Di(µ-2,2´,2´´-nitrilotris(ethanol)-diperchlorato)dinatrium

Epidemiological data

Administrative data

Endpoint:

epidemiological data

Type of information:

other: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Justification for Read Across is given in Section 13 of IUCLID.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2002

Materials and methods

Study type:

other: study in health adult volunteers

Endpoint addressed:

repeated dose toxicity: oral

other: Thyroidal iodide uptake inhibition

Principles of method if other than guideline:

The inhibition of thyroidal iodide uptake was assessed after daily administration of perchlorate in the drinking water for 14 days.

GLP compliance:

no

Test material

Test material information

Constituent 1

Reference substance name:

Potassium perchlorate

EC Number:

231-912-9

EC Name:

Potassium perchlorate

Cas Number:

7778-74-7

Molecular formula:

ClHO4.K

IUPAC Name:

Potassium perchlorate

Method

Type of population:

general

Ethical approval:

not specified

Details on study design:

SETTING: the logistics of the study were such that only three subjects could be started each week. To preclude skewing the dose response through potential temporal variations in measured parameters, in the main study each of the three subjects started each week was given a different perchlorate dose (0.02, 0.1, or 0.5 mg/kg-day), deviating from this schedule only to balance the sexes in each dose group as necessary. The first three subjects were signed in the uptake study the 0.007 mg/kg-day dose and thereafter rotated subject enrollment through the four doses tested (0.007, 0.02, 0.1 and 0.5 mg/kg-day).

STUDY POPULATION
- Selection criteria: candidates having a history of thyroid disease, recent ingestion of any iodine-containing pharmaceutical (including thyroid hormone), or significant thyroid enlargement were excluded. All 37 subjects who presented themselves for the screening visit met the screening criteria and were entered into the study. The screening visit consisted of a history, a physical examination, blood sampling (for complete blood count, routine serum chemistry, and serum thyroid function tests), and urine sampling (for routine urinalysis, screening for drug abuse, and pregnancy testing for women who had not had a hysterectomy or tubal ligation). Following one woman’s enrollment and assignment to the 0.007- mg/kg-day dose group (uptake study), laboratory analysis of her screening-visit blood draw revealed elevated TSH (18 mIU/l). Because her thyroid hormone levels and physical examination were normal, she was kept in the study to evaluate, in a subject with subclinical hypothyroidism, the effect of perchlorate at a dose predicted to be the NOEL.
- Total number of subjects participating in study: 37.
- Total number of subjects at end of study: 37.
- Age: 18-57 years old (mean ± SD, 38 ± 12 years; median 40 years)
- Study size rationale: considering that Lawrence et al. found highly significant inhibition of uptake in nine subjects given perchlorate at 10 mg/day, a dose approximately the same as the 0.1 mg/kg-day dose and 4-fold lower than the 0.5 mg/kg-day dose, it was considered as highly probable that a comparable group size (8–10 per dose) would yield a signsignificant degree of inhibition at these two doses.

MAIN STUDY:
- The first 24 subjects were tested according to the initial (main study) protocol; four subjects of each sex received a perchlorate dose of 0.02, 0.1, or 0.5 mg/kg-day.
- Ingestion of 123I at 0900 hr on 4 days: the baseline visit (1 day before the start of perchlorate exposure), E2, E14, and postexposure day 15 (P15); measurement of RAIU at 1700 hr on the day of 123I ingestion and 0900 hr the following morning.
- In addition to the blood draw at the screening visit, a total of 22 blood draws on 11 days throughout the study period of 35 days.
- Collection of 24-hour urine in five pooled collections on 5 days (the day before the baseline visit, E1, E2, E8, and P1); collection of 24-hr pooled urine on 3 days (E14, P2, and P14); recording the time of urine collection and the amount of any discarded urine on a preprinted log sheet.

UPTAKE STUDY
- The next 13 subjects were tested according to the modified (uptake-study) protocol; six women and one man received a dose of 0.007 mg/kg-day and one additional subject of each sex received a dose of 0.02, 0.1, or 0.5 mg/kg-day. Even though no statistically significant difference between men and women at the three doses tested in the main study was found, when testing the new dose in the uptake study more women were selected because protecting the developing fetus is of primary concern.
- Ingestion of 123I at 0900 hr on 3 days: the baseline visit (1 day before the start of perchlorate exposure), E14, and P15. Baseline visit and E14: measurement of RAIU at 1700 hr on the day of 123I ingestion and 0900 hr the following morning. P15: measurement of RAIU only at 0900 hr on the day following 123I ingestion.
- In addition to the blood draw at the screening visit, a blood draw on E8 and on E14.
- Collection of 24-hr pooled urine on the day before the baseline visit and on E14.

RADIOIODINE STUDY: although no difference was expected to be found between the 8- and 24-hr relative uptakes once a quasi-steady-state equilibrium between perchlorate absorption and elimination was established (i.e., in which perchlorate concentrations in blood fluctuated with the dosing schedule in a regular daily pattern, with no further net perchlorate accumulation over time), both 8- and 24-hr uptake measurements were chosen primarily because it was uncertain whether such an equilibrium would be achieved by Exposure day 2 (E2). All radioiodine studies were conducted in the Nuclear Medicine facility of the OHSU Hospital. 123I capsules (nominal specific activity, 100 μCi) were obtained from Mallinckrodt Inc. (Portland, OR). An Atomlab 950 Thyroid Uptake System (Biodex Medical Systems, Inc., Shirley, NY) was programmed to measure and record the 123I counts per minute (cpm) in the capsule just before ingestion (time zero). The instrument was also programmed to measure and record the cpm over the thyroid, the cpm over the thyroid adjusted for radioactive decay of the ingested 123I, and the percentage uptake of the ingested 123I by the thyroid since time zero. The instrument was calibrated each day before use.

THYROID FUNCTION TEST:
- Main study, serum TT4, FT4, TT3, and TSH were analyzed in blood drawn on 16 occasions: the screening (unspecified time) and baseline (0800 hr) visits, E1 (1200 and 1600 hr), E2 (0800, 1200, and 1700 hr), E3 (0900 hr), E4 (0800 and 1200 hr), E8 (0900 hr), E14 (0800, 1200, and 1700 hr), P1 (0900 hr), and P15 (0900hr). Serum Anti-TPO levels were measured in blood drawn at the screening visit and on P15 for all subjects. Anti-Tg results were reported in both samples for only eight mainstudy subjects and in the P15 sample alone for the remaining 16 main-study subjects.
- Uptake study: serum TT4, FT4, TT3, and TSH were analyzed only in blood drawn at the screening visit (unspecified time) and on E14 (0800 hr). Anti-Tg results were reported only in the P15 sample alone for the one uptake-study subject (the woman with abnormally elevated TSH).

SERUM CHEMISTRY AND HEMATOLOGY:
- Main study: a serum chemistry panel (analysis of sodium, potassium, calcium, chloride, total CO2, glucose, urea nitrogen, total bilirubin, albumin, TBG, total protein, creatinine, aspartate transaminase, and alkaline phosphatase) and a complete blood count (including differential) were performed on blood samples drawn at the screening visit and on E2, E14, and P15. Analysis of alanine aminotransferase (ALT) in the same samples was requested, but the test was inadvertently omitted from the serum chemistry panel until midway through the study; only 16 main-study subjects were tested for ALT at one or more scheduled time points.
- Uptake study: the above tests (excluding analysis of TBG) were performed only in blood samples drawn at the screening visit.

QUALITY ASSURANCE/QUALITY CONTROL: the study data were subjected to an intensive quality assurance/quality control (QA/QC) audit by outside auditors. The RAIU data were audited under the management of the AFRL. All study data other than the RAIU data were audited by Toxicology/ Regulatory Services, Inc. (TRS; Charlottesville, VA) under the management of Toxicology Excellence for Risk Assessment (Cincinnati, OH). The TRS audit led to the discovery of five FT4 data points that had been entered into the database in error. All results reported reflect the database corrected to exclude these five data points.

OTHER DESCRIPTIVE INFORMATION ABOUT STUDY: during an initial interview, each prospective subject was given verbal information about the study’s purpose, exclusionary criteria, and requirements. At that time an appropriate consent form was provided that the subject took home to review. The two consent forms, one for each protocol, were approved by the institutional review board at OHSU, where the study was conducted. Both forms listed the study’s sponsor and explained the purpose, procedures, risks and discomforts, benefits, alternatives to participation, confidentiality, compensation provided ($ 1,200 for the main-study protocol, $ 650 for the uptake-study protocol), and the voluntary nature of participation. The consent form was signed at the screening visit by the subject, the principal investigator, and a witness.

Exposure assessment:

not specified

Details on exposure:

TYPE OF EXPOSURE: via drinking water

DOSING SOLUTIONS: the contents of one or more capsules were emptied into a beaker, the powder was mixed with 0.5–1.0 ml lemon juice to make a slurry, and sufficient commercial spring water was added to make a stock solution containing 50 mg KClO4/100 ml. Dosing solutions were prepared by appropriate dilution of the stock solution with spring water to a volume of 400 ml in a half-liter plastic bottle. The concentration of perchlorate in some of the stock solutions and in each subject’s individual dosing solution was analyzed and confirmed at the Air Force Research Laboratory (AFRL; Wright-Patterson Air Force Base, OH). The stock solution samples were found by AFRL to contain perchlorate at 98 % of the nominal concentration, indicating essentially complete solubility. Samples of the unadulterated spring water were also analyzed by AFRL to ensure the absence of perchlorate contamination. Some of the filler in the capsule was insoluble and remained visible as a fine white sediment in the stock solution. Bottled dosing solutions were prepared several times for each subject during the 14 days of perchlorate administration.

ADMINISTRATION OF DOSING SOLUTIONS: subjects were given two 250-ml clear plastic cups that had been marked with a horizontal line to indicate the 100-ml level, one to keep at home and one to leave at work. They were instructed to drink 100 ml at 0800, 1200, 1600, and 2000 hr on each scheduled perchlorate ingestion day and to record the time and volume of each ingestion on a preprinted log sheet for additional verification. They were also instructed to empty the bottle completely into their 2000 hr aliquot to ensure that the entire prescribed perchlorate dose was ingested each day.

EXPOSURE LEVELS: 0.007, 0.02, 0.1, and 0.5 mg/kg-day.

RATIONALE FOR DOSE SELECTION: based on the observation by Lawrence et al. that a mean perchlorate dose of 0.12 mg/kg-day inhibits RAIU to about 60 % of baseline, the doses of 0.02, 0.1, and 0.5 mg/kg-day were chosen to broadly span the effective inhibitory range, thinking it possible that a dose of 0.02 mg/kg-day would be a NOEL. By the time the first 14 subjects had been tested at these three doses, it was clear that 0.02 mg/kg day was not a NOEL. Regression analysis of the E14 uptake relative to baseline for the first 14 subjects predicted that a perchlorate dose of 0.007 mg/kg-day would produce no inhibition of RAIU; this became the fourth dose tested.

EXPOSURE PERIOD: 14 days.

POSTEXPOSURE PERIOD: 15 days.

Lawrence J.E., Lamm, S.H, Pino, S., Richman, K., Braverman, L.E., 2000, Thyroid 10:659-663

Statistical methods:

Linear regression and correlation analysis to reveal regression slopes and correlation coefficients (r values) were used. RAIU data from the main and uptake studies were combined. Analysis of variance (ANOVA) was used to investigate the dose dependence of outcome variables, dose was entered as a categorical variable. For pairwise comparisons with baseline values, the two-tailed t-test for dependent samples was used and the nonparametric Wilcoxon matched pairs test. All statistical analyses were run on Statistica (StatSoft, Tulsa, OK). The selected criterion for statistical significance was p < 0.05; however, all p values < 0.1 were also reported. Results are given as mean ± SE (or regression fit ± SE).

Results and discussion

Results:

THYROIDAL RADIOIODINE UPTAKE: given its 13-hr half-life, no 123I was detectable in the thyroid just before administration of any subsequent 123I dose.
- Baseline uptake: thyroidal radioiodine uptake at baseline varied widely among subjects: 5.6–25.4 % for the 8-hr uptake and 9.8–33.7 % for the 24-hr uptake.
- Effect of perchlorate ingestion: the suppression of radioiodine uptake was linearly related to the logarithm of perchlorate dose. Pairwise comparison revealed no statistically significant difference between the suppression of radioiodine uptake on E2 and E14 at either 8 or 24 hr after radioiodine administration (p > 0.7), indicating the achievement of quasi-steadystate inhibition by E2 and the absence of a cumulative effect. The lowest dose producing no statistically significant inhibition of uptake was 0.007 mg/kg-day. Thus, 0.007 mg/kg-day (7 μg/kg-day) was a NOEL for inhibition of RAIU.
- Recovery from perchlorate ingestion: on P15, the 8- and 24-hr uptakes were statistically indistinguishable from their respective baseline uptakes (p > 0.4 by pairwise comparison), indicating complete recovery from the inhibitory effect of perchlorate.The mean (±SE) 8-hr uptakes relative to baseline were 111.7 ± 8.1 %, 103.5 ± 10.4 %, and 107.7 ± 11.3 % in the 0.02-, 0.1-, and 0.5-mg/kg-day dose groups, respectively. The mean (±SE) 24-hr uptakes relative to baseline were 100.3 ± 8.4 %, 105.3 ± 5.5 %, 106.6 ± 9.1 %, and 104.6 ± 9.4 % in the 0.007-, 0.02-, 0.1-, and 0.5-mg/kg-day dose groups, respectively. The woman with elevated TSH at the screening visit had a 24- hr uptake of 10 % at the baseline visit, the lowest value observed in her dose group (0.007 mg/kg-day) or any group. Her 24-hr uptake was 13.9 % on E14, or 139 % of baseline. For subjects with low baseline uptake, small changes in absolute uptake, up or down, appear large in terms of relative uptake.
- Effect of sex: in a two-way model testing the effect of sex and dose on the uptake relative to baseline for each of the six postbaseline measurements (8- and 24-hr uptakes on E2, E14, and P15), ANOVA revealed no effect of sex for any measurement (p > 0.4). Similarly, two-way ANOVA revealed no effect of sex on the 8- and 24-hr raw uptakes at the baseline visit (p > 0.7) or on E2, E14, or P15 (p > 0.3).
- Correlation of 8- and 24- hr uptakes and relative uptakes: over all measurement days and dose groups, the 8- and 24-hr uptakes were linearly related (r = 0.975, n = 121). Pairwise comparison of the U24/U8 ratio at baseline with the U24/U8 ratios for measurements made during perchlorate administration (E2 and E14 combined) or on P15 revealed no significant difference, indicating that the ratio is independent of perchlorate treatment. The 8- and 24-hr relative uptakes for E2, E14, and P15 combined were likewise linearly related (r = 0.967, n = 84).
- Perchlorate elimination rate: the rate of perchlorate elimination from levels in serum following perchlorate withdrawal was calculated. For the eight main-study subjects in the 0.5-mg/kg-day dose group, t1/2 values ranged from 6.0 to 9.3 hr (average, 8.1 hr). Fewer data points were available for the 0.1- mg/kg-day subjects because levels fell more rapidly below the analytical detection limit of 5 ppb after perchlorate withdrawal. Serum levels in the 0.02-mg/kg-day dose group were almost entirely below detection throughout the exposure period.

SERUM HORMONES
- Effect of perchlorate ingestion: TSH, FT4, TT4, and TT3 levels throughout the study were in the normal range for all subjects except one woman in the 0.007-mg/kg-day dose group (uptake study) who had abnormally high TSH (18 and 15 mIU/l) on both occasions at which the hormone was measured (screening visit and E14, respectively). Two-way ANOVA applied to the exposure and dose variables revealed no significant dependence of any serum hormone on the exposure variable. By chance, the 0.5-mg/kg-day group had significantly higher baseline visit TSH levels than did the 0.1-mg/kg-day group (p < 0.014, t-test for independent samples). Similarly, the 0.5-mg/kg-day group had significantly higher screening-visit (p < 0.046) and marginally higher baseline-visit (p <0.058) TT3 levels than did the 0.02-mg/kg day group. The disparity in pre-exposure values skewed the comparison of TSH and TT3 across dose groups. To eliminate the problem of intergroup differences in pre-exposure serum hormone values and elucidate any possible effects of perchlorate, oneway ANOVA was performed for each dose group separately, once against the exposure variable and once against blood-draw event (16 draws between the screening visit and P15). The results revealed no association of FT4, TT4, or TT3 with blood-draw event in any dose group. However, a marginally significant association of TSH with blood-draw event in the 0.5-mg/kg-day dose group (p = 0.09) was found.
- Influence of circadian variation: to eliminate any potential dampening of the above association by circadian variation in TSH levels, a time-of-day variable with three categories was defined: morning (before 1030 hr), mid-day (1030–1359 hr), and late afternoon (1400 hr or later); these categories refer to the recorded time of each blood draw, not the target (nominal) time. ANOVA performed separately within each dose group for each time-of-day category (a total of 3 × 3 = 9 analyses) yielded a significant relationship between TSH and blood-draw event only in the morning draws of the 0.5-mg/kg-day dose group (p = 0.03). The data suggest an overall downward trend during exposure with recovery by P15. Analysis of the dependence of FT4, TT4, and TT3 on blood-draw event (ANOVA performed separately within each dose group for each time-of-day category) indicated that TT3 was significantly related to blood-draw event in the late-afternoon draws of subjects in the 0.1-mg/kg-day dose group (p = 0.03). However, the effect appears to be attributable to the fact that two of the screening-visit draws for the 0.1-mg/kg-day group fell into the late-afternoon category and both happened to be low (80 and 84 ng/dl). All other late-afternoon draws in the 0.1-mg/kg-day group occurred at the scheduled late-afternoon events on E1, E2, and E14; mean TT3 values (± SE) for these draws in the eight subjects tested were 102.9 ± 3.9 ng/dl, 104.9 ± 2.9 ng/dl, and 106.3 ± 3.4 ng/dl, respectively. For the two subjects with respective TT3 values of 80 and 84 ng/dl at the screening visit, TT3 values on E1, E2, and E14 were 92, 91, and 95 ng/dl in the first subject and 100, 99, and 112 ng/dL in the second. TT3 levels in the 0.5- mg/kg-day dose group were independent of blood-draw event in every time-of-day category (p > 0.7). Consideration of all the available data does not suggest an effect of perchlorate on TT3 in this study.
- Serum anti-Tg and anti-TPO: serum anti-Tg levels were below detection (< 40 IU/ml) in all samples tested. Levels of anti- TPO were above normal (> 20 IU/ml) in two subjects. One was a 56-year-old male in the 0.02-mg/kg-day group whose anti-TPO levels at the screening visit and on P15 were 63 and 59 IU/mL, respectively. The other was the previously mentioned 49-year-old female in the 0.007-mg/kg-day group with elevated TSH. Her screening-visit anti-TPO level was 75 IU/ml; she was not tested a second time. Both subjects were clinically euthyroid, and their other thyroid function test results remained within normal limits throughout the study.
- Serum chemistry and hematology: serum chemistry and hematology results were within normal limits throughout the study in all subjects. No significant change were found in any of these parameters during or after perchlorate administration.

Strengths and weaknesses:

The power of the study to detect a 20 % difference between males and females in the uptake relative to baseline was tested. The highest power values were found for E2: 83 % and 95 % for the respective 8-hr and 24-hr measurements in the 0.5-mg/kg-day dose group and 99 % for the 8-hr measurement in the 0.1-mg/kg-day dose group.

Any other information on results incl. tables

The descriptive statistics for the 8- and 24 -hr raw uptakes (expressed as a percentage of ingested ¹²³I) and the 8- and 24 -hr relative uptakes (expressed as a percentage of the baseline uptake).

Table: descriptive statistics for the 8- and 24 -hr thyroidal RAIU by dose

	8-hr uptake (mean ± SE)			24-hr uptake (mean ± SE)
Dose	No	Raw (% ^123I)	Percent of baseline	No	Raw (% ^123I)	Percent of baseline
0.5 mg/kg-day
Baseline visit	10	14.1 ± 1.4	-	10	21.6±2.0	-
E2	8	4.4 ± 0.4	31.6 ± 2.9**	8	6.5±0.6	30.6 ± 2.6**
E14	10	4.5 ± 0.5	32.6 ± 3.3**	10	6.9±0.9	32.9 ± 3.8**
P15	8	14.7 ± 1.4	107.7 ± 11.3	10	21.7±2.0	104.6 ± 9.4
0.1 mg/kg-day
Baseline visit	10	12.8 ± 1.5	-	10	19.9 ± 2.1	-
E2	8	7.7 ± 1.1	59.4 ± 2.0**	8	11.8 ± 1.7	59.2 ± 3.5**
E14	9	7.4 ± 1.3	56.7 ± 5.2	10	11.0 ± 1.6	55.3 ± 3.9**
P15	8	12.9 ± 1.8	103.5 ± 10.4	10	20.8 ± 2.2	106.6 ± 9.1
0.02 mg/kg-day
Baseline visit	10	11.8 ± 1.0	-	10	18.4 ± 1.2	-
E2	8	10.2 ± 1.0	83.8 ± 6.3*	8	15.7 ± 1.4	82.8 ± 5.6*
E14	10	9.4 ± 0.7	81.8 ± 4.2**	10	15.2 ± 1.1	83.6 ± 4.1**
P15	8	13.5 ± 1.2	11.7 ± 8.1	10	19.1 ± 1.3	105.3 ± 5.5
0.007 mg/kg-day
Baseline visit	7	12.6 ± 2.5	-	7	18.1 ± 3.1	-
E14	7	10.6 ± 1.1	93.8 ± 9.0	7	16.5 ± 1.6	98.2 ± 8.3
P15	-	-	-	7	17.3 ± 2.5	100.3 ± 8.4

*p < 0.05; **p < 0.005 (pairwise comparison to baseline).

True NEL values of 5.2 and 6.4 μg/kg-day were estimated based on the dose–response for inhibition of the respective 8-hr (n = 36) and 24-hr (n = 37) RAIU on E14 in all subjects tested one with subclinical autoimmune hypothyroidism, the rest euthyroid.

Based on the variability observed in these subjects, there is a 95 % probability that thyroidal iodide uptake will be inhibited by no more than 8.3–9.5 % at a dose of 5.2–6.4 μg/kg-day.

NOEL is indicated as the highest exposure level tested at which inhibition of thyroidal iodide uptake is not statistically or biologically significant.

Applicant's summary and conclusion

Conclusions:

NOEL = 0.007 mg/kg bw-day, based on radioactive iodine uptake.

Executive summary:

The dose response in humans for test substance inhibition of thyroidal iodide uptake and any short-term effects on thyroid hormones was evaluated. For this reason, 37 male and female volunteers were exposed to the substance in drinking water at 0.007, 0.02, 0.1, or 0.5 mg/kg-day for 14 days. In 24 subjects 8 - and 24 -hr measurements of thyroidal ¹²³I uptake (RAIU) were performed before exposure, on exposure days 2 (E2) and 14 (E14), and 15 days postexposure (P15). In another 13 subjects both E2 studies and the 8-hr P15 study were omitted.

A strong correlation between the 8- and 24-hr RAIU over all dose groups and measurement days was observed. No difference between E2 and E14 in the inhibition of RAIU produced by a given perchlorate dose was found nor any sex difference. On both E2 and E14, the dose response was a negative linear function of the logarithm of dose. Given default body weight and exposure assumptions, these doses would be ingested by an adult if the drinking-water supply contained perchlorate at concentrations of approximately 180 and 220 μg/l (ppb), respectively. On P15, RAIU was significantly indistinguishable from baseline indicating complete recovery from the inhibitory effect of the substance. Serum levels of thyroxine (total and free), triiodothyronine, and thyrotropin in blood sampled 16 times throughout the study were in the normal range for all subjects besides a woman that had abnormally TSH during the screening visit and E14. Only the 0.5 mg/kg-day dose group showed any effect on serum hormones: a slight downward trend in thyrotropin levels in morning blood draws during perchlorate exposure, with recovery by P15. Serum chemistry and hematology results were within the normal limits throughtout the study.

The lowest dose producing no statistically significant inhibition of uptake was 0.007 mg/kg-day. Thus, 0.007 mg/kg-day (7 μg/kg-day) was a NOEL for inhibition of RAIU. A more refined estimate of the true no-effect level (NEL) was estimated based on the dose response for inhibition of the 8- and 24-hr RAIU on E14 in all subjects; it was found to be 5.2 and 6.4 μg/kg-day, respectively.