Supporting Materials for a 31-Day Study of Cobalt(II)chloride Ingestion in Humans: Pharmacokinetics and Clinical Effects

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1 Supporting Materials for a 31- Study of Cobalt(II)chloride Ingestion in Humans: Pharmacokinetics and Clinical Effects Brent L. Finley a, Kenneth M. Unice b, Brent D. Kerger c, Joanne M. Otani a, Dennis J. Paustenbach a, David A. Galbraith a, and Brooke E. Tvermoes d a Cardno ChemRisk, 11 2nd St. Suite 7, San Francisco, CA 9415, United States; b Cardno ChemRisk, 2 Stanwix St. Suite 5, Pittsburgh, PA 15222, United States; c Cardno ChemRisk, 13 Vantis Suite 17, Aliso Viejo, CA United States; d Cardno ChemRisk, 48 Pearl East Circle, Boulder, CO 831, United States. Supplement Table of Contents TABLES Table S1 Table S2 Table S3 Table S4 Table S5 Table S6 Whole Blood Cobalt Measurements (µg/l) Following Dietary Supplementation with 1 mg Co Daily for an Average of 31 s. Serum Cobalt Measurements (µg/l) Following Dietary Supplementation with 1 mg Co Daily for an Average of 31 s. Cobalt Serum Measurements for Select s. Computed Parameters Describing the Retention of Co in Whole Blood and Serum. Blood Clinical Indices for Females. Blood Clinical Indices for Males. FIGURES Figure S1 Figure S2 Figure S3 Figure S4 Figure S5 Figure S6 Figure S7 Figure S8 Figure S9 Figure S1 Figure S11 Figure S12 Figure S13 Figure S14 Figure S15 Figure S16 Figure S17 Figure S18 Figure S19 Figure S2 Figure S21 Time Course of the Average Whole Blood and Serum Cobalt Measurements Following Dietary Supplementation with Approximately 1. mg Co/day for an Average of 31 days. Relationship Between Whole Blood and Serum Cobalt Concentration During Dosing. Time Course of Estimated RBC Concentration Categorized by Gender Red Blood Cell (RBC) Counts for Each Participant Throughout the Study. Serum Thyroid Stimulating Hormone (TSH) Levels for Each Participant Throughout the Study. Serum Tree Thyroxine (T4) Levels for Each Participant Throughout the Study. Albumin Concentrations for Each Participant Throughout the Study. Total Protein Concentrations for Each Participant Throughout the Study. White Blood Cell (WBC) Counts for Each Participant Throughout the Study. Hematocrit (Hct) Percentages for Each Participant Throughout the Study. Hemoglobin (Hgb) Concentrations for Each Participant Throughout the Study. Total Iron Concentrations for Each Participant Throughout the Study. Ferritin Concentrations for Each Participant Throughout the Study. Creatine Kinase-Myocardial Band (CK-MB) concentrations for Each Participant Throughout the Study. Alanine Aminotransferase (ALT) Concentrations for Each Participant Throughout the Study. Aspartate Aminotransferase (AST) Concentrations for Each Participant Throughout the Study. Creatinine Concentrations for Each Participant Throughout the Study. Glucose Concentrations for Each Participant Throughout the Study. Normalized Lymphocyte Proliferation Response to Various Metals Before and After Co Dietary Supplementation with Approximately 1 mg Co/day Time Course of Observed Whole Blood Cobalt Measurements Compared to Cobalt Biokinetic Model Predictions. Effect of 12-Month Whole Blood or RBC Donation Frequency on the Geometric Mean of Plasma. 1

2 Table S1. Whole Blood Cobalt Measurements (µg/l) Following Dietary Supplementation with 1 mg Co Daily for an Average of 31 s. Study 1 wk Predose Pre-dose/ 1 4/5 8/9 14/16 23/24 29/33 1 wk Postdose 2 wk Postdose 6 wk Postdose 1 wk Post-dose Males A <.5 < B <.5 < d d d C <.5 < d d d D <.5 < d d d E <.5 < Average wk Post-dose Females F.5 < G <.5 <.5 a d d d H I b < / 6.3 c d d d J a a d d d Average a Missing data (-) due to error in specimen collection and/or processing. b Missing data (-) due to volunteer being unavailable for blood draw appointment. c provided both a 29 and 33 blood draw because she continued to ingest supplement after 29, which was initially scheduled to be the last day of dosing. Dosing ceased on 33. d did not participate in the additional 6 wk, 1 wk and 16 wk post-dose follow up blood draws. 2

3 Table S2. Serum Cobalt Measurements (µg/l) Following Dietary Supplementation with 1 mg Co Daily for an Average of 31 s. Study 1 wk Predose Pre-dose/ 1 4/5 8/9 14/16 23/24 29/33 1 wk Postdose 2 wk Postdose 6 wk Postdose 1 wk Post-dose Males A <1. < B <1. < d d d C <1. < a d d d D <1. < d d d 16 wk Post-dose E <1. < Average Females F a < G <1. < d d d H <1. < a I b < / 8.2 c d d d J a a a a d d d Average a Missing data (-) due to error in specimen collection and/or processing. b Missing data (-) due to volunteer being unavailable for blood draw appointment. c provided both a 29 and 33 blood draw because she continued to ingest supplement after 29, which was initially scheduled to be the last day of dosing. Dosing ceased on 33. d did not participate in the additional 6 wk, 1 wk, and 16 wk post-dose follow up blood draws. 3

4 Table S3. Cobalt Serum Measurements for Select s. Co serum samples analyzed by Quest Diagnostics were sent to a third party analytical lab for Co analysis. In general, there was good agreement between the Co concentrations reported by the two laboratories. Study Study Date Outside Laboratory Concentration (µg/l) Quest Concentration (µg/l) E 29/ H 29/ H 23/ C 1 wk post-dose I 2 wk post-dose B 2 wk post-dose F 2 wk post-dose G 2 wk post-dose J 2 wk post-dose D 2 wk post-dose

5 Table S4. Computed Parameters Describing the Retention of Co in Whole Blood and Serum. The rate of Co elimination from whole blood and serum fit best to a two-phase exponential decay model with a biologic half-life of T 1 andt 2 for the fast and slow elimination phase, respectively. The elimination rate constant for phase one and phase two of the model is represented by λ 1 and λ 2, respectively. The fraction eliminated by each component of the model is represented by a 1 and a 2, respectively. Based on the reporting limits of the assay and known background concentrations of Co in serum and whole blood of unexposed individuals, we set the constraints of the exponential decay curve to plateau at.5 µg/l and.4 µg/l for Co in whole blood and serum, respectively, in order to calculate an approximate elimination half-life. Matrix Whole blood a 1 (unitless) λ 1 (day -1 ) T 1 (days) a 2 (unitless) λ 2 (day -1 ) T 2 (days) A H E F Serum A H E F Whole Blood Average Serum Average

6 Table S5. Blood Clinical Indices for Females. Data are represented as the mean ± standard deviation of individual results. Student s two-tailed paired t-test was used to assess the effects of dietary Co supplementation on blood chemical indices, and significant differences were not observed unless noted by superscript b. When calculating the mean, one-half of the reporting limit was used for values reported to be less-than the reporting limit. Parameter Unit Average Baseline (n = 9) 14/16 (n = 5) 29/33 (n = 5) 1 wk post-dose (n = 5) 2 wk post dose (n = 5) Reference Range* Albumin U/L 4.5 ± ± ± ±.18 b 4.4 ±.8 b Total Protein mg/dl 7.2 ± ± ± ± ±.23 b WBC 1 3 /μl 5.77 ± ± ± ± ± RBC 1 6 /μl 4.42 ± ± ± ± ± Hct % 39.8 ± ± ± ± ± Hgb g/dl 13.2 ± ± ± ± ± TSH mlu/l 2.27 ± ± ± ± ± Free T4 ng/ml 1.4 ± ± ± ± ± Total Iron μg/dl 12 ± 28.3 c 72.4 ± ± ± 28.9 b 86.8 ± Ferritin ng/ml 26 ± ± 1.6 b 16.6 ± 11.4 b 14.8 ± 1.5 b 17. ± 11.2 b CK-MB ng/ml.93 ± ± ±.19. ± ±.6-5. ALT U/L 12.7 ± ± ± 1.48 b 1. ± 3. b 12.4 ± AST U/L 15.2 ± ± ± ± ± Creatinine mg/dl.79 ±.9.85 ±.6.81 ±.8.85 ±.1.81 ± HDL Cholesterol mg/dl 66.4 ± ± Total Cholesterol mg/dl 2 ± ± Triglycerides mg/dl 12 ± ± <1 Glucose mg/dl 91.6 ± ± ± ± ± *The reference range is defined as the minimum and the maximum reference value from Quest Diagnostic Laboratories as determined in adult females. a Individual baseline values for 1 wk pre-dose and pre-dose/day 1 draw were averaged unless otherwise noted. b Significant difference in blood chemical indices before and after Co dosing at specific time point (p <.5), however the mean value after dosing was within the reference range. c The average baseline is the mean ± SD of the 1 wk pre-dose draw because there was a significant difference between the 1 wk pre-dose draw and the pre-dose/ day 1 draw; as such the two baseline values were not averaged. 6

7 Table S6. Blood Clinical Indices for Males. Data are represented as the mean ± standard deviation of individual results. Student s two-tailed paired t-test was used to assess the effects of dietary Co supplementation on blood chemical indices, and significant differences were not observed unless noted by superscript b. When calculating the mean, one-half of the reporting limit was used for values reported to be less-than the reporting limit. Parameter Unit Average Baseline (n = 1) 14/16 (n = 5) 29/33 (n = 5) 1 wk post-dose (n = 5) 2 wk post dose (n = 5) Reference Range* Albumin U/L 4.7 ± ± ± ± ± Total Protein mg/dl 7.1 ± ± ± ± ± WBC 1 3 /μl 4.91 ± ± ± ± ± RBC 1 6 /μl 4.62 ± ± ± ±. b 4.63 ± Hct % 43.2 ± ± ± ± 2.9 b 43.3 ± Hgb g/dl 14.4 ± ± ± ± 1.15 b 14.5 ± TSH mlu/l 2.8 ± ± ± ± ± Free T4 ng/ml 1.28 ±.16 c 1.35 ± ± ±.18 b 1.24 ± Total Iron μg/dl 113 ± ± 23.4 b 67.4 ± 11.6 b 11 ± ± Ferritin ng/ml 18 ± 13 c 142 ± 94.9 b 156 ± ± ± 82.1 b 2-38 CK-MB ng/ml 1.7 ± ± ± ± ± ALT U/L 36.4 ± ± ± ± ± AST U/L 25.9 ± ± 4.88 b 23.6 ± ± 4.83 b 23. ± Creatinine mg/dl 1.6 ± ± ± ± ± HDL Cholesterol mg/dl 54.8 ± ± Total Cholesterol mg/dl 198 ± ± Triglycerides mg/dl 261 ± ± <1 Glucose mg/dl 9.3 ± ± ± ± ± *The reference range is defined as the minimum and the maximum reference value from Quest Diagnostic Laboratories as determined in adult females. a Individual baseline values for 1 wk pre-dose and pre-dose/day 1 draw were averaged unless otherwise noted. b Significant difference in blood chemical indices before and after Co dosing at specific time point (p <.5), however the mean value after dosing was within the reference range. c The average baseline is the mean ± SD of the 1 wk pre-dose draw because there was a significant difference between the 1 wk pre-dose draw and the pre-dose/ day 1 draw; as such the two baseline values were not averaged. 7

8 Cobalt Concentration ( g/l) Cobalt Concentration ( g/l) Figure S1. Time Course of the Average Whole Blood and Serum Cobalt Measurements Following Dietary Supplementation with Approximately 1. mg Co/day for an Average of 31 s. A. Average Co Whole Blood ( ) and Serum Concentrations ( ) in Males. B. Average Co Whole Blood ( ) and Serum ( ) Concentrations in Females. The error bars represent the standard error of Co whole blood or serum concentration. A) Males 8 Whole Blood Serum // // Elapsed Time Since Beginning of Dosing (s) 8 B) Females Whole Blood Serum 6 // 2 // Elapsed Time Since Beginning of Dosing (s) 8

9 Cobalt Serum (µg/l) Figure S2 Relationship Between Whole Blood and Serum Cobalt Concentration During Dosing y = 1.392x R² = Cobalt Whole Blood (µg/l) 9

10 Figure S3. Time Course of the Average Estimated Cobalt RBC Concentration Categorized by Gender. Average Estimated Co RBC Concentration in Males ( ) and Females ( ). The error bars represent the standard error of Co whole blood or serum concentration. Dietary supplementation was approximately 1. mg Co/day for 3-31 days, and RBC concentration was estimated from whole blood concentration, serum concentration and average hematocrit values for each volunteer. The error bars represent the standard error of Co whole blood or serum concentration. 1

11 Free T4 (ng/dl) TSH (mlu/l) TSH (mlu/l) RBCs ( x 16 /µl) RBCs ( x 16 /µl) Figure S4. Red Blood Cell (RBC) Counts for Each Participant Throughout the Study. A. Males; B. Females. RBC counts show no significant change during dosing for males or females and all data are within the normal reference ranges during dosing. The upper and lower end of the reference range is denoted by the broken black lines. A. B. A B C D E Figure S5. Serum Thyroid Stimulating Hormone (TSH) Levels for Each Participant Throughout the Study. A. Males; B. Females. TSH counts show no significant change during dosing for males or females and all data are within the normal reference ranges during dosing. The upper and lower end of the reference range is denoted by the broken black lines. A. A B C D E B Figure S6. Serum Free Thyroxine (T4) Levels for Each Participant Throughout the Study. A. Males; B. Females. T4 counts show no significant change during dosing for males or females and all data are within the normal reference ranges during dosing. The upper and lower end of the reference range is denoted by the broken black lines. A. A B C D E B

12 Total Protein (g/dl) Total Protein (g/dl) Albumin (U/L) Albumin (U/L) Figure S7. Albumin Concentrations for Each Participant Throughout the Study. A. Males; B. Females. The upper and lower end of the reference range is denoted by the broken black lines. A. 5.5 A B C D E B Figure S8. Total Protein Concentrations for Each Participant Throughout the Study. A. Males; B. Females. The upper and lower end of the reference range is denoted by the broken black lines. A. A B C D E B

13 Hgb (gm/dl) Hgb (gm/dl) Hct (%) Hct (%) WBC (1 3 /µl) WBC (1 3 /µl) Figure S9.White Blood Cell (WBC) Counts for Each Participant Throughout the Study. A. Males; B. Females. The upper and lower end of the reference range is denoted by the broken black lines. A. A B C D E B Figure S1. Hematocrit (Hct) Percentages for Each Participant Throughout the Study. A. Males; B. Females. The upper and lower end of the reference range is denoted by the broken black lines. A. B. A B C D E Figure S11. Hemoglobin (Hgb) Concentrations for Each Participant Throughout the Study. A. Males; B. Females. The upper and lower end of the reference range is denoted by the broken black lines. A B C D E

14 CK-MB (ng/ml) CK-MB (ng/ml) Ferritin (ng/ml) Ferritin (ng/ml) Total Iron (µg/dl) Total Iron (µg/dl) Figure S12. Total Iron Concentrations for Each Participant Throughout the Study. A. Males; B. Females. The upper and lower end of the reference range is denoted by the broken black lines. A B C D E Figure S13. Ferritin Concentrations for Each Participant Throughout the Study. A. Males; B. Females. The upper and lower end of the reference range is denoted by the broken black lines. A B C D E Figure S14. Creatine Kinase-Myocardial Band (CK-MB) Concentrations for Each Participant Throughout the Study. A. Males; B. Females. The upper and lower end of the reference range is denoted by the broken black lines. Results below the limit of detection are plotted as 1/2 the detection limit. A B C D E * B was asymptomatic at his two week post-dose draw, however, follow up cardiac test were performed; the additional labs did not suggest cardiac damage. 14

15 Creatinine (mg/dl) Creatinine (mg/dl) AST (U/L) AST (U/L) ALT (U/L) ALT (U/L) Figure S15. Alanine Aminotransferase (ALT) Concentrations for Each Participant Throughout the Study. A. Males; B. Females. The upper and lower end of the reference range is denoted by the broken black lines. A B C D E Figure S16. Aspartate Aminotransferase (AST) Concentrations for Each Participant Throughout the Study. A. Males; B. Females. AST concentrations show no significant change during dosing for males or females and all data are within the normal reference ranges during dosing. The upper and lower end of the reference range is denoted by the broken black lines Voluntee r A B C D E Figure S17. Creatinine Concentrations for Each Participant Throughout the Study. A. Males; B. Females. The upper and lower end of the reference range is denoted by the broken black lines. A B C D E

16 Glucose (mg/dl) Glucose (mg/dl) Figure S18. Glucose Concentrations for Each Participant Throughout the Study. A. Males; B. Females. The upper and lower end of the reference range is denoted by the broken black lines. 1 A B C D E

17 Stimulation Index Figure S19. Normalized Lymphocyte Proliferation Response to Various Metals Before and After Cobalt Dietary Supplementation with Approximately 1 mg Co/day. Proliferation assays were performed using separated peripheral blood mononuclear cells (PBMCs) collected from 3- milliliters of peripheral blood. Proliferation was determined by [3H]-thymidine (Amersham International, Arlington Heights, IL) incorporation rates. The average proliferation measurement for each metal treatment was normalized to the proliferation measurement of non-treated control cells isolated from that same individual, generating a stimulation index (SI). The SI was then used to compare lymphocyte reactivity (sensitivity) to various metals before and after dosing. According to Orthopedic Analysis, the manufacturer of the assay, a stimulation index (SI) greater than two indicates a positive result, meaning that lymphocytes were reactive to the metal.the graph illustrates the averaged normalized lymphocyte transformation response to each metal. The lymphocyte transformation test shows no significant change in lymphocyte response before and after dosing (paired Student s t-test, p >.5). Ten volunteers are included in each group; five males and five females Average Pre-dsoe Average Post-dose

18 Cobalt Whole Blood Concentration (µg Co/L) Figure S2. Time Course of Observed Whole Blood Cobalt Measurements Compared to Cobalt Biokinetic Model Predictions for Whole Blood Concentrations. Mean Co whole blood concentrations measured during dosing compared to the biokinetic model predictions assuming a 61% GI absorption rate for all volunteers, a 24% GI absorption rate for males, and 81% GI absorption rate females. The best fit GI absorption rate for each group was determined using the least squared errors approach. 6 All s (mean) Males (mean) Females (mean) All (best fit oral absorption during dosing = 61%) Males (best fit oral absorption during dosing = 24%) Females (best fit oral absorption during dosing = 81%) s 18

19 Percent Decrease in Ferritin From Figure S21. Effect of 12-Month Whole Blood or RBC Donation Frequency on the Geometric Mean of Plasma Ferritin by Males and Females from the Cable et al. (211) Study Compared to Ferritin Loss in the Male and Female s from This 31- Study. The percent decrease is a comparison of ferritin levels at the 1 week pre-dose draw and two week post-dose draw. Each gray circle represents one male from the 31-day study and each gray square represents a female from the 31-day study. A comparable decrease in ferritin levels was observed in the males and females from the 31-day study as was observed in the volunteers studied by Cable et al. (211). % 1% 2% 3% % % 6% 7% 8% Females < Females >= Males 31- Males 31-day Females 9% 1% Donations in last 12 months 19