Revertants/Plate Dose –S9 +hamster S9 Strain (

### μ g/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 3,333 c 236 ± 11.0 237 ± 3.0 272 ± 25.0 275 ± 15.0 447 ± 5.0 357 ± 23.0 6,666 c 356 ± 43.0 345 ± 16.0 10,000 c 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 3,333 c 287 ± 6.0 277 ± 39.0 10,000 c 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 3,333 c 444 ± 15.0 261 ± 8.0 340 ± 10.0 427 ± 13.0 320 ± 5.0 373 ± 31.0 10,000 c 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

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### T ABLE 

### E3 Mutagenicity of Chromium Picolinate in Salmonella typhimurium Revertants/Plate Dose –S9 +hamster S9 Strain (

### μ g/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 ± 6.0 c 110 ± 5.0 130 ± 8.0 c 127 ± 6.0 c 105 ± 2.0 c 119 ± 2.0 6,666 c 112 ± 4.0 104 ± 6.0 10,000 c 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 ± 18.0 c 106 ± 6.0 c 146 ± 6.0 6,666 c 135 ± 11.0 10,000 c 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 ± 1.0 c 10 ± 1.0 c 10 ± 1.0 c 8 ± 1.0 10 ± 0.0 c 6,666 c 6 ± 1.0 7 ± 1.0 10,000 c 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

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### T ABLE 

### E3 Mutagenicity of Chromium Picolinate in Salmonella typhimurium Revertants/Plate Dose +rat S9 Strain (

### μ g/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 ± 3.0 c 9 ± 0.0 13 ± 2.0 c 6,666 c 9 ± 1.0 10,000 c 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 3,333 c 159 ± 8.0 134 ± 5.0 141 ± 15.0 188 ± 7.0 152 ± 12.0 192 ± 11.0 10,000 c 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 3,333 c 26 ± 3.0 18 ± 2.0 21 ± 4.0 38 ± 1.0 21 ± 3.0 40 ± 4.0 10,000 c 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.

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### T ABLE 

### E4 Induction of Micronuclei in Bone Marrow Polychromatic Erythrocytes of Male Rats Treated with Chromium Picolinate by Gavage a


### Number of Rats Compound Dose with Erythrocytes Micronucleated PCEs/ P Value c 

### PCEsb 

### (%) (mg/kg) Scored 1,000 PCEsb

Corn oil d 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=0.385 e Cyclophosphamide f 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 signific ant

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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### NOT FOR DISTRIBUTION OR ATTRIBUTION F-1

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# APPENDIX F CLINICAL PATHOLOGY RESULTS

T ABLE 

### 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



### T ABLE 

### F1 Hematology and Clinical Chemistry Data for Rats in the 3-Month Feed Study of Chromium Picolinate Monohydrate a 

### 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 (10 6/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 (10 6/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 (10 3 /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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### NOT FOR DISTRIBUTION OR ATTRIBUTION Chromium Picolinate Monohydrate, NTP TR 556 F-3



### T ABLE 

### 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 (10 3 /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 (10 3 /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 (10 3 /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 (10 3 /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 (10 3 /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 (10 3 /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 (10 3 /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



### T ABLE 

### 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 ± 1 b 14 ± 1 b 16 ± 0b 17 ± 1 b 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

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### NOT FOR DISTRIBUTION OR ATTRIBUTION Chromium Picolinate Monohydrate, NTP TR 556 F-5



### T ABLE 

### 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 (10 6/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 (10 6/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 (10 3 /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



### T ABLE 

### 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 (10 3 /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 (10 3 /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 (10 3 /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 (10 3 /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 (10 3 /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 (10 3 /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 (10 3 /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

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### NOT FOR DISTRIBUTION OR ATTRIBUTION Chromium Picolinate Monohydrate, NTP TR 556 F-7



### T ABLE 

### 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

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### T ABLE 

### F2 Hematology Data for Mice in the 3-Month Feed Study of Chromium Picolinate Monohydrate a 

### 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 (10 6/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 (10 6/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 (10 3 /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 (10 3 /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 (10 3 /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 (10 3 /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 (10 3 /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 (10 3 /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 (10 3 /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 (10 3 /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 (10 6/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 (10 6/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 (10 3 /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 (10 3 /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 (10 3 /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 (10 3 /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 (10 3 /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 (10 3 /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 (10 3 /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 (10 3 /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

T ABLE 

### 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 Chromium Picolinate Monohydrate, NTP TR 556 G-2

NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### G1 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Rats in the 3-Month Feed Study of Chromium Picolinate Monohydrate a 

### 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) aregiven as mg organ weight/g body weight (mean ± standard error).

### G-3 Chromium Picolinate Monohydrate, NTP TR 556

Board Draft NOT FOR DISTRIBUTION OR ATTRIBUTION 

### T ABLE 

### G2 Organ Weights and Organ-Weight-to-Body-Weight Ratios for Mice in the 3-Month Feed Study of Chromium Picolinate Monohydrate a 

### 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) aregiven as mg organ weight/g body weight (mean ± standard error).

### Board Draft Chromium Picolinate Monohydrate, NTP TR 556 G-4

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### NOT FOR DISTRIBUTION OR ATTRIBUTION H-1

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# APPENDIX H REPRODUCTIVE TISSUE EVALUATIONS AND ESTROUS CYCLE CHARACTERIZATION

T ABLE 

### 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

### H-2 Chromium Picolinate Monohydrate, NTP TR 556

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft 

### T ABLE 

### H1 Summary of Reproductive Tissue Evaluations for Male Rats in the 3-Month Feed Study of Chromium Picolinate Monohydrate a 

### 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 (10 7/g testis) 109.47 ± 4.91 111.75 ± 3.94 100.41 ± 3.45 120.74 ± 7.93 Spermatid heads (10 7/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 (10 7/g cauda epididymis) 625.59 ± 62.68 576.71 ± 33.76 570.01 ± 32.04 595.47 ± 26.03 Sperm (10 7/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).



### T ABLE 

### H2 Estrous Cycle Characterization for Female Rats in the 3-Month Feed Study of Chromium Picolinate Monohydrate a 

### 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 ± 0.47 c 4.75 ± 0.21 4.44 ± 0.15 d 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 groupare 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.

### NOT FOR DISTRIBUTION OR ATTRIBUTION Chromium Picolinate Monohydrate, NTP TR 556 H-3

Board Draft 

### T ABLE 

### H3 Summary of Reproductive Tissue Evaluations for Male Mice in the 3-Month Feed Study of Chromium Picolinate Monohydrate a 

### 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 (10 7/g testis) 204.15 ± 6.18 190.85 ± 13.69 214.29 ± 14.32 221.64 ± 8.15 Spermatid heads (10 7/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 (10 7/g cauda epididymis) 802.99 ± 56.27 814.71 ± 52.23 850.90 ± 57.47 824.25 ± 47.63 Sperm heads (10 7/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).



### T ABLE 

### H4 Estrous Cycle Characterization for Female Mice in the 3-Month Feed Study of Chromium Picolinate Monohydrate a 

### 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 groupare 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

### H-4 Chromium Picolinate Monohydrate, NTP TR 556

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### NOT FOR DISTRIBUTION OR ATTRIBUTION I-1

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# APPENDIX I CHEMICAL CHARACTERIZATION AND DOSE FORMULATION STUDIES

P ROCUREMENT AND 

### C HARACTERIZATION OF 

### C HROMIUM 

### P ICOLINATE 

### M ONOHYDRATE 

### . . . . . . . . . . . . . . . . . . I-2 P REPARATION AND 

### ANALYSIS OF 

### D OSE 

### F ORMULATIONS 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I-3 F IGURE 

### I1 Infrared Absorption Spectrum of Chromium Picolinate Monohydrate . . . . . . . . . . . . . . . . I-4 F IGURE 

### I2 Proton Nuclear Magnetic Resonance Spectrum of Chromium Picolinate Monohydrate . . I-5 F IGURE 

### 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

### I-2 Chromium Picolinate Monohydrate, NTP TR 556

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# CHEMICAL CHARACTERIZATION AND DOSE FORMULATION STUDIES
P ROCUREMENT AND 

# C HARACTERIZATION OF 

# C HROMIUM 

# P ICOLINATE 

# M ONOHYDRATE 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

### NOT FOR DISTRIBUTION OR ATTRIBUTION Chromium Picolinate Monohydrate, NTP TR 556 I-3

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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.


# P REPARATION AND 

# ANALYSIS OF 

# D OSE 

# F ORMULATIONS 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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### F IGURE 

### I1 Infrared Absorption Spectrum of Chromium Picolinate Monohydrate

### NOT FOR DISTRIBUTION OR ATTRIBUTION Chromium Picolinate Monohydrate, NTP TR 556 I-5

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### F IGURE 

### I2 Proton Nuclear Magnetic Resonance Spectrum of Chromium Picolinate Monohydrate

### I-6 Chromium Picolinate Monohydrate, NTP TR 556

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### F IGURE 

### I3 X-Ray Diffraction Spectrum of Chromium Picolinate Monohydrate

### NOT FOR DISTRIBUTION OR ATTRIBUTION Chromium Picolinate Monohydrate, NTP TR 556 I-7

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### T ABLE 

### I1 High-Performance Liquid Chromatography Systems Used in the Feed Studies of Chromium Picolinate Monohydrate a 

### 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 DInductively 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® C 18 , 150 mm × 4.6 mm, 5 μm (Supelco, Bellefonte, PA)

Supelco Discovery® C 18 , 150 mm × 4.6 mm, 5 μm (Supelco)

Supelco Discovery® C 18 , 150 mm × 4.6 mm, 5 μm (Supelco)

Dionex IonPac ® AS7, 250 mm × 4.4 mm, 10 μm (Dionex, Sunnyvale, CA)

Supelco Discovery® C 18 , 150 mm × 4.6 mm, 5 μm (Supelco)

Supelco Discovery® C 18 , 150 mm × 4.6 mm, 5 μm (Supelco)

Supelco Discovery® C 18 , 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).

### I-8 Chromium Picolinate Monohydrate, NTP TR 556

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### T ABLE 

### 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)

### NOT FOR DISTRIBUTION OR ATTRIBUTION Chromium Picolinate Monohydrate, NTP TR 556 I-9

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### T ABLE 

### 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 a 

### 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

### I-10 Chromium Picolinate Monohydrate, NTP TR 556

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### T ABLE 

### 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

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### T ABLE 

### 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 a 

### 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, 2003 c 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

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### T ABLE 

### 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, 2003 b 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

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### T ABLE 

### 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 1,939 d –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, 2003 b 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

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### T ABLE 

### 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

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### T ABLE 

### 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, 2003 b 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

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### T ABLE 

### 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 1,939 d –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, 2003 b 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

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### T ABLE 

### 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

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### NOT FOR DISTRIBUTION OR ATTRIBUTION J-1

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# APPENDIX J FEED AND COMPOUND CONSUMPTION IN THE 2-YEAR FEED STUDIES OF CHROMIUM PICOLINATE MONOHYDRATE

T ABLE 

### 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

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### T ABLE 

### 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

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### T ABLE 

### 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



### T ABLE 

### 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

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft

### NOT FOR DISTRIBUTION OR ATTRIBUTION Chromium Picolinate Monohydrate, NTP TR 556 J-5



### T ABLE 

### 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



### Board Draft

### J-6 Chromium Picolinate Monohydrate, NTP TR 556

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft

### NOT FOR DISTRIBUTION OR ATTRIBUTION K-1

Board Draft 

# APPENDIX K INGREDIENTS, NUTRIENT COMPOSITION, AND CONTAMINANT LEVELS IN NTP-2000 RAT AND MOUSE RATION

T ABLE 

### 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

### K-2 Chromium Picolinate Monohydrate, NTP TR 556

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### T ABLE 

### 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



### T ABLE 

### K2 Vitamins and Minerals in NTP-2000 Rat and Mouse Ration a


### 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 B 12 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

### NOT FOR DISTRIBUTION OR ATTRIBUTION Chromium Picolinate Monohydrate, NTP TR 556 K-3



### T ABLE 

### 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) 1,000 a 

### " -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 B 12 (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



### T ABLE 

### K4 Contaminant Levels in NTP-2000 Rat and Mouse Ration a


### 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. 

### Chromium Picolinate Monohydrate, NTP TR 556

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### NOT FOR DISTRIBUTION OR ATTRIBUTION 

# 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 

# R

ATS 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 Chromium Picolinate Monohydrate, NTP TR 556

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### NOT FOR DISTRIBUTION OR ATTRIBUTION Chromium Picolinate Monohydrate, NTP TR 556 L-3



### 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.

Board Draft

### L-4 Chromium Picolinate Monohydrate, NTP TR 556

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### NOT FOR DISTRIBUTION OR ATTRIBUTION M-1



# APPENDIX M CHROMIUM TISSUE DISTRIBUTION STUDY

Board Draft 

### MATERIALS AND 

### METHODS 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M-2 T ABLE 

### 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

### M-2 Chromium Picolinate Monohydrate, NTP TR 556

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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 CO 2

### /O 2 

### 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 CO 2

### /O 2 

### 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.

### NOT FOR DISTRIBUTION OR ATTRIBUTION Chromium Picolinate Monohydrate, NTP TR 556 M-3

Board Draft 

### 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.

### M-4 Chromium Picolinate Monohydrate, NTP TR 556

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft 

### T ABLE 

### M1 Tissue Concentrations and Excreta Content of Chromium in Male Rats in the 2-Year Feed Study of Chromium Picolinate Monohydrate a 

### 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 o f 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 

### T ABLE 

### M2 Tissue Concentrations and Excreta Content of Chromium in Female Mice in the 2-Year Feed Study of Chromium Picolinate Monohydrate a 

### 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 ± 14.2041 c 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 thefirst two samples resulted in similar chromium concentrations (measured at 5.518 and 44.05 μg/g forestomach) and there were three samplesanalyzed 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 D ISCUSSION 

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-7 REFERENCES

### . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-7 T ABLE 

### N1 Excretion of Radioactivity and Total Chromium by Male Rats After a Single Gavage Dose of 15.3 mg/kg [ 14 

### C]-Chromium Picolinate in Propylene Glycol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . N-8 T ABLE 

### N2 Urinary Excretion of Chromium Picolinate and Metabolites by Male Rats After a Single Gavage Dose of 15.3 mg/kg [ 14 

### C]-Chromium Picolinate in Propylene Glycol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . N-8 T ABLE 

### N3 Distribution of Radioactivity in Tissues of Male Rats at 52 Hours After a Single Gavage Dose of 15.3 mg/kg [ 14 

### C]-Chromium Picolinate in Propylene Glycol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . N-9 T ABLE 

### N4 Excretion of Radioactivity and Total Chromium by Male Rats After a Single Gavage Dose of 17.4 mg/kg [ 14 

### C]-Chromium Picolinate as an Aqueous Slurry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-9 T ABLE 

### N5 Distribution of Radioactivity in Tissues of Male Rats 48 Hours After a Single Gavage Dose of 17.4 mg/kg [ 14 

### C]-Chromium Picolinate as an Aqueous Slurry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-10 T ABLE 

### N6 Excretion of Radioactivity and Total Chromium by Male Mice After a Single Gavage Dose of 20.5 mg/kg [ 14 

### C]-Chromium Picolinate as an Aqueous Slurry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-10 T ABLE 

### N7 Urinary Excretion of Chromium Picolinate and Metabolites by Male Mice After a Single Gavage Dose of 20.5 mg/kg [ 14 

### C]-Chromium Picolinate as an Aqueous Slurry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . N-11 T ABLE 

### N8 Excretion of Radioactivity and Total Chromium by Male Mice After a Single Gavage Dose of 19.0 mg/kg [ 14 

### C]-Chromium Picolinate in Propylene Glycol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . N-11 T ABLE 

### N9 Distribution of Radioactivity in Tissues of Male Mice 48 Hours After a Single Gavage Dose of 19.0 mg/kg [ 14 

### C]-Chromium Picolinate in Propylene Glycol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . N-12 T ABLE 

### N10 Urinary Excretion of Chromium Picolinate and Metabolites by Male Mice After a Single Gavage Dose of 19.0 mg/kg [ 14 

### C]-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 

## [ 14 

## C]-Chromium Picolinate Radiolabeled chromium picolinate, labeled with 14 

### C 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 [ 14 

### C]-chromium picolinate was supplied as a neat solid and its radiochemical purity was determined by HPLC. Following injection of [ 14 

### C]-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 [ 14 

### C]-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 MS 3 

### 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 CO 2

### ).

### N-4 Chromium Picolinate Monohydrate, NTP TR 556

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft 

## Preparation of Aqueous Oral Dose Formulations of [ 14 

## C]-Chromium Picolinate A known amount of [ 14 

### C]-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 [ 14 

## C]-Chromium Picolinate in Propylene Glycol to Rats A dose formulation of [ 14 

### C]-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 CO 2 

### 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 [ 14 

## C]-Chromium Picolinate to Rats as an Aqueous Slurry A slurry of [ 14 

### C]-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 [ 14 

## C]-Chromium Picolinate to Mice as an Aqueous Slurry A slurry of [ 14 

### C]-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 [ 14 

## C]-Chromium Picolinate in Propylene Glycol to Mice A dose formulation of [ 14 

### C]-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 14 

### C 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 [ 14 

### C]-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 [ 14 

### C]-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 [ 14 

## C]-Chromium Picolinate in Propylene Glycol to Rats Excretion of radioactivity in rats following administration of [ 14 

### C]-chromium picolinate is shown in Table N1. The rats dosed orally with [ 14 

### C]-chromium picolinate dissolved in propylene glycol excreted an average of 53% of the 14 

### C dose in urine, 39% in feces, and 1.5% as CO 2 

### in breath in 24 hours. No measurable dose was collected as volatiles in breath. An additional 8% of the 14 

### C 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 14 

### C dose received was excreted in 24 hours as N-picolinoylglycine. An average of 1.9% ± 0.4% and 1.1% ± 0.7% of the 14 

### C 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 14 

### C dose was found in these tissues. 

## 17.4 mg/kg Oral Dose of [ 14 

## C]-Chromium Picolinate to Rats as an Aqueous Slurry Excretion of radioactivity in rats sacrificed at 48 hours following administration of [ 14 

### C]-chromium picolinate is shown in Table N4. The rats dosed orally with [ 14 

### C]-chromium picolinate water slurry excreted an average of 41%

Board Draft

### N-6



### of the 14 

### C dose in urine, 47% in feces, and 1.4% as CO 2 

### in breath in 24 hours. An additional 4% of the 14 

### C 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 14 

### C 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 [ 14 

## C]-Chromium Picolinate to Mice as an Aqueous Slurry Excretion of radioactivity in mice sacrificed at 48 hours following administration of [ 14 

### C]-chromium picolinate is shown in Table N6. The mice excreted an average of 26% of the 14 

### C dose in urine and 59% in feces in 48 hours. Less than 0.1% of the 14 

### C dose was excreted as CO 2 

### in breath in 48 hours, and an average of 0.2% of the 14 

### C 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 14 

### C 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 14 

### C 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 14 

### C dose. 

## Single 19.0 mg/kg Oral Dose of [ 14 

## C]-Chromium Picolinate in Propylene Glycol to Mice Excretion of radioactivity in mice sacrificed at 48 hours following administration of [ 14 

### C]-chromium picolinate is shown in Table N8. The mice excreted an average of 42.0% ± 7.8% of the 14 

### C 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 14 

### C 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 14 

### C 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 14 

### C 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 14 

### C dose. 

## Isolation and Identification of the Urinary Metabolite Only a single radiolabeled metabolite (Ur1) was excreted in rat urine following an oral dose of [ 14 

### C]-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/ 14 

### C 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 CHO 2

### ). 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


# D ISCUSSION 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 

### T ABLE 

### N1 Excretion of Radioactivity and Total Chromium by Male Rats After a Single Gavage Dose of 15.3 mg/kg [ 14 

### C]-Chromium Picolinate in Propylene Glycola 

### Time (hours) Urine Feces CO 2 

### 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 applica ble. b Not collected



### T ABLE 

### N2 Urinary Excretion of Chromium Picolinate and Metabolites by Male Rats After a Single Gavage Dose of 15.3 mg/kg [ 14 

### C]-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 

### T ABLE 

### N3 Distribution of Radioactivity in Tissues of Male Rats 52 Hours After a Single Gavage Dose of 15.3 mg/kg [ 14 

### C]-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



### T ABLE 

### N4 Excretion of Radioactivity and Total Chromium by Male Rats After a Single Gavage Dose of 17.4 mg/kg [ 14 

### C]-Chromium Picolinate as an Aqueous Slurrya 

### Time (hours) Urine Feces CO 2 

### 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 applica ble. b Not collected

### N-10 Chromium Picolinate Monohydrate, NTP TR 556

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft 

### T ABLE 

### N5 Distribution of Radioactivity in Tissues of Male Rats 48 Hours After a Single Gavage Dose of 17.4 mg/kg [ 14 

### C]-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



### T ABLE 

### N6 Excretion of Radioactivity and Total Chromium by Male Mice After a Single Gavage Dose of 20.5 mg/kg [ 14 

### C]-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 

### T ABLE 

### N7 Urinary Excretion of Chromium Picolinate and Metabolites by Male Mice After a Single Gavage Dose of 20.5 mg/kg [ 14 

### C]-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.



### T ABLE 

### N8 Excretion of Radioactivity and Total Chromium by Male Mice After a Single Gavage Dose of 19.0 mg/kg [ 14 

### C]-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

NOT FOR DISTRIBUTION OR ATTRIBUTION Board Draft 

### T ABLE 

### N9 Distribution of Radioactivity in Tissues of Male Mice 48 Hours After a Single Gavage Dose of 19.0 mg/kg [ 14 

### C]-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



### T ABLE 

### N10 Urinary Excretion of Chromium Picolinate and Metabolites by Male Mice After a Single Gavage Dose of 19.0 mg/kg [ 14 

### C]-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.