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Registration Dossier
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EC number: 235-183-8 | CAS number: 12124-97-9
Specific investigations: other studies
Administrative data
Link to relevant study record(s)
- Endpoint:
- specific investigations: other studies
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1982
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Study investigated the mechanism of the formation of wavy ribs and no guideline is available for the conduct of mechanistic studies
- Qualifier:
- no guideline available
- Deviations:
- not applicable
- Remarks:
- there is no guideline available for this special investigation
- Principles of method if other than guideline:
- study was performed according to good experimental practice
- GLP compliance:
- no
- Type of method:
- in vivo
- Endpoint addressed:
- developmental toxicity / teratogenicity
- Species:
- rat
- Strain:
- other: SD-JCL
- Sex:
- female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Not indicated
- Age at study initiation: 12 weeks
- Weight at study initiation: 230 ± 25 g - Route of administration:
- oral: gavage
- Vehicle:
- other: Distilled water (bunitrolol) or distilled water 0.05% Tween-80 solution (fenoterol)
- Details on exposure:
- The test substances were dosed by gavage at a constant volume of 1 ml/100 g.
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- Fenoterol:
-Day 7-17 of gestation to study dose dependency, development, repairability, serum biochemistry and β-adrenoceptor-blocking
-Day 7-12 or 13-17 to study the period of susceptibility
Bunitrolol:
- Day 7-17 to study β-adrenoceptor-blocking - Frequency of treatment:
- Please refer to "duration of treatment/exposure"
- Post exposure period:
- Day 18-21 (or Day 13-21) of gestation; Caesarean section was performed on Day 21 of gestation.
- Remarks:
- Doses / Concentrations:
20, 100, 300, 500 and 1000 mg/kg;
Basis:
actual ingested
Fenoterol hydrobromide - Remarks:
- Doses / Concentrations:
150 mg/kg bw
Basis:
actual ingested
Bunitrolol hydrochloride - No. of animals per sex per dose:
- Not indicated
- Control animals:
- yes
- Examinations:
- General:
Nr. of dead Foetuses, foetal weight
Skeleton:
Yes; foetal skeleton was stained with alizarin red and alcian blue and examined for abnormalities. Radiographs were taken on Days 1, 3, 7, 14 and 21 from litters that were allowed to grow up until Day 21 of lactation - Details on results:
- Teratogenic effects:
Dose dependency:
Litter parameters were comparable among the groups, except a slight decrease in fetal weight of the 1000 mg/kg group. The number of fetuses showing wavy ribs was none among controls and at 20 mg/kg, 2.6% at 100 mg/kg, 14.1% at 300 mg/kg, 51.9% at 500 mg/kg and 77.3% at 1000 mg/kg and followed a clear dose-response relationship. Calcareous deposits around veins in chorionic membrane of placentae and edema of the labyrinth were observed at 1000 mg/kg only. Eleven fetuses of the 1000 mg/kg group had an edematous skin (anasarca). Edematous fetuses mostly had wavy ribs, too.
Period of susceptibility:
In the group dosed at 1000 mg/kg bw/day from Day 7-12 of gestation, only one case (1.1%) of wavy ribs occurred, while 23 fetuses 31.6%) with wavy ribs and six (6.9%) anasarca were observed in the group treated at Days 13-17 of gestation.
Development:
In control fetuses ossification of ribs started on Day 16 of gestation gradually from the middle third of the rib. One day later, ossification became more distinct and spread towards both ends of the rib. On Day 18, ribs were ossified. Pathologic ribs in fetuses of the 1000 mg/kg group did not differ visibly from ribs of controls on Day 16. On Day 17 and 18, ribs had separated or single, shortenedossification centres. Curving of sufficiently ossified ribs was observed on Day 19, and on Day 20, curves were ossified.
Repairability:
The rate of the total wavy ribs observed by alizarin staining did not differ significantly between fetuses and the various age groups of pups post partum, but the incidence was reduced at weaning. The severity of the finding after administration of 1000 mg/kg bw/day gradually decreased with aging through the lactation period and the ribs were almost normalized at weaning .The mortality of pups was high after treatment with 1000 mg/kg bw/day but X-ray examination of individual pups, distorsion of ribs which was distinctly visible on 1st day post partum, gradually disappeared during the lactation period or was hardly visible at weaning.
Serum biochemistry:
Values of maternal serum did not differ significantly between the three groups investigated. At the dose level investigated (1000 mg/kg), alkaline phosphatase (AP) and total protein were significantly decreased in fetal serum and the decrease was more distinct in the group treated through Day 20 compared to the group treated through Day 17. Calcium and phosphorus values were comparable between the groups. The amount of fetuses with wavy ribs was 40% in the group treated from Day 7-17 and 53.4% in the group treated from Day 7-20, respectively. A number of edematous fetuses was also observed.
Beta-adrenoceptor blocking:
The rate of fetuses showing “wavy ribs” was significantly decreased in the group treated at 300 mg/kg fenoterol and 150 mg/kg bunitrolol on Days 7-17 of gestation. The group treated with two doses of 300 mg/kg bunitrolol and 300 mg/kg fenoterol from Day 7 to 17 of gestation, showed amounts of fetuses with wavy ribs comparable to control group. - Conclusions:
- Both investigation methods, alizarin staining and X-ray examination, clearly showed that wavy ribs which were distinctly visible in fetuses obtained by caesarean section and in pups during first week post partum, gradually normalized during the lactation period until weaning, when wavy ribs were hardly visible or apparently disappeared.
From the results, wavy ribs are considered not to belong to true malformations or variations, but to be a reversible pathologic finding acquired during the perinatal period of foetal life. - Executive summary:
Materials and Methods
In this investigation, the origin and repairability of wavy ribs in the offspring after treatment of pregnant rats with fenoterol at dose levels of 0, 20, 100, 300, 500 and 1000 mg/kg was examined. Dose dependency, period of susceptibility, development, repairability, serum biochemistry and beta-adronoceptor blocking were studied using different approaches (variations in amounts of fenoterol, days when the test substance was administered and day of investigation).
Results and Discussion
In the present investigation it became evident that wavy ribs are mostly related to the treatment from Day 13-17 of gestation. The results confirm a statement made before, that wavy ribs are due to stimuli acting between the period of true teratogenic susceptibility and the immediate prenatal period. The ribs did not show alterations on Day 16 before ossification started. One day later, retarded ossification was observed followed by distortion of the ribs. Therefore, the wavy ribs do not belong to the basic developmental disturbances in organogenesis of the bone, but are caused by retarded ossification. The mortality of pups after treating the dams with 1000 mg/kg bw/day was high in the first week post partum only, showing that the disappearance of distinctly visible wavy ribs was not merely related to the death of abnormal pups. In one group of pups which was observed between Day 1 and 21 post partum after treatment at 1000 mg/kg bw/day during Days 7-17 of gestation, gradual disappearance of wavy ribs until weaning was seen by periodically taken radiographs. Wavy ribs were also found in 0.02% of rat foetuses in the background control data. Other investigators had reported 0.2% and >2% foetuses with wavy ribs within the control group. The reasons for the retarded ossification are not yet completely understood. Considering the large variation in chemical structure and biological activity of the various compounds causing wavy ribs, this side effect is not likely to be associated with a specific pharmacological activity.
- Endpoint:
- specific investigations: other studies
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1974
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: No official guidelines were available at the time of study conduct and for this kind of special investigations.
- Qualifier:
- no guideline available
- Deviations:
- not applicable
- Remarks:
- no official guidelines were available at the time of study conduct
- GLP compliance:
- no
- Type of method:
- in vivo
- Endpoint addressed:
- not applicable
- Species:
- dog
- Strain:
- not specified
- Sex:
- female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Not indicated
- Age at study initiation: not indicated in the study
- Weight at study initiation: average weight of 16 kg - Route of administration:
- infusion
- Vehicle:
- not specified
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Concentration in vehicle: 4, 6 and 8 M (corresponding to 213.96, 320.94 and 427.92 g/L ammonium chloride)
- Amount applied: 50-125 µM/kg/min (corresponding to 2.67-6.69 mg NH4Cl/kg/min or 160.2-401.4 mg NH4Cl/kg in total over one hour infusion period) as infusion into the renal artery. - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 60-110 minutes
- Frequency of treatment:
- Single treatment
- Post exposure period:
- 2 days
- Remarks:
- Doses / Concentrations:
4, 6 and 8 M
Basis:
nominal in water - No. of animals per sex per dose:
- 40 animals in total
- Control animals:
- yes, concurrent no treatment
- Details on study design:
- Ammonium chloride was infused into the renal artery at different concentrations.
- Examinations:
- Mortality: daily
Clinical Chemistry: pH of blood, as well as concentrations of sodium, potassium, chloride, bicarbonate and ammonia were measured. No other parameters were investigated.
Gross and Histopathology: kidneys were examined only. No other organs were subjected to gross and histolopathological examination.
Measurement of renal blood flow : during ammonium cloride infusion in four experiments.
Urinalysis - Details on results:
- Mortality:
No mortalities observed.
Clinical Chemistry:
Changes included decreases in pH, HCO3- and Na+ and increases in K+, Cl- and NH4. After 90-minutes infusion of NH4Cl, arterial NH4+ concentration was 1-2 mM/L (18-36 mg/L) and pH approximately 7.0. The observed decreases in pH with NH4Cl were in the presence of marked hyperventilation: PCO2 decreased to 20 mm Hg from a pre-infusion level of 37 mm. Arterial pH showed less regular change with ammonium acetate: decreased to 7.2 and 7.1 during repiratory depression in two experiments in which pH was measured.
Renal venous blood from the infused kidney was sampled through the ovarian vein in eight experiments. Renal arteriovenous pH difference was small and difficult to measure accurately prior to infusion, but renal venous blood became significantly more acid than arterial blood by approximately pH 0.1 during NH4Cl infusion. No significant arterial-venal pH difference was seen with NH4 acetate. Renal venous NH4+ concentrations were of the expected magnitude (5-40 mM/L), but measurements of these and blood flow were not of sufficient accuracy to estimate renal NH4+ extraction. Renal O2 arterial-venal difference, measured in one experiment, remained unchanged at 5 cm3/100 ml during the infusion.
Urinalysis:
Urine flow, osmolality and creatinine clearance of kidneys with definite necrotic changes were regularly decreased as compared with the contralateral control organ. Because urine Na concentration from the damaged kidney was increased, excretion rates of sodium from the two kidneys were approximately equal. Comparison of data from kidneys which did not show significant necrosis, showed that infused and control kidneys were very similar in urine flow and creatinine clearance; but the infused kidney had a lower urine osmolality. When necrosis with impairment creatinine clearance followed NH4Cl infusion, impairment was permanent.
Gross and Histopathology:
Histological changes were observed in some kidneys. The eight animals infused with 4 M NH4Cl for 90 minutes showed few or no changes, while those infused with 6 M NH4Cl for 90 minutes regularly showed extensive changes.
NH4+ acetate infused under similar conditions (8 mM/kg in 90 min) or intravenous bicarbonate (5 % at 3-4 mL/min) during intrarenal infusion of NH4Cl was not associated with significant histological changes.
Two apparently distinct histological lesions were discerned: one was arterial damage with or without thrombosis. In seven experiments thrombosis extended into the renal artery and occurred with or without addition of heparin (166 U/ml) to the infusate. These kidneys were anuric, showed widespread necrosis and were rejected from the further consideration. In other kidneys, occasional thrombosed small arteries or arterioles could be found, usually with patchy cortical areas of necrosis and interstitial hemorrhage. Many arteries which were not thrombosed showed loss of nuclear detail and fibrinoid changes in the intima and media. Medullary necrosis was found in one kidney. The second lesion was tubular necrosis. This had the appearance of homogenous, pink-staining proximal segments with absent nuclei and lumina, again with a patchy distribution in the less severely affected kidneys. Glomeruli were usually well preserved although endothelial cell swelling tended to diminish capillary lumina in all glomeruli of perfused kidneys. Whether the second lesion (tubular necrosis with preservation of glomeruli and blood vessels) was related to impairment of blood flow following the infusion could not be determined. Contralateral, non-infused control kidneys were invariably normal indicating that systemic acidosis, alone, did not cause tubular necrosis. - Conclusions:
- The experiments show that exposure of the kidney to 4 M NH4Cl for 90 minutes results in few or no changes while infusion with 6 M NH4Cl for 90 minutes showed extensive changes. Thus, animals receiving less than 6 mM/kg (321 mg NH4Cl/kg) showed few or no changes, while those receiving 8 mM/kg (428 mg NH4Cl/kg) regularly showed histological changes in the kidneys. Correspondingly, the degree of renal and systemic acidosis was proportional to the quantity infused. Similarly, NH4+ acetate infused under similar conditions (8 mM/kg in 90 min corresponding to 616 mg/kg in 90 min) was not associated with significant histological changes. Two distinct histological lesions were discerned: arterial damage with or without thrombosis and tubular necrosis.
It is concluded that renal ammonium acetate concentrations up to 40 µM/L for 90 minutes does not cause tubular necrosis but does impair urine concentration. Severe tissue damage followed renal exposure to high ammonium concentrations in the presence of metabolic or renal acidosis.
LOAEL: 6M NH4Cl due to histological changes within the kidney, which were arterial damage with or without thrombosis and tubular necrosis.
NOAEL: Renal ammonia concentration of 40 µM NH4Cl/L corresponding to 192.6 mg NH4Cl/kg bw during 90 minutes - Executive summary:
Materials and Methods
The study was performed to investigate if increased renal ammonia concentration might adversely affect renal function or produce renal damage. Experiments were designed to supply additional information on renal function and histology after ammonium chloride infusion. One or both kidneys of 40 phenobarbital-anesthezied female dogs with an average weight of 16 kg were approached through flank or abdominal incisions. In most experiments, NH4+ was infused as the 0.6 M chloride salt at 50-125 µM/kg/min for 60-110 minutes into the left renal artery through a recurved needle. This exposure time was adopted because it encompasses the time usually required to induce oligomeric normothermic tubular necrosis in the dog. In five experiments, NH4+ was infused similarly as the acetate. These infusion rates were calculated to produce a renal arterial blood concentration of 20-30 mM/L. In some experiments, renal venous blood was sampled via the ovarian vein for NH4+ concentration and pH. In most experiments, the wound was closed following the infusion and the animals returned to their cages. 48 hours later, the animals were re-anesthezied, the right and left ureters cannulated through a suprapubic incision for assay of renal function and the animals sacrificed for histological examination after hematoxylin-eosin staining. Prior to removing the kidneys, urine specimens were obtained from each ureter to compare sodium, potassium, chloride, osmotic and creatinine concentrations. This procedure was supplemented in a few experiments each as follows: hemorrhagic hypotension during ammonium infusion; infusion of sodium bicarbonate or sodium sulphate intravenously; and studies of renal function and histology 10-50 days after ammonium infusion in trigone-explant dogs.
Results and Discussion
After infusion with NH4Cl a decreases in pH, HCO3- and Na+ and increases in K+, Cl- and NH4+ were observed along with marked hyperventilation. Arterial pH showed less regular change with ammonium acetate. Renal blood flow was decreased in flow to 50 % of control at 10-20 minutes of infusion followed by an increase to an average of 114 % of control after 60 min of infusion. A rapid decrease in urine flow from the infused kidney to anuria was demonstrated which continued into the period of increasing blood flow. Urine flow from the control kidney showed little change. After infusion of 4 M NH4Cl for 90 minutes few or no histological changes were noted in the kidney, while at 6 M NH4Cl for 90 minutes extensive changes were regularly evident. Correspondingly, the degree of renal and systemic acidosis was proportional to the quantity infused. NH4+ acetate infused under similar conditions. Two apparently distinct histological lesions were discerned: arterial damage with or without thrombosis and tubular necrosis. Urine flow, osmolarity and creatinine clearance of kidneys with definite necrotic changes were regularly decreased as compared with the control organ. Because urine Na-concentration from the damaged kidney was increased, excretion rates of sodium from the two kidneys were approximately equal. Comparison of data from kidneys which did not reveal significant necrosis, showed that infused and control kidneys were very similar in urine flow and creatinine clearance; but the infused kidney had a lower urine osmolarity.
- Endpoint:
- specific investigations: other studies
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1985
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: study investigated the mechanism of the formation of wavy ribs and as no guideline available for the conduct of mechanistic studies
- Qualifier:
- no guideline available
- Deviations:
- not applicable
- Remarks:
- no guideline available for this special investigation
- Principles of method if other than guideline:
- study was performed according to good experimental practice
- GLP compliance:
- no
- Type of method:
- in vivo
- Endpoint addressed:
- developmental toxicity / teratogenicity
- Species:
- rat
- Strain:
- Crj: CD(SD)
- Sex:
- female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River, Japan
- Age at study initiation: 10 weeks
- Weight at study initiation: 225-252 g - Route of administration:
- oral: unspecified
- Vehicle:
- not specified
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- Rats were treated on Day 16 of gestation only
- Frequency of treatment:
- Single treatment
- Post exposure period:
- From Day 17 to 21 of gestation, each day 2-3 rats from the treated and the control group were sacrificed and foetuses removed for examination. Some dams were allowed to give birth spontaneously and rear their pups. Pups were killed serially from Day 0 to 4 post partum for skeletal examination.
- Remarks:
- Doses / Concentrations:
300 mg/kg
Basis:
no data - No. of animals per sex per dose:
- For each time-point investigated, 2-3 treated and control animals were examined
- Control animals:
- yes, concurrent no treatment
- Examinations:
- Skeleton:
Skeletons were examined in the cartilage and bone specimens were stained with alcain blue and alizarin red. Of the pups that were killed serially from Day 0 to 14 post partum skeletons were examined in cartilage and bone specimens. Observation was focused on the ribs. - Details on results:
- Teratogenic effects:
In contrast to the untreated foetuses, azosemide-treated foetuses on Day 17 of gestation showed delayed enchondral ossification. In the area corresponding to ossification centres in the untreated foetuses, the periosteal band appeared, but bone depositions in the interior of cartilagemodel were not observed in most cases. Portions stained with neither alcain blue nor alizarin red were scattered within the cartilage, and were large and irregular compared with the untreated foetuses. A bend was not yet seen in ribs at this stage. On Day 18 of gestation, the well ossified zone was recognized near the vertebrae. A zone stained with neither alizarin rad nor alcain blue was observed between ossified and cartilage zones, while the periosteal band was forming on the unstained zone. The bend consisting of cartilage and unstained portion first appeared ventral to the unstained zone. The cartilage existed at the convex surface of the bend, and the unstained portion at the concave surface. The convex surface was much swollen, and the concave surface was relatively flat. On Day 19 of gestation, the ossified area extended, and became as dense and even as in the untreated foetuses. The unstained zone was also ossified. In the bent region, ossification began, but showed an irregular pattern; the cartilaginous, ossified and unstained portions coexisted. In rostral ribs, the ventral area adjacent to the bent zone was also ossified densely. On Day 20 of gestation, the bent region was almost completely ossified. From Day 21 of gestation onward, the ossification extended toward both sides.
Wavy ribs were seen in 93-100% of treated foetuses on Days 17-21 of gestation. The change in ribs 2-10 were observed from Day 17, while that in ribs 11-13 was noted from Day 18.
After birth, the prominence of the convex surface gradually disappeared with age. Concurrently with the change on the convex surface, the concave surface flattened out. On Days 6-8 post partum, the bend of ribs was much reduced in degree, and could not be seen in most treated pups on Days 10-14 post partum. In some ribs, the bent region showed a nodule-like appearance.
A bend of the scapula was also observed in treated foetuses with high frequency (97-100%). The sequence from appearance and disappearance of the bend in the scapula was almost similar to that in wavy ribs. - Conclusions:
- In conclusion, the present study showed that after single oral administration of azosemide on day 16 which is considered to be a critical day with resepect to the development of cartilage and bone of rats, the bend of ribs first appeared on Day 18 of gestation, i.e. 2 days after azosemide treatment and might be caused by mechanical action. The bend disappeared about 10-14 days after birth and surface remodelling of the bones might straighten the bend.
The present finding suggests that wavy ribs and bent scapula induced by azosemide are temporal, transient changes in the process of bone development and not real skeletal malformations. - Executive summary:
Materials and Methods
The aim of the study was to investigate the morphological sequence from appearance to disappearance of wavy ribs in pregnant rats after azosemide treatment. Examinations were performed in cartilage-bone double stained specimens of foetuses and pups from mothers treated with azosemide on Day 16 of gestation only, which was considered to be a critical time point with respect to developmental changes in cartilage and bone since skeletal abnormalities were produced with the highest frequency in preceding studies. Two to three dams were sacrificed on each day from Days 17-21 of gestation and foetuses removed for examination. Some dams were allowed to give birth spontaneously and rear the newborns. Pups were killed serially from Day 0-14 post partum and the skeletons were examined. Untreated rats served as control.
Results and Discussion
Differential staining of cartilage and bone of rat foetuses revealed that the cartilage models of ribs were already established on Day 16 of gestation when azosemide was administered to mothers. The first pathological change observed in azosemide-treated fetuses on Day 17 of gestation was inhibition of enchondral ossification. Large and irregular portions stained with neither alcain blue nor alizarin red were noted in the presumptive regions of primary ossification centres in treated foetuses. The results obtained by differential staining of cartilage and bone suggest that the bend itself may be caused by a mechanical action. The bend first appeared the second day after treatment. It consisted of cartilage at the convex surface of the bend and unstained portion at the concave surface in most cases. The convex surface was more prominent than the concave surface. In the normal development of long bones, the ends of the cartilage model continue to elongate and broaden by proliferation of chondrocytes and elaboration of new matrix after establishment of the primary ossification center. The cartilage in the bent region is considered to be normal and continue to grow, whereas growth in the unstained portion might be arrested or delayed. It is postulated that the difference in the growth between cartilage and unstained portion resulted in the bend. The bent long bone such as wavy ribs produced by azosemide was encountered in rat foetuses and pups during the perinatal and neonatal periods, but disappeared during infancy. Normally growing bones are constantly changing their internal organisation. The shape of a bone is maintained during growth by a continual remodelling of its surface, which involves bone deposition in some areas of the periosteum and bone absorption in other areas. In a bone of the bent region, remodelling mainly with resorption at the swelling convex surface and bone deposition at the concave surface might take place during the postnatal period, resulting finally in a straightening of the bend.
- Endpoint:
- endocrine system modulation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1994
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- other: publication is a review, no details given.
- Qualifier:
- no guideline available
- Deviations:
- not applicable
- Remarks:
- review article on bromide
- Principles of method if other than guideline:
- Not applicable, as publication is a review article on bromide.
- GLP compliance:
- no
- Type of method:
- in vivo
- Endpoint addressed:
- repeated dose toxicity: oral
- Species:
- dog
- Strain:
- not specified
- Sex:
- not specified
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- No details given on test animals within the review article. - Route of administration:
- oral: feed
- Vehicle:
- not specified
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- Not indicated
- Frequency of treatment:
- Daily; potassium bromide doses were given over 12 hours
- Post exposure period:
- Not indicated
- Remarks:
- Doses / Concentrations:
10 mg/kg bw
Basis:
no data
As a starting dose over a period of 12 hours. - No. of animals per sex per dose:
- Not indicated
- Control animals:
- not specified
- Examinations:
- No examinations are stated within the review article.
- Details on results:
- No details on results are given; publication is a review article on bromide.
- Conclusions:
- Bromide can be used in anticonvulsant therapy of dogs, in addition to phenobarbital or as the only anticonvulsant drug used when phenobarbital control is insufficient. As a starting dose 10 mg/kg bodyweight of potassium bromide should be administered via food. Serum bromide concentrations should be measured regularly to prevent bromide toxicosis. Because bromide does not undergo hepatic metabolism, bromide does not affect hepatic enzymes and, therefore, is useful in dogs with liver disease.
LOAEL: Not indicated
NOAEL: 10-20 mmol/L (serum concentration) as the therapeutic range corresponding to 800 – 1600 mg/L bromide or 80 – 160 mg/kg bw/day (assuming a total blood volume of 1L and a body weight of 10 kg for a dog). - Executive summary:
Materials and Methods
Potassium bromide is the oldest and, chemically, the simplest of the anticonvulsant drugs. Bromide has become popular as a second anticonvulsant drug in dogs that continue to have seizures despite adequate phenobarbital concentrations. Bromide replaces chloride in all body fluids and stabilizes neuronal cell membranes by interfering with chloride transport and potentiating the effect of GABA (gamma-aminobutyric acid), the inhibitory neurotransmitter in the central nervous system.
Results and Discussion
Potassium bromide is well adsorbed from the gastrointestinal tract in dogs, with peak adsorption in 1.5 hours. Maximum bromide concentration in cerebrospinal fluid (CSF) occurs 2 hours after oral administration. The elimination half-life is extremely long (24 days), therefore it takes approximately 4 months to achieve steady-state concentrations. Bromide is eliminated by the kidneys. Because it does not undergo hepatic metabolism, bromide does not affect hepatic enzymes and, therefore, is useful in dogs with liver disease. Bromide toxicosis (bromism) may be seen if serum concentrations exceed the therapeutic range. Neurological signs of bromism include lethargy, generalized ataxia, disorientation and delirium. Some animals may show signs of sedation for the first 3 weeks of therapy. Other common side effects include polyuria/polydipsia, erythematous dermatitis, conjunctivitis, nausea and anorexia. Sensitivity may be affected by the dog´s physical condition, food and salt intake, hydration, vomiting, diarrhea or renal insufficiency. To prevent bromide toxicosis (bromism) serum bromide concentration should be taken regularly. The therapeutic range for bromide is considered to be 10-20 mmol/L. Bromide should be considered to be added to anticonvulsant therapy when phenobarbital control is insufficient. Potassium bromide can be formulated in double distilled water. The recommended starting dose is 10 mg/kg bodyweight over 12 hours, administered via food. This allows the dog to gradually adapt to the cumulative sedative effects of phenobarbital and bromide. Bromide concentrations should be monitored at 30 days, 120 days and every 6 months after initiating the therapy. Therapy must be individually tailored, as serum bromide concentrations vary among dogs and each dog adjusts to bromide differently. Once bromide concentrations reach steady-state serum concentrations in the therapeutic range, the clinician can attempt to decrease the total daily phenobarbital dose. Some dogs can be successfully maintained on bromide therapy alone.
- Endpoint:
- endocrine system modulation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 2003
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: No guideline available for this type of special investigations
- Qualifier:
- no guideline available
- Deviations:
- not applicable
- Remarks:
- no guideline available for this type of special investigations
- Principles of method if other than guideline:
- Study is a publication and was performed according to good experimental practice.
- GLP compliance:
- no
- Type of method:
- in vivo
- Endpoint addressed:
- repeated dose toxicity: oral
- Species:
- dog
- Strain:
- other: Laboratory hound dog
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Not indicated
- Age at study initiation: 1-2 years
- Weight at study initiation: 24 kg - Route of administration:
- oral: drinking water
- Vehicle:
- water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Concentration in vehicle: 200 mg/mL - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 182 days
- Frequency of treatment:
- daily
- Post exposure period:
- None; animals were killed on termination of the study on Day 182
- Remarks:
- Doses / Concentrations:
100 mg/kg bw
Basis:
nominal conc.
Loading dose over 12 hours for a period of 2 days - Remarks:
- Doses / Concentrations:
30 mg/kg bw
Basis:
nominal conc.
Maintenance dose over 24 hours for a period of 180 days. - Remarks:
- Doses / Concentrations:
35 mg/kg bw
Basis:
nominal conc.
Adjustment on Day 120; if serum bromide concentrations were less than 250 mg/dL, dose was increased for the remaining study - No. of animals per sex per dose:
- 2 males and 3 females for treatment and control group each
- Control animals:
- yes, concurrent vehicle
- Examinations:
- Clinical signs: daily
Bodyweight: at study initiation and on Day 177; no subsequent body weight determinations were performed
Haematology: at start and termination of study haematological examination was undertaken. The parameters examined are not further specified in the report since no differences were observed between values at start and termination of the study.
Clinical Chemistry: on Days 3, 30, 120 and 177 serum bromide concentration, T4, free T4 and TRH levels were examined and serum biochemical analysis was performed (the details of parameters determined were not further specified in the report sine no differences were observed at start and termination of the study in serum biochemical parameters).
Total T4 and TSH concentrations were measured using chemiluminescent enzyme immunoassays validated for use in the dog. Free T4 concentrations were measured by equilibrium dialysis validated for use in dogs.
Serum bromide concentration was measured using a gold colorimetric assay. All blood samples were processed on the day of collection. Serum was removed from each blood tube within 45-60 minutes. Serum samples were divided into 4-5 aliquots and stored at -20°C until analysis was performed.
Urinalysis: at start and termination of study (urine specific gravity, USG)
Organ weights: Only thyroid tissue wet weights were recorded immediately after removal and expressed as g/kg bodyweight. No other organ weights were taken.
Gross and Histopathology: Thyroids were subjected to gross and histopathological examinations. Hematoxylin and eosin-stained sections of all thyroids were evaluated by light microscopy. No further organs/tissues were examined.
Other examinations: Physical examinations were performed daily for the first week, every 2 weeks for the first 28 days, and then once every 30 days until completion of the study.
Serum basal total T4 (TT4) and thyroid stimulating hormone (TSH) concentrations, serum free T4 (fT4) concentration, anticanine thyroglobulin antibody (TgA) optical density (OD) and a thyrotropin-releasing hormone (TRH) stimulation test were evaluated in each dog at baseline. Basal TT4 and TSH concentrations were measured in addition on Days 3, 30, 120 and 177. A fT4 by equilibrium dialysis concentration and TRH stimulation test were repeated on Day 177.
Thyroid tissue was assessed for activation referring to the following criteria that occur in response to hormone stimulation: enhanced vascularisation, increased microfollicular development (MFD), decreased intrafollicular colloid staining (IFC), increased number of columnar follicular epithelial cells, and increased follicular epithelium mitotic figures.
Statistics: The independent and joint influences of group (experimental versus control) and time on serum hormone concentration or body weight were evaluated by repeated measures analysis of variance (ANOVA). Thyroid wet-weights and thyroid histopathology scores were analyzed by the Wilcoxon´s signed rank sum test. Significance was defined as P< 0.05 for all statistical analysis. - Details on results:
- Clinical signs:
Neither clinical signs of hypothyroidism nor evidence of bromism were identified in any of the dogs.
Mortality:
No mortalities were reported in this study.
Bodyweight gain:
There was a significant (P< 0.0001) gain in body weight over time, but the weight change did not differ between experimental and control groups. However, the interaction for group x time was significant (P< 0.01), reflected by more rapid weight gain of dogs in the experimental group.
Haematology:
Hematological analysis was within reference ranges in each dog at initiation and termination of the study.
Clinical Chemistry:
Serum biochemical analysis was within reference ranges in each dog at initiation of the study. Abnormalities identified in KBr-treated dogs were consistent with those expected due to administration of KBr (falsely elevated chloride and low anion gap).
From day 30 to completion of the study, all serum bromide concentrations in KBr-treated dogs were within or exceeded the therapeutic range (88-300 mg/dL) recommended for epileptic dogs treated with KBr monotherapy. Three dogs exceeded the target serum bromide concentration (250-300 mg/dL) on Day 120. One of theses dogs was within the target range, one exceeded it on Day 177 and the third one had a serum bromide concentration within the therapeutic range but below the target range. At the end of the study, four of five dogs were within the therapeutic range, one of which was within the target range (250-300 mg/dL). The remaining dog had a serum bromide concentration of 313 mg/dL. On Day 177, serum bromide concentrations in three of five control dogs measured 0 mg/dL. Two control dogs had serum bromide concentrations of 8 and 9 mg/dL, but on repeated analysis bromide concentrations were 0 mg/dL in both dogs.
Urinalysis:
Urine specific gravity (USG) was within reference ranges in each dog at initiation of the study.
Organ weights:
No significant difference in thyroid wet-weight (P> 0.05) was found between experimental and control dogs. Thyroid wet-weights of the experimental group were 0.037 ± 0.017 g/kg bodyweight and for the control group 0.045 ± 0.015 g/kg bodyweight.
Gross and Histopathology:
In the scored categories of microfollicular development (MFD) and intrafollicular colloid staining (IFC), no significant difference was found between the KBr-treated and control dogs. However, most dogs (9/10 for MFD, 8/10 for IFC) scored outside of normal for these categories. The degree of vascularity identified in thyroid sections was considered appropriate and as not different between the treatment and control groups. Calculation of a mitotic index for thyroid sections was not possible because of the difficulty of differentiating apoptotic cells from mitotic cells. In general, mitotic figures appeared primarily in areas of microfollicular development; however, they were rarely seen in these regions. With respect to the prevalence of columnar epithelium in thyroid sections, no morphological differences were identified when microfollocular structures were compared with normal-appearing follicles. Neither normal follicular nor microfollicular regions of thyroid sections were different between the treatment and control groups. No significant inflammatory infiltrates consistent with thyroiditis were found in any of the thyroid sections.
Other results:
The median anticanine thyroglobulin antibody (TgA) OD for all dogs was 0.037 (range 0.009-0.153). Eight dogs were negative for TgA, having ODs less than twice the OD of the negative control. Two dogs (one each fro the treatment and control groups) were weakly positive for TgA.
There was no significant difference between experimental and control groups for serum basal TT4, basal TSH or results of TRH (thyrotropin-releasing hormone) stimulation tests. There were significant decreases in TT4 (P< 0.0001) and fT4 (P= 0.0002) over time, but these changes did not differ between the experimental and control groups. At baseline, one control dog had a basal TT4 concentration less than the established reference range (1.3-4 µg/dL) and a TSH concentration in the reference range (0-0.65 ng/mL). The postTRH TT4 concentration in this dog was consistent with euthyroidism (1.7 µg/dL). On Day 177, five dogs (three KBr-treated and two control dogs) had basal TT4 concentrations below the reference range. The fT4 of one KBr-treated dog was below the reference range (9-40 pmol/L) on Day 177. Thyroid-stimulating hormone concentrations in all dogs were within the reference range (0-65 ng/mL). Four of the five KBr-treated dogs gad postTRH TT4 concentrations > 1.5 µg/dL. One KBr-treated dog had a postTRH TT4 concentration of 1.3 µg/dL. On Day 0, percent TSH concentration change following TRH stimulation was < 100 % in all dogs. All KBr-treated dogs and four of five control dogs had a percent TSH concentration change following TRH stimulation > 100 % on Day 177. - Conclusions:
- In this study, KBr administration for 6 months to young, healthy adult dogs did not have a significant effect on the function or morphology of the canine thyroid gland compared to the control group. Although there was no difference in thyroid function between the groups at any time, the fact that both control dogs and KBr-treated dogs exhibited a significant decline in TT4 and fT4 over time complicated the results of this study. It was not possible to determine the cause of the decreased thyroid hormone concentration over the course of the study; however, because the decrease occurred in both the control and the experimental group, the change did not appear to be related to bromide administration. Based on the results of this investigations it may be further concluded that serum bromide levels in the therapeutic range of 250 – 300 mg/dL are not associated with adverse side effects.
LOAEL: > 300 mg bromide/dL (serum concentration)
NOAEL: 300 mg bromide/dL (serum concentration) corresponding to about 240 mg/kg bw/day assuming a body weight of 25 kg/dog and a (total) blood volume of about 2 L. - Executive summary:
Materials and Methods
A placebo-controlled experiment was performed to evaluate the effect of potassium bromide on the canine thyroid gland. Basal total thyroxine (TT4), free thyroxine (fT4), and basal thyrotropin serum (TSH) concentrations were evaluated over a 6-month period in potassium bromide-treated and control dogs. A thyrotropin-releasing hormone (TRH) stimulation test was also performed in all dogs at the beginning and conclusion of the study. Thyroid histopathology was compared between treated and control dogs at the end of the study. Serum bromide levels were collected from KBr-treated dogs on Days 3, 30, 120 and 177, and from control dogs on Day 177. Unilateral thyroidectomy was performed in all dogs on Day 182. Thyroid tissue wet weights were recorded and expressed as g/kg bodyweight. Hematoxylin and eosin-stained sections were evaluated and each slide assessed for evidence of thyroid activation. Increased microfollicular development (MFD) and decreased intrafollicular colloid staining (IFC) were scored for each slide on a scale of 1 to 4. Vascularity was scored by averaging the number of intermediate-sized blood vessels. To evaluate the prevalence of mitotic figures in thyroid sections, calculation of a mitotic index (number of mitotic figures per follicle/total number of epithelial cells per follicle) was attempted. The predominance of columnar follicular epithelial cells was subjectively assessed.
Results and Discussion
Neither clinical signs of hypothyroidism nor evidence of bromism were identified in any of the dogs. From day 30 to completion of the study, all serum bromide concentrations in KBr-treated dogs were within or exceeded the therapeutic range (88-300 mg/dL) recommended for epileptic dogs treated with KBr monotherapy. Three dogs exceeded the target serum bromide concentration (250-300 mg/dL) on Day 120. Hematological and serum biochemical analyses and USG were within reference ranges in each dog at initiation and as well at termination of the study. Abnormalities identified on serum biochemical analyses of KBr-treated dogs were consistent with those expected due to administration of KBr (falsely elevated chloride and low anion gap). The ability of the thyroid to concentrate iodide and subsequently incorporate this halide into thyroglobulin is essential to thyroid hormone synthesis. Experimental studies in vitro demonstrated that the thyroid was able to concentrate other ions of the group IV elements (ie fluorine, chlorine, bromine, iodine, astatine). In addition to the relative lack of anion specificity of the iodide uptake mechanism, halogen ions were also found to act as competitive inhibitors of iodide accumulation and cause release of accumulated iodide. Based on these findings it was hypothesized that bromide treatment might decrease thyronine synthesis. Experimental studies performed in rats confirmed that administration of bromide salts resulted in significantly decreased serum T4 and triiodothyronine (T3) concentrations with concurrent goitrous alterations of the thyroid gland. Further investigations suggested that in addition to inhibiting iodide uptake, bromide strongly inhibited the oxidation of iodide to iodine by hydrogen peroxide and, to a lesser degree, inhibited iodinated tyrosine residue coupling to thyronine. For the present study a target serum concentration of 250-300 mg/dL was chosen to ensure maximal effect on thyroid function. There was no significant difference between experimental and control groups for serum basal TT4, basal TSH or results of TRH (thyrotropin-releasing hormone) stimulation tests. This is in agreement with a previous study (Kantrowitz, et al, 1992) that also failed to find an effect of bromide administration on canine thyroid function. Bromide is a relatively weak competitive inhibitor of iodide uptake by the thyroid gland, and compared to iodide, it is concentrated by the thyroid to a much smaller degree. The figures measured in KBr-treated and control dogs for total and free T4 are in line with the background figures reported elsewhere for dogs (total T4 levels in the range of 1.5-4.5 µg/dL and free T4 levels of 7.7-47.6 pmol/L are reported as reference ranges in a textbook of veterinary medicine (“Klinische Labordiagnostik in der Tiermedizin”, Schattauer Verlag, 5.Auflage)). Although in a different study, bromide administration in rats caused depression of thyroid hormone production, this was not observed in dogs in this study. Reasons for this may include species differences in iodide uptake, thyroglobulin organification, tyrosine residue coupling, or thyroid hormone metabolism. The duration of exposure to bromide may have been inadequate and in addition, most studies in rats utilized higher doses of bromide than were used in this study. Another possible explanation for the differences in the effect of bromide on the thyroid function between rats and dogs is the fact that the majority of thyroid-hormones in dogs are plasmaprotein-bound which is not the case in rats. Thyroid hormones in rats are found free in the plasma and might, thus, be more sensitive to bromide than when protein-bound. Both control and experimental groups of dogs of the study described herein developed a statistically significant decrease in serum TT4 and fT4 concentrations over time but were within the reference ranges, consistent with euthyroidism. No significant difference in thyroid wet-weight (P> 0.05) and in thyroid (histo)pathology was found between experimental and control dogs and no significant inflammatory infiltrates consistent with thyroiditis were found in any of the thyroid sections. The difference in body weights between dogs in the experimental and control groups at day 177 was not significant (P= 0.15).
- Endpoint:
- mechanistic studies
- Type of information:
- other: review article
- Adequacy of study:
- supporting study
- Study period:
- 1995
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: publication is a summary of various investigations
- Qualifier:
- no guideline available
- Deviations:
- not applicable
- Remarks:
- publication is a summary of various investigations
- Principles of method if other than guideline:
- Study is a publication and was performed according to good experimental practice.
- GLP compliance:
- no
- Type of method:
- in vivo
- Endpoint addressed:
- basic toxicokinetics
- Species:
- dog
- Strain:
- not specified
- Sex:
- not specified
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
No details on test animals are given in the report. - Route of administration:
- other: in capsules or in drinking water
- Vehicle:
- other: capsules or sterile solution
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- Not indicated
- Frequency of treatment:
- Not indicated
- Post exposure period:
- Not indicated
- Remarks:
- Doses / Concentrations:
50-80 mg/kg bw/day
Basis:
actual ingested - No. of animals per sex per dose:
- Not indicated
- Control animals:
- not specified
- Examinations:
- Clinical signs: several time points over the study period which are not further specified.
Clinical Chemistry: serum bromide concentrations were determined only. No other clinical chemical parameters were determined. - Details on results:
- Clinical signs:
Common adverse effects of bromide in dogs are polyuria, polydipsia and polyphagia. During the first few weeks of treatment, sedation, ataxia and hind limb weakness are often seen, particularly in dogs given Phenobarbital or in dogs given loading doses of bromide. These neurologic effects can be resolved by lowering the dosage of phenobarbital to 10-30 %. Adverse neurologic effects can also be seen with serum bromide concentrations greater than the recommended therapeutic range. These signs can progress to stupor, inability to walk, or even coma.
Bromide:
Bromide may falsely increase serum chloride determinations by some methods, such as the thiocyanate colorimetric method used in many automated analysers. This false hypochloremia can be distinguished from true hypochloremia because it is not associated with hypernatremia or metabolic acidosis. Because bromide is not subject to metabolism and is not protein bound, this compound is not involved in a lot of drug interactions. However dietary chloride loads, intravenous or subcutaneous administration of chloride-containing fluids, or drug formulations containing chloride would be expected to lower serum bromide concentrations. In addition, loop diuretics such as ethacrynic acid may also enhance bromide elimination by blocking reabsorption of bromide through renal tubular chloride channels.
Bromide is released during halothane metabolism, leading to small but detectable increases in serum bromide concentrations in dogs and also humans after halothane anesthesia. This release of bromide from halothane is mediated by hepatic P 450 enzymes and is enhanced by Phenobarbital administration. - Conclusions:
- Because of the ease of once-a-day administration, low cost, apparent lack of hepatotoxicity and demonstrated efficacy in dogs, bromide offers an important therapeutic alternative in the management of epilepsy in dogs. Therapeutic monitoring should be performed and serum bromide concentrations checked regularly to avoid toxicosis. Dogs overdosed with sodium bromide at 100-600 mg/kg bw/day for up to 25 weeks had no clinical evidence of either renal or hepatic damage, although histologic results were not reported.
LOAEL: Not applicable
NOAEL: Oral doses of 50-80 mg/kg bw/day - Executive summary:
Materials and Methods
Several studies are summarised to describe mode of action, metabolism, pharmacokinetics, dosage and adverse effects of bromide treatment mainly in epileptic dogs. Clinicals adverse effects of bromide treatment, blood bromide concentrations and treatment of toxicosis are reported.
Results and Discussion
Mechanism of action:
Bromide is actively transported out of the central nervous system by the choroid plexus, but with pharmacologic doses, this active transport system is overwhelmed, and bromide accumulates in the cerebrospinal fluid and interstitial tissues of the brain. Like chloride, bromide distributes mostly to the extracellular space, but bromide does accumulate intracellularly in neurons and appears to cross neuronal chloride channels more readily than chloride. It appears that bromide´s major mechanism of anticonvulsant action is to hyperpolarize neuronal membranes and thereby stabilize them against excitatory input from epileptic foci.
Metabolism and pharmacokinetics:
Bromide is distributed like chloride throughout the body and as such, its volume of distribution approximates the extracellular fluid space. Bromide is concentrated in fluids such as sweat, saliva and gastric juice (as hydrobromic acid). Bromide is not subject to hepatic metabolism and is not bound to plasma proteins. Unchanged bromide is eliminated predominantly by glomerular filtration, whereas other routes of excretion, such as sweat, saliva and faeces are quite minor. Bromide is extensively reabsorbed by the renal tubules in competition with chloride. Thus extensive reabsorption leads to an unusually long elimination half-life of about 9-12 days in humans and 25-46 days in dogs. Rate of bromide elimination varies directly with chloride intake, because increased chloride loads will increase urinary loss of bromide. In dogs, a 6-fold increase in chloride intake markedly shortens the elimination half-life of bromide and leads to a pronounced decrease in predicted steady state serum bromide concentrations.
Formulation and dosage:
Potassium bromide can be formulated into sterile solutions or capsules. For dogs treated with bromide alone doses of 50-80 mg/kg bw/day of potassium bromide may be necessary for seizure control (in combination with phenobarbitone, doses of 22-40 mg/kg bw/day are recommended). When therapeutic bromide doses must be reached rapidly an oral loading dose of 400-600 mg of potassium bromide/kg bw can be given in several divided doses over 24-48 hours. Bromide can also be administered as sodium bromide. This substance appears to be less irritating to gastric mucosa but is a poor choice in dogs with congestive heart failure, hepatic diseases or hypertension. If sodium bromide is used the dosage should be reduced by 15 % to account for the higher bromide content of the sodium salt.
Therapeutic monitoring:
In several studies epileptic dogs refractory to Phenobarbital alone had improvement in seizure control when serum bromide concentrations were targeted in the range of 1000-2000 mg/L. Higher concentrations might be necessary for treatment with bromide alone. It is recommended that serum bromide concentrations be determined 6-8 weeks after initiation of treatment with bromide. If concentrations are in therapeutic range, monitoring can be performed every 6-12 months unless problems arise.
Effect of chloride intake:
Bromide and chloride compete for renal elimination, therefore high chloride intake has been shown to increase bromide elimination in humans, rats and dogs. One study showed that beagles receiving an experimental diet with high chloride content led to predicted steady state bromide concentrations below the therapeutic range. In a different study several dogs showed decreases in serum bromide concentrations and increases in seizure activity resulting from a change to a higher chloride diet.
Treatment of toxicosis:
Serum bromide concentrations greater than 2000-2500 mg/L are often associated with clinically important adverse effects like stupor, inability to walk or even coma. In these dogs bromide should be discontinued temporarily and if sedation is profound, chloride loading in the form of short-term intravenous administration of 0.9% NaCl solution will lower serum bromide concentrations rapidly because of the competition of the ions for renal elimination.
- Endpoint:
- specific investigations: other studies
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1997
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: no guideline available for this special investigation
- Qualifier:
- no guideline available
- Deviations:
- not applicable
- Remarks:
- no guideline available for this special investigation
- Principles of method if other than guideline:
- study was performed according to good experimental practice
- GLP compliance:
- no
- Type of method:
- in vivo
- Endpoint addressed:
- repeated dose toxicity: oral
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Top Velaz Ltd, Prague
- Age at study initiation: 41 days
- Weight at study initiation: 100-120 g. - Route of administration:
- oral: drinking water
- Vehicle:
- water
- Details on exposure:
- DIET
The mean iodine content of the diet was determined by kinetic photometry ans amounted to 0.52 mg/kg diet. The mean bromide content of the diet was assayed by determining the radionuclide 82Br by instrumental neutron activation analysis (INAA) in a long-term activation regime and amounted to 10.04 mg/kg diet.
VEHICLE
- Concentration in vehicle:
0, 10, 50 and 100 mg Br-L for 16 and 66 days;
0, 100, 200 or 400 mg Br-/L for 133 days - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 16, 66 or 133 days
- Frequency of treatment:
- daily
- Post exposure period:
- No postexposure period. The animals were sacrificed after the treatment period (16, 66 or 133 days) and thyroid lobes were removed for examination.
- Remarks:
- Doses / Concentrations:
0, 0.15, 0.75 and 1.5 mg Br-/day
Basis:
actual ingested
for 16 and 66 days; based on a measured water intake of 15 mL/animal/day - Remarks:
- Doses / Concentrations:
0, 1.5, 3 or 6 mg Br-/day
Basis:
actual ingested
for 133 days; based on a measured water intake of 15 mL/animal/day - No. of animals per sex per dose:
- 10/group
- Control animals:
- yes, concurrent vehicle
- Examinations:
- Food- and Water-Consumption
Gross and Histopathology of the thyroid
Mitotic activity of follicular cells: evaluated by the immunohistochemical assay of PCNA on sections from the paraffin-embedded thyroid. Sections were transferred to microscopic slides coated with 0.1 % gelatine in water containing 0.01 % chromium potassium sulphate and dried in a thermostat at 37°C for 20 hours. Paraffin was removed by xylene (2-15 min) and the slides were transferred through an alcohol series into water. The endogenous peroxidase activity was blocked with 0.3 % H2O2 for 30 minutes. Slides were then washed in distilled water and incubated in 0.05 M Tris-HCl buffer, pH 7.6 containing 1 % bovine albumin, monoclonal antibody mouse anti-PCNA diluted 1:50 and sodium azide (50 mg/50 ml) in a moist chamber at 4°C for 16-24 hours. After washing with Tris-buffered saline (TBS, pH 7.6; 3x5 min) the sections were overlaid with swine-anti-mouse-gamma-globulin labelled with peroxidase (diluted 1:500) and incubated in a moist chamber at room temperature for 60 minutes. After washing in TBS (3x5 min) the reaction was visualized by a solution 3,3´-diaminobenzidinetetrahydrochloride. Thereafter 20 µl of 30% H2O2 were added. After 10-minutes incubation at room temperature the specimens were washed in distilled water, counter-stained with hematoxylin, dehydrated and mounted in Entellan.
Statistics: Statistical evaluation for proliferation activity test results was done by the Kruskal-Wallis test. - Details on results:
- Food consumption and compound intake:
The average amount of consumed diet was 20 g/animal/day. The amount of consumed water was measured and corresponded approximately to 15 ml/animal/day. Bromide intake was calculated to be 0.15, 0.75, 1.5, 3 and 6 mg bromine/animal/day or 0.5, 2.5, 5, 10 and 20 mg Br-/kg bodyweight based on an assumed average bodyweight of 300 g/rat over the study period.
Gross and Histopathology of the thyroid:
The thyroid of rats exposed to bromide exhibited microfollicular rearrangement of the follicular epithelium and a reduction of the amount of colloid. Control animals showed follicles of varying size the lumina of which were filled with PAS (periodic acid Schiff stain) -positive colloid. PCNA positive nuclei were stained diffusely brown and were larger in size. PCNA-L1 is higher in experimental animals. The values of mitotic index increased with increasing bromide concentrations. The square area in the thyroid sections from control animals contained maximally one PCNA-positive nucleus only. The thyroid of young rats after 16 days of bromide application contained more positive nuclei in the square. - Conclusions:
- Based on the findings made in this investigations it was concluded that morphological and functional changes in the thyroid correlated with concentration and length of period of bromide treatment when administered via the drinking water. Thyroids from rats treated with bromide showed increased mitotic activity of follicular cells. In addition, an increase in bromine content and a concomitant decrease of the I/Br molar ratio in the thyroid tissue after administration of Br- in drinking water was demonstrated.
LOAEL: 10 mg bromide/L (corresponding to 0.5 mg/kg bw)
NOAEL: Not applicable - Executive summary:
Materials and Methods
Preceding experiments had shown that microfollicular rearrangement of the rat thyroid, accompanied by enlargement of follicular cells, can be induced by low bromine concentrations (10-100 mg Br-/L drinking water).The aim of the study was to find out to which extent the hyperplasia resulting from increased mitotic activity of follicular cells participates in the changes observed in these previous experiments. The experiments were carried out in three series: (1) Four groups of ten animals each received 0, 10, 50 and 100 mg Br-/L drinking water (corresponding to 0, 0.5, 2.5, 5, 10 and 20 mg Br-/kg bodyweight based on an assumed average bodyweight of 300 g/rat over the study period and taking into account a water consumption of 15 mL/animal/day). Exposure time 16 days. (2) Four groups of ten animals each received 0, 10, 50 and 100 mg Br-/L drinking water. Exposure time 66 days. (3) Four groups of ten animals each received 0, 100, 200 and 400 mg Br-/L drinking water. Exposure time 133 days. After termination of potassium bromide administration, the animals were sacrificed and thyroid lobes were excised, weighed and fixed in Bouin´s fluid for 24 hours. Mitotic activity of follicular cells was evaluated by the immunohistochemical assay of PCNA on sections from the paraffin-embedded thyroid. Sections incubated without anti-PCNA were used as negative controls. Proliferation activity was evaluated by counting diffusely brown-coloured nuclei and PCNA-L1 index calculated according to the following formula: number of PCNA-positive nuclei x 100/total number of nuclei in the square. The number of nuclei evaluated in each specimen was 4025-6407.
Results and Discussion
The thyroid tissue of control animals contained follicles of varying size, the lumina of which were filled with a PAS (periodic acid Schiff stain)-positive colloid. The follicles were lined with cuboid or low-cylindrical epithelium. By contrast, the thyroid of animals exposed to bromide exhibited microfollicular rearrangement of the follicular epithelium and a reduction of the amount of colloid. PCNA positive nuclei were stained diffusely brown and were larger in size. PCNA-L1 is higher in experimental animals. The values of mitotic index increased with increasing bromide concentrations. The Kruskal-Wallis test showed that the PCNA-L1 differences were significant. The square area in the thyroid sections from control animals contained maximally one PCNA-positive nucleus only. The thyroid of young rats after 16 days of bromide application contained more positive nuclei in the square. In the synthesis of thyroid hormones a conversion of inorganic iodine into an organic form occurs. The transport of iodine and its organification can be blocked by Br- ions due to a mutual competition between bromide and iodide anions. This results in a thyroxin and triiodothyronine deficiency which in turn increases secretion of TSH (thyrotropic hormone). Previous as well as present experiments also demonstrated an increase in bromine content and a concomitant decrease of the I/Br molar ratio in the thyroid tissue after administration of Br- in drinking water. This finding is important in connection with the data showing an increasing exposure of living organisms to environmental bromine which represents an important environmental factor contributing to the development of endemic goitre; this is caused by a decreased utilization of the consumed iodine which produces a relative iodine deficiency even when the iodine intake is sufficient. The experiments of this study demonstrated that the goitrogenic action is exerted even by low concentrations of bromine in drinking water. Changes of the thyroid had the character of a parenchymal goitre and were thus similar to the findings described previously by other groups. The extent and degree of morphological and functional changes found in this investigation correlates with the bromide concentration and the length of period of its administration. The immunohistochemical demonstration of PCNA also correlated with the changes in the thyroid tissue and showed an increased mitotic activity of follicular cells. The values of the PCNA-L1 index in rats exposed to bromide for 16 days were higher than those in animals exposed to bromide for 66 and 133 days. A decrease of mitoses with increasing age was found also in control groups of animals.
- Endpoint:
- specific investigations: other studies
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1997
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- other: no guideline available for the particular investigations performed
- Qualifier:
- no guideline available
- Deviations:
- not applicable
- Remarks:
- there is no guideline available for this special investigation
- Principles of method if other than guideline:
- study was performed according to good experimental practice
- GLP compliance:
- no
- Type of method:
- in vivo
- Endpoint addressed:
- repeated dose toxicity: oral
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Top Velaz Ltd., Prague
- Age at study initiation: 41 days
- Weight at study initiation: 100-120 g - Route of administration:
- oral: drinking water
- Vehicle:
- water
- Details on exposure:
- DIET
The mean content of bromine and iodine in the diet was determined to amount to 10.04 mg bromide/kg and 0.52 mg iodine/kg
VEHICLE
- Concentration in vehicle: 10, 50 or 100 mg/L - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 16 and 66 days
- Frequency of treatment:
- daily
- Post exposure period:
- No postexposure period; animals were sacrificed at the end of the treatment period.
- Remarks:
- Doses / Concentrations:
10, 50 or 100 mg/L
Basis:
nominal in water
corresponding to 1.25, 6.25 and 12.5 mg/kg bw/day (calculation based on the following assumptions: drinking water consumption: 15 mL/animal; average animal weight: 120 g/animal) - No. of animals per sex per dose:
- 6/group
- Control animals:
- yes, concurrent vehicle
- Examinations:
- Food consumption and compound intake
Clinical Chemistry:
Free T4, total T4, total T3 and plasma TSH levels were determined at termination of the investigation
Organ weights:
Weights of the thyroid gland were taken only
Gross and Histopathology:
Thyroid gland was examined only
Immunohistochemistry:
The assay of thyroglobulin was carried out by an indirect immunoperoxidase technique with 3,3´-diaminobenzidine tetrahydrochloride visualisation. Rabbit-anti-human-thyroglobulin was used as primary, and swine-anti-rabbit as secondary antibody.
Morphometric analysis:
The morphometric and immunohistochemical evaluation was performed with a Nikon Microphot FXA microscope and the Lucia M image analysis program Lucia 3.5. The proportion of the colloid area relative to the remaining parenchyma, as well as the size distribution of colloid deposits in the follicular lumen and the circularity of the colloid deposits, were measured and evaluated.
Radioimmunoassay (RIA):
For the plasma concentration determination of thyroid hormones and TSH in all experimental animals, 2-3 mL of blood were collected at the end of the experiment by cardiac puncture under ether anaesthesia. Thyroid hormones in nonextracted plasma were determined using the RIA T3 and RIA T4 kits. The RIA of TSH in the rats was assayed with the following reagents: rat TSH-PR-3, AFP-5512B, TSH-antibodies anti-rat TSH-RIA-6, AFP-329 661 Rb.
Tissue and diet bromine and iodine levels:
Bromine and iodine levels in the thyroid gland dry weight of experimental animals were determined by instrumental neutron activation analysis (INAA) during a short-term irradiation regime in a nuclear reactor. The bromine level in the diet was determined by INAA during long-term irradiation regime using the 82Br radionuclide, and the iodine level by means of kinetic photometry.
Statistics:
The differences between the mean values were tested by variance analysis, followed by the F-test, the ANOVA test and the Kruskal-Wallis test. - Details on results:
- Food consumption and compound intake:
Approximately 20 g of diet were consumed per animal per day. The amount of drinking water containing potassium bromide was approximately 15mL/animal/day.
Clinical Chemistry:
The following results were obtained by radioimmunoassay. The plasma thyroxin concentration was significantly decreased in animals exposed to all bromide concentrations as compared with the T4 level in the controls (P<0.001). The plasma T3 concentration in animals exposed to 10, 50 and 100 mg bromide/L for 66 days was significantly lowered. Treatment for 16 days did not bring about significant changes in the T3 plasma concentration. The administration of increased bromide concentrations brought about only a slight and statistically insignificant increase in the TSH level after 66 days at 10-100 mg bromide/L. Furthermore, no significant change in the TSH level was observed after a 16-day administration of bromide.
Organ weights:
Thyroid dry weights were taken for determination of I/BR concentration ratio. No changes in thyroid weights is reported in the investigation
Gross and Histopathology:
The administration of bromide resulted in definite morphological changes in the thyroid gland, in contrast to the control animals. The extent of the changes rose with the concentration of bromine. The extent of changes after 16- and 66-days treatment did not differ conspicuously for any given concentration of bromide.
The tissue of the thyroid gland of animals exposed to bromide displayed a marked growth activation of the follicular epithelial component, and mitoses in the follicular cells were more frequent. Microfollicular reorganisation, increased height of the follicular cells and increased vascularisation of the parenchyma were observed.
Image analysis demonstrated a significant lowering in the proportion of colloid in the thyroid tissue of rats treated with potassium bromide.
Immunohistochemistry:
The assay of thyroglobulin (Tg) in the colloid of most of the follicles in the control animals over the 16- and 66-day periods was strongly or moderately positive, whereas the colloid in the smaller follicles in the central parts of the tissue exhibited moderate but definitely perceptible positivity.
Tissue concentration of bromine and iodine:
The increasing bromide intake resulted in a concentration and/or duration of treatment-dependent rise in the bromine level in the thyroid tissue and a concomitant decrease in the I/Br molar concentration ratio. - Conclusions:
- The results of this investigation indicate that even the lowest amount of bromide administered (10 mg/L corresponding to 150 µg/animal/day or 1.25 mg/kg bw/day) can induce changes comparable with parenchymatous goitre in humans. The decrease in thyroid hormone level (T3, T4) detected after 16 and 66 days of treatment was accompanied by definite morphological changes. The plasma TSH level of bromide-exposed animals did not significantly differ from that in the controls after administration for a period of 16 days while after 66 days TSH was statistically significantly increased. The results of this investigation demonstrate that rats appear to react in a sensitive manner to the administration of even low amounts of bromide with the drinking water as demonstrated by a modulation of thyroid hormones and thyroid morphology.
LOAEL: 10 mg/L corresponding to 1.25 mg/kg bw based on morphological changes in the thyroid gland and decreased serum thyroxine and triiodithyronine levels.
NOAEL: < 10 mg/L - Executive summary:
Materials and Methods
In order to establish the functional and morphological effects of bromine on the thyroid, experiments were performed on male rats, which in addition to a standard diet with an estimated iodine/bromine content, were fed for periods of 16 and 66 days with the small quantities of bromide expected to be encountered in the environment (10, 50 and 100 mg/L drinking water).
Animals were divided into 6 experimental and two control groups, each containing 6 animals. Rats were fed 0, 10, 50 or 100 mg of bromide/L in drinking water for 16 or 66 days. Important to note is the fact, that each animal consumed approximately 15 mL water per day, corresponding to bromide amounts of 0, 150, 750 and 1500 µg bromide/animal/day or 1.25, 6.25 and 12.5 mg bromide/kg bw/day. The diet fed during the investigation contained 10.04 mg bromine/kg and 0.52 mg iodine/kg; since the animals consumed 20 g food/day, this corresponds to 200 µg bromine/animal/day (about 1.7 mg/kg bw/day) and 10.4 µg iodine/animal/day (about 0.087 mg/kg bw/day).
After the experiments were completed, the animals were killed and both thyroid lobes were excised. The tissue of one part of the lobe was fixed and embedded in paraffin, and used for preparation of serial sections which were stained with haematoxylin-eosin and processed by the PAS method for morphometric assessment. The tissue of the other part of the lobe was fixed and further processed for electron microscopy.
Immunohistochemistry was carried out with an anti-human-thyroglobulin primary antibody. For morphometric analysis the proportion of the colloid area relative to the remaining parenchyma, as well as the size distribution of colloid deposits in the follicular lumen and the circularity of the colloid deposits, were measured and evaluated.
Radioimmunassay for determination of thyroid hormone concentrations (T3, T4, TSH) in plasma were carried out in all animals.
Instrumental neutron activation analysis (INAA) was performed to determine the bromine and iodine content of the thyroid glands. For determination of the bromine content in the diet, also INAA was performed; and the iodine levels were determined by means of kinetic photometry.
Results and Discussion
Morphology:
The administration of bromide resulted in definite morphological changes in the thyroid gland, in contrast to the control animals. The extent of the changes rose with the concentration of bromide. It was largest at a concentration of 100 mg/L, but changes induced by 50 and 10 mg/L were also well pronounced. The extent of changes after 16 and 66 days treatment did not differ conspicuously for any given concentration of bromide. The tissue of the thyroid gland of animals exposed to bromide displayed a marked growth activation of the follicular epithelial component, and mitoses in the follicular cells were more frequent. Microfollicular reorganisation occurred in up to more than two thirds of the parenchyma, and was accompanied by the formation of minute follicles with diameters ranging from 6 to 20 µm. The strongly reduced slit-like constricted or round lumina exhibited a substantially reduced amount of PAS positive colloid, frequently non-homogeneous in nature. The walls of medium-sized follicles sometimes exhibited very small daughter follicles, which retained PAS positive colloid in their lumina. In most animals, the microfollicles were also found in the peripheral parts of the lobe tissue. The height of the follicular cells was increased relative to the diminished lumina. In a number of medium sized and larger follicles, groups of follicular cells with increased height had formed protuberances within the lumen. The cytoplasm of follicular cells featured multiple PAS positive spherical vacuoles. Increased vascularisation of the parenchyma was observed.
The electron microscopic findings in the thyroid after 16 and 66 days exposure revealed bromide concentration-dependent changes in the configuration of the follicles. Within the follicular cells, changes of the rough endoplasmic reticulum and lysosomes were particularly apparent.
Image analysis:
Image analysis demonstrated a significant lowering in the proportion of colloid in the thyroid tissue of rats treated with potassium bromide. During the 16-day experiment, the proportion of the colloid dropped from 15 ± 3% to 11 ± 3%, 10 ± 4% and 9 ± 3% for bromide concentrations of 10, 50 and 100 mg/L respectively. The corresponding values found in the 66-day experiment were 23 ± 6% in the controls and 12 ± 4%, 7 ± 2% and 8 ± 3% in experimental animals. The lowest concentration of administered bromide during the 16-day experiment evoked a response comparable to that found with higher concentrations. During the 66-day experiment this concentration induced a response similar to that found in the 116-day experiment but substantially different from the response elicited by higher concentrations. Statistical analysis showed the differences in the proportions of the colloid-occupied area to be significant in the 16-day experiment (P<0.05) and highly significant in the 66-day experiment (P<0.001). The lowering of the amount of colloid in the tissue was accompanied by microfollicular reorganisation of the tissue. The size distribution of the colloid deposits in the follicular lumen exhibited a conspicuous rise in the number of the smallest-lumen follicles with colloid in all experimental groups of animals exposed to bromide for 16 and 66 days. The colloid deposits with the largest area were fewer. The variations in the effect, depending on the bromide concentration and length of administration in these groups were obviously due to the generally small number of large-area colloid deposits. An evaluation of the circularity of the colloid deposits based on image analysis did not show any significant difference between bromide-exposed rats and control animals. The occurrence of slit-like follicular lumina was accompanied, in terms of the microfollicular reorganisation of the tissue, by an increase in the number of almost completely circular follicular lumina.
Immunohistochemistry:
The assay of thyroglobulin (Tg) in the colloid of most of the follicles in the control animals over the 16- and 66-day periods was strongly or moderately positive, whereas the colloid in the smaller follicles in the central parts of the tissue exhibited moderate but definitely perceptible positivity. Animals exposed to 10 mg bromide/L for 16 or 66 days exhibited mildly positive evidence of Tg in the colloid of microfollicles, and in medium-sized and large follicles. After 16 days of 50 mg/L of bromide administration, the Tg in the colloid of the microfollicles was mildly positive, whereas the colloid of the medium-sized and large follicles was moderately positive. After 66 days the Tg assay in the colloid of microfollicles was moderately or strongly positive, but only in a narrow peripheral rim of the colloid. The colloid of most larger follicles showed no substantial reduction in positivity. Animals exposed to 100 mg bromide/L for 16 days exhibited a weakly positive or even negative reaction to Tg in the colloid of the microfollicles, whereas in the colloid of the larger follicles it was weakly or moderately positive. Animals exposed for 66 days exhibited moderately or strongly positive evidence of Tg in the colloid of some follicles, but predominantly in the narrow peripheral rim adjacent to the apical surface of follicular cells only. The demonstration of Tg in the larger follicles was mainly moderately positive.
Radioimmunoassay:
The plasma thyroxin concentration was significantly decreased in animals exposed to all bromide concentrations as compared with the T4 level in the controls (P<0.001). The plasma T3 concentration in animals exposed to 10, 50 and 100 mg bromide/L for 66 days was significantly lowered. Treatment for 16 days did not bring about significant changes in the T3 plasma concentration. The administration of increased bromide concentrations brought about only a slight and statistically insignificant increase in the TSH level after 66 days at 10-100 mg bromide/L. Furthermore, no significant change in the TSH level was observed after a 16-day administration of bromide.
Tissue concentration of bromine and iodine:
The increasing bromide intake resulted in a concentration and/or duration of treatment-dependent rise in the bromine level in the thyroid tissue and a concomitant decrease in the I/Br molar concentration ratio.
- Endpoint:
- endocrine system modulation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1998
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: no guideline available for this special mechanistic investigation
- Qualifier:
- no guideline available
- Deviations:
- not applicable
- Remarks:
- no guideline available for this special mechanistic investigation
- Principles of method if other than guideline:
- Study was performed according to good experimental practice.
- GLP compliance:
- no
- Type of method:
- in vivo
- Endpoint addressed:
- repeated dose toxicity: oral
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Top Velaz, Prague
- Age at study initiation: 42 days
- Weight at study initiation: 100-130 g - Route of administration:
- oral: drinking water
- Vehicle:
- water
- Details on exposure:
- DIET
- The bromine level in the diet was assessed by INAA in a long-term irradiation regime using the 82Br radionuclide and the iodine level by kinetic photometry.
VEHICLE
- Concentration in vehicle: 100, 200 and 400 mg/L - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 133 days
- Frequency of treatment:
- daily
- Post exposure period:
- No postexposure period; animals were sacrificed at the end of the treatment period.
- Remarks:
- Doses / Concentrations:
100, 200 or 400 mg/L
Basis:
nominal in water
corresponding to 11.6, 23.1 and 46.2mg/kg bw/day (calculation based on the following assumptions: drinking water consumption:15 mL/animal, average animal weight:130 g/animal ) - No. of animals per sex per dose:
- 10/group
- Control animals:
- yes, concurrent vehicle
- Examinations:
- Bodyweight: at termination of the study
Water consumption: measured in a random manner by weighing the drinking vessel after filling and 24 hours later.
Clinical chemistry: The concentration of thyroid hormones and TSH in plasma was determined by radioimmunoassay (RIA). Blood was collected from animals of all experimental groups at the end of the treatment period by cardiac puncture.
No further clinical chemical parameters were determined in this investigation.
Organ weights
Gross and Histopathology: thyroid lobes were examined only. Sections were stained with haematoxylin-eosin and by the PAS method. Small samples from the other part of the lobe were fixed in 2,5% glutardialdehyde/0.1 M phosphate buffer for 2 hours. After washing with 5% glucose/0.1 M phosphate buffer and post-fixation in 2% osmium tetroxide for 1 hour, the material was dehydrated and embedded in Durcupan and further processed for electron microscopy.
Immunohistochemistry: The assay of thyroglobulin (Tg) was carried out in paraffin sections by an indirect immunoperoxidase technique followed by 3,3´-diaminobenzidine tetrahydrochloride visualisation. Rabbit anti-human thyroglobulin was used as primary, and swine-anti-rabbit as secondary antibody.
Morphometry: The morphometric and immunohistochemical evaluation was performed by using the Nikon microphot FXA microscope and the Lucia M image analysisd programme Lucia 3.5.
Determination of tissue and diet bromine and iodine concentrations: Bromine and iodine levels in the thyroid gland dry weight were determined by instrumental neutron activation analysis (INAA) in a short-term irradiation regime applied to dried thyroid glands in a nuclear reactor. The bromine level in the diet was assessed by INAA in a long-term irradiation regime using the 82Br radionuclide and the iodine level by kinetic photometry.
Statistics: Differences between mean values were tested by variance analysis followed by F-test and Kruskal-Wallis test. - Details on results:
- Bodyweight:
The bodyweight taken at termination of the study is not further specified in the investigation.
Food consumption and compound intake:
Food consumption was approximately 20 g/animal/day. Consumption of drinking water, and with that intake of the test substance, was approximately 10-15 mL/animal/day.
Clinical chemistry:
Exposure of the rats to 100 mg bromide/L evoked a slight increase in TSH level after 133 days. Treatment with 200 and 400 mg bromide/L did not increase TSH concentration but led rather to a slight decrease. The thyroxin (T4) concentration in the plasma of animals exposed to different bromine concentrations decreased significantly as compared to the T4 level in controls. The plasma concentration of 3,5,3´-triiodothyronine (T3) in bromine-exposed animals did not differ from control values.
Organ weights:
Dry weights from thyroid lobes were taken only and used for calculation of bromine and iodine content.
Gross and Histopathology:
In comparison to control rats the thyroid glands of animals exposed to a concentration of 100 mg bomide/L dinking water for 133 days exhibited a striking follicular rearrangement characterized by the presence of a large number of very small follicles (6-20 µm) with round lumina of very small diameter (1-10 µm) or with slit-like constricted lumina.
The height of the thyrocytes was increased in medium-sized follicles at the thyroid periphery. Mitoses of thyrocytes were more frequent than in the controls. Animals exposed to 200 and 400 mg/L showed the same changes. In addition, the microfollicular tissue of the 200 mg-group occupied a smaller part of the lobe and the proportion of medium-sized follicles appeared larger.
Electron microscopy revealed changes in the localisation and extent of the golgi apparatus, rough endoplasmic reticulum, lysosomes and microvilli in all treatment groups.
Immunohistochemistry of thyroglobulin (Tg):
Animals exposed to 100 mg bromide/L drinking water for 133 days exhibited an overall decrease in Tg immunoreactivity.
A complete loss of Tg immunoreactivity occurred in animals exposed to a bromide concentration of 400 mg/L.
Morphometry:
The amount of intrafollicular colloid was significantly decreased in thyroids of rats treated with bromide in comparison with the control animals. Surprisingly, the lowest concentration of the bromide ion administered in this experiment (100 mg/L) lowered the colloid volume more than the next higher dose (200 mg/L). The changes obtained with 100 and 400 mg/L were similar.
Bromine and iodine tissue concentrations:
Animals exposed to bromine for 133 days exhibited changes in bromine and iodine concentration in the thyroid tissue. With an increasing concentration of bromide, the amount of bromine in the thyroid dry weight showed a concomitant decrease in the molar I/Br ratio from 17.9 to 0.39, and the extent of the decrease were again dependent on bromide concentration. - Conclusions:
- Bromide administration for 133 days caused an increased growth activity of the thyrocytes accompanied by symptoms of hypothyroidism, decreased T4 plasma concentration, lowered Tg immunoreactivity and a decrease in the I/Br molar ratio in the thyroid. The results of this investigation demonstrate that rats appear to react in a sensitive manner to the administration of even low amounts of bromide with the drinking water as demonstrated by a modulation of thyroid hormones in the plasma and changes in thyroid morphology.
LOAEL: was estimated to be 100 mg/L (about 12.5 mg/kg bw/day) based on follicular rearrangements in the thyroid and decrease of TSH, T4 and Tg immunoreactivity.
NOAEL: was estimated to be < 100 mg/L. - Executive summary:
Materials and Methods
This investigation was a continuation of previous experiments which revealed a positive effect of 16- and 66-day administration of low bromide concentrations on the rat thyroid which displayed a marked growth activation of the follicular epithelial component, and in addition, mitoses in the follicular cells were more frequent. Also, microfollicular reorganisation occurred. The aim of this investigation was to show if the most effective concentration used in the previous study (100 mg bromide/L) and further multiples of this concentration would evoke morphological and functional changes in the rat thyroid after a long-term administration. Male rats were divided into four groups, each consisting of 10 animals, and treated for 133 days with 0, 100, 200 or 400 mg bromide/L drinking water corresponding to 0, 1-1.5, 2-3 and 4-6 mg/animal/day (consumption of drinking water: 10-15 mL/animal/day) or 0, 7.7-11.5, 15.4-23.1 and 30.8-46.2 mg/kg bw/day (based on a weight of 130 g/rat). After the termination of the experiments the animals were killed, weighed, and the thyroid lobes excised. A portion of one lobe (fixed in Bouin´s fluid and embedded in paraffin) was used for preparation of serial sections (200 sections/animal). These sections were stained, fixed and further processed for electron microscopy. The assay of thyroglobulin (Tg) was carried out in paraffin sections by an indirect immunoperoxidase technique followed by 3,3´-diaminobenzidine tetrahydrochloride visualisation. Rabbit anti-human thyroglobulin was used as primary antibody. The concentration of thyroid hormones and TSH in plasma was determined by radioimmunoassay (RIA). Bromine and iodine levels in the thyroid gland dry weight were determined by instrumental neutron activation analysis (INAA) in a short-term irradiation regime. The bromine level in the diet was assessed by INAA in a long-term irradiation regime using the 82Br radionuclide and the iodine level by kinetic photometry.
Results and Discussion
Light microscopy:
In comparison to control rats the thyroid glands of animals exposed to a concentration of 100 mg bomide/L dinking water for 133 days exhibited a striking follicular rearrangement characterized by the presence of a large number of very small follicles (6-20 µm) with round lumina of very small diameter (1-10 µm) or with slit-like constricted lumina. The lumina contained a small amount of PAS-positive colloid. Some follicles had no lumina. In other follicles, the thyrocytes appeared higher. These changes were usually diffuse and occurred less frequently in foci, usually in the central part o the lobe. Their presence in the periphery of the lobe, however, was also found. The height of the thyrocytes was increased in medium-sized follicles at the thyroid periphery. Groups of such cells protruded into the lumina of the follicles. The apical cytoplasm of thyrocytes showed multipled PAS-positive spherical vacuoles. Mitoses of thyrocytes were more frequent than in the controls. The capillaries were numerous and dilated. The thyroid of animals exposed to 200 mg/L for 133 days exhibited a similar histological picture but the microfollicular tissue occupied a smaller part of the lobe, and the proportion of medium-sized follicles appeared larger. In animals exposed to 400 mg bromide/L drinking water for 133 days the pattern was essentially identical with that in animals receiving 100 mg/L.
Electron microscopy:
Electron microscopy revealed changes in the localisation and extent of the golgi apparatus, rough endoplasmic reticulum, lysosomes and microvilli in thyrocytes of all treatment groups.
Immunohistochemistry of thyroglobulin:
In control animals thyroglobulin (Tg) immunoreactivity in the colloid of most follicles was medium to strongly positive particularly in the peripheral rim adjacent to the apical pole of the thyrocytes. By contrast, animals exposed to 100 mg bromide/L drinking water for 133 days exhibited an overall decrease in Tg immunoreactivity. In some follicles a medium to strong immunoreaction was concentrated partly at the border between the thyrocyte and peripheral rim of colloid of both microfollicles and larger follicles. In some follicles immunoreactivity was absent. A complete loss of Tg immunoreactivity occurred in animals exposed to a bromide concentration of 400 mg/L. The central part of the thyroid tissue of these animals had medium Tg reactivity concentrated mostly in the narrow peripheral colloid rim in the lumina of part of the follicles, while other parts were negative. The periphery of the thyroid reacted less intensively.
Morphometry:
The amount of intrafollicular colloid was significantly decreased in thyroids of rats treated with bromide in comparison with the control animals. Surprisingly, the lowest concentration of the bromide ion administered in this experiment (100 mg/L) lowered the colloid volume more than the next higher dose (200 mg/L). The changes obtained with 100 and 400 mg/L were similar.
Statistical evaluation showed differences in the volume proportion of colloid in the thyroid tissue of rats exposed to 100, 200 and 400 mg bromide/L for 133 days relative to controls that were highly significant (P < 0.001). The distribution of colloid deposits in the follicular lumina in sections showed a marked increase in the number of the smallest follicles (colloid area in the section up to 100 and 100-300 µm2) in all groups of animals exposed to bromine. By contrast, the proportion of larger-area colloid deposits (500-1000 µm2) was lower. The form of follicular lumina of bromine-exposed rats did not differ from that in controls.
RIA determination of T4, T3 and TSH:
Exposure of the rats to 100 mg bromide/L evoked a slight increase in TSH level after 133 days. Treatment with 200 and 400 mg bromide/L did not increase TSH concentration but led rather to a slight decrease. The thyroxin (T4) concentration in the plasma of animals exposed to different bromine concentrations decreased significantly as compared to the T4 level in controls. The plasma concentration of 3,5,3´-triiodothyronine (T3) in bromine-exposed animals did not differ from control values. The T4 decrease in the plasma of bromine-exposed animals correlated well with the persisting morphological changes and with changes in the thyroglobulin content of follicles.
Bromine and iodine tissue concentrations:
Animals exposed to bromine for 133 days exhibited changes in bromine and iodine concentration in the thyroid tissue. With an increasing concentration of bromide, the amount of bromine in the thyroid dry weight showed a concomitant decrease in the molar I/Br ratio from 17.9 to 0.39, and the extent of the decrease were again dependent on bromide concentration.
- Endpoint:
- specific investigations: other studies
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 2004
- Reliability:
- 2 (reliable with restrictions)
- Qualifier:
- no guideline available
- Deviations:
- not applicable
- Remarks:
- no guideline available for this special investigation
- Principles of method if other than guideline:
- study was performed according to good experimental practice
- GLP compliance:
- no
- Type of method:
- in vivo
- Endpoint addressed:
- repeated dose toxicity: oral
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Top Velaz Ltd, Prague
- Age at study initiation: 41 days
- Weight at study initiation: 100-120 g - Route of administration:
- oral: drinking water
- Vehicle:
- water
- Details on exposure:
- DIET PREPARATION
-The mean content of bromide and iodide in the diet was 10.04 mg bromide/kg and 0.52 mg iodide/kg respectively.
VEHICLE
- Concentration in vehicle:
0, 10, 50 and 100 mg Br-L for 16 and 66 days
0, 100, 200 or 400 mg Br-/L for 133 days - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 16, 66 or 133 days
- Frequency of treatment:
- daily
- Post exposure period:
- No postexposure period. The animals were sacrificed after the treatment period (16, 66 or 133 days) and thyroid lobes were removed for electron microscopic examination.
- Remarks:
- Doses / Concentrations:
0, 1.5, 3 or 6 mg Br-/day
Basis:
actual ingested
for 133 days (based on a measured water intake of 15 mL/animal/day) - Remarks:
- Doses / Concentrations:
0, 0.15, 0.75 and 1.5 mg Br-/day
Basis:
actual ingested
for 16 and 66 days (based on a measured water intake of 15 mL/animal/day) - No. of animals per sex per dose:
- 10/group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- Performance of electron microscopy:
Thyroid glands of rats were dissected free of fibrous fatty tissue and muscles; small pieces of tissue (approximately 1 mm3) were fixed in 2.5 % glutardialdehyde in a 0.3 M phosphate buffer, pH 7.2 at 4°C for 2 hours, rinsed in the same buffer with 5 % glucose, and post-fixed in 2 % osmium tetroxide in the same buffer at room temperature for 1 hour. Afterwards the material was dehydrated in ethanol, embedded in Durcupam ACM and processed for electron microscopy. Semithin and ultrathin sections were cut using ultramicrotomes and Ultracut. Semithin sections were stained in aqueous solution of Azur B in borax buffer, pH 8. Ultrathin sections were contrasted using uranyl-acetate, lead-citrate and examined using electron microscope. - Examinations:
- Food consumption and compound intake
Organ weights: Weights of thyroid lobes were taken only.
Gross and Histopathology (Elektron microscopy): Both thyroid lobes were stained by haematoxylin und eosin and by the PAS reaction.
Statistics: Statistical evaluation for proliferation activity test results was done by the Kruskal-Wallis test. - Details on results:
- Food consumption and compound intake:
The diet was available ad libitum. The mean amount of food consumed was approximately 20 g/animal/day. Water consumption was measured to be approximately 15 mL/animal/day.
Gross and Histopathology:
The thyroid gland was examined in detail via electron microscopy. No other organs were investigated for changes.
The thyroid gland of the control animals consisted of follicles predominantly round in shape, containing colloid in their lumen. The follicular size varied to some extent and measured 30-50 µm on average. The walls of these follicles were lined with a simple cuboid epithelium of thyrocytes. The nucleus was usually round or ovoid and the nuclear chromatin of medium density, the medium and basal parts of the cytoplasm contained a well developed granular endoplasmic reticulum (ER) and numerous of free ribosomes. The Golgi complex consisted of a varying number of flattened cisterns with small pockets and vacuoles at the periphery and was usually located between the nucleus and luminal plasma membrane. Oval and elongated mitochondria with marked cristae were scattered thoughout the entire cytoplasm. In the luminal cytoplasm dark or medium density granules with a homogenous core were found close to the surface membrane. The cytoplasmic matrix also contained numerous primary and secondary lysosomes, including polyphagosomes, colloid droplets and residual myelin corpuscles. The luminal plasma membrane of the thyrocytes showed coated pits, and branching microvilli. Between the lateral membranes of the thyrocytes, zonulae occludents, zonulae adhaerentes, desmosomes and nexus were found.
After 16 days of 10 mg bromide application the size of follicles varied, many small follicles with very small lumina were found. Thyrocytes with hypertrophic granular ER and dilated cisterns with light material were observed. Proliferated microvilli protruded into the luminal colloid. The nuclei were often irregular in shape with incisions, and the density of nuclear chromatin was higher than in the controls. The well developed Golgi apparatus showed small, transport granules of a density similar to that of some subapical granules and minute spherical structures and was mostly enlarged. The number of secondary lysosomes including phagolysosomes was increased only in some cells. Colloid droplets were rarely found. Sporadic thyrocytes with signs of necrosis were seen. Complex intercellular contacts were typical between membranes of adjacent cells even in very small follicles. Similar findings were obtained after 66 days administration of bromide.
After 16 days of 50 mg bromide application the overall picture of the tissue was dominated by numerous very small follicles with very small lumen containing a smaller amount of colloid. ER was markedly increased with highly dilated cisterns not located only in central and basal parts of cytoplasm but also in the subapical region of many cells. Microvilli protruding into the luminal colloid were significantly increased. The nuclei were of irregular shape with condensed nuclear chromatin. In the supranuclear cytoplasm of the follicular cells the small, subapical granules wth dark or medium-dark homogeneous core substance and coated pits were increased. Lysosomes (size: 400-1500 nm) filled with a fine granulated substance were also present sometimes fusing with small granules. Colloid droplets were rare. Similar findings were obtained after 66 days of 50 mg bromide administration.
After 16 days of 100 mg bromide administration the thyroid gland showed the highest proliferation rate of the thyrocytes. Newly formed intracellular cavities resembling microfollicles were frequently found containing normal colloid or fragments of degraded follicular cells separated from the wall of the follicle. The cytoplasm was filled with dilated ER cisterns. Remnants of former thyrocytes were also found in the colloid of some follicles after their separation from the follicular wall. The nucleus of the thyrocytes was usually smaller, often of irregular shape, with increased chromatin density. Even in apical parts of the cytoplasm the ER cisterns were significantly delated. Golgi complex was usually enlarged. The number of larger round lysosomes including phagolysosomes appeared to be unchanged; however, their contents were mostly more dense. Colloid droplets were rarely found. In some cases clusters of cells with spongy cytoplasm and bizarre-shaped nuclei were seen. Two types of cells were present in the follicular epithelium. One type was characterized by many small round subapical granules of medium and small density, and flat ER cisterns. The other type had only a few subapical granules and dilated ER cisterns with a low density content. Similar results were obtained 66 days after administration of 100 mg bromide.
In the group receiving 100 mg bromide over 133 days the overall picture micro-follicular restructure predominated and the mean follicular size was around 8-20 µm and less. Nuclei were of irregular shape with higher density chromatin. In the basal parts of the thyrocytes various amounts of dilated ER cisterns, often parallel in arrangement were found. Dense bodies were also present. As in the previous groups of animals (receiving 100 mg bromide/L for 16 and 66 days), two types of thyrocytes were present. Between the lumino-lateral plasma membranes, well developed junctional complexes were found and signs of necrosis were observed in the cytoplasm of some thyrocytes.
In the group treated with 200 mg bromide/L for 133 days, microfollicles predominated. The amount of colloid in the small lumina was markedly reduced in most follicles. An increased number of microvilli and coated pits were frequently seen. The luminal cytoplasm contained more spherical structures and granules, cisterns of ER and dense bodies (lysosomes). The nucleus was of irregular shape and with higher density chromatin. The golgi apparatus, desmosomes and junctional complexes between the neighbouring thyrocytes were usually well developed.
The thyroid gland of the 400 mg/L-group (133 days) was characterized by small follicles with reduced lumina. Their wall consisted of thyrocytes, the ultrastructure of which displayed smaller changes than in the other groups of bromide-treated rats. One common finding were medium dilated cisterns of ER and increased number of spherical structures and granules in the subapical cytoplasm. - Conclusions:
- The ultrastructural findings in this investigation after bromide administration were similar to those caused by well known goitrogens used in experiments and clinical practice. It may be suggested that the changes caused by bromide treatment of rats in the thyrocytes point to a defect in transport and probably also synthesis of thyroidal hormones caused by increased bromide levels which inhibit active absorption of iodide by the thyroid.
LOAEL: 10 mg/L drinking water/day, corresponding to 0.15 mg/animal/day or 0.6 mg/kg bw/day
NOAEL: Not applicable - Executive summary:
Materials and Methods
The present investigation is a continuation of previous investigations in which environmental concentrations of bromide have been used. It extends the earlier findings on the rat thyroid gland to the electron microscopic level using the same experimental design. Male Wistar rats were divided into 9 experimental and 3 control groups, consisting of 10 animals each. The animals received bromide in drinking water: 0, 10, 50 and 100 mg/L for 16 and 66 days and 0, 100, 200 and 400 mg/L for 133 days. The amount of water consumed by the animals was measured and was approximately 15 ml/animal/day.
Results and Discussion
The most important finding in the cytoplasm of thyrocytes was the hypertrophy and hyperplasia of ER, combined with dilated cisterns and tubules not only in the central and basal but also in the apical cytoplasm where, in addition, the dilated cisterns were often of ovoid shape. The hypertrophic ER, together with other ultrastructural findings, may indicate the degradation of protein biosynthesis of components of iodine containing thyroglobulin in thyrocytes. The shape, size and number of mitochondria did not significantly differ from the control groups, with the exception of those animals receiving 100 mg Br-/L for 133 days. In these rats the mitochondria were markedly enlarged. Colloid droplets indicating an increased resorption of the colloid from the lumen were hardly seen in the test material. This finding was also described by other groups after high bromide doses. The dilatation or vesiculation of ER may relect enzymatic or mechanical defects in the ER of neoplastic cells, which was noticed for thyroid tumours. The cistern patterns in he ER are comparable to observations in follicular cells of experimentally induced goitre by low-iodine diet. The thyrocytes also showed nuclei with irregular shape and condensed chromatin. This was also found in hamsters fed a low-iodine diet.
- Endpoint:
- mechanistic studies
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 2002
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: no guideline available for this particular investigation
- Qualifier:
- no guideline available
- Deviations:
- not applicable
- Remarks:
- no guideline available for this particular investigation
- Principles of method if other than guideline:
- Study was performed according to good experimental practice
- GLP compliance:
- no
- Type of method:
- in vivo
- Endpoint addressed:
- repeated dose toxicity: oral
- Species:
- dog
- Strain:
- Beagle
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Not indicated
- Age at study initiation: Not indicated
- Weight at study initiation: Mean bodyweight was 11.3 kg (range 9.6-13.7 kg) - Route of administration:
- oral: feed
- Vehicle:
- water
- Details on exposure:
- DIET PREPARATION
- Mixing appropriate amounts with (Type of food): Test substance was dissolved in water and mixed with the diet
VEHICLE
- Concentration in vehicle: 200 mg/mL distilled water, mixed with canned food - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 115 days
- Frequency of treatment:
- daily
- Post exposure period:
- Not indicated
- Remarks:
- Doses / Concentrations:
30 mg/kg bw/day
Basis:
actual ingested - No. of animals per sex per dose:
- 3/sex
- Control animals:
- no
- Examinations:
- Clinical signs: once per week
Bodyweight: once per week
Haematology: complete blood count was performed during the acclimatisation period only (prior to start of the experiment).
Clinical Chemistry: bromide concentration was determined periodically in order to determine attainment of a steady-state level. After the steady-state was attained, venous blood samples were collected every two hours over a 12-hour dosing interval in all dogs to determine if serum bromide concentrations underwent fluctuations between doses.
Urinalysis: 24-hour collection every three days
Gross and Histopathology: Muscle and nerve biopsies were collected at the termination of the study. Quadriceps, cranial tibial and triceps muscle and sciatic nerve were submitted for histochemical evaluation. No further gross and histopathological examinations were performed.
Bromide concentrations in cerebrospinal fluid: Samples were collected on Days 0, 9, 45 and 115. A complete neurologic examination was performed weekly and the degree of ataxia and/or paresis was scored. Electrodiagnostic testing was performed on Days 0, 9, 45 and 115.
Statistics: One- compartment model for all sample results assuming a constant rate of administration of bromide. PK Analyst software was used to perform nonlinear, least-squares regression analysis. - Details on results:
- Clinical signs and mortality:
No mortalities reported in this investigation.
There were no adverse neurologic side-effects in any of the dogs at an oral dose of 30 mg/kg bw/day. Following dose adjustment, two of the dogs exhibited grade III-IV caudal paresis and ataxia characterized by a wide-based and/or crouched pelvic limb stance, difficulty rising from a sitting position and decreased hemistanding and flexor withdrawal reflexes in the pelvic limbs. Neither the magnitude of nor the relative change in the serum bromide concentration was related to the development of neurologic deficits. Two of the dogs were agitated to hyperexcitable following the dose adjustment phase but neither showed signs of weakness or ataxia.
Bodyweight/Bodyweight gain:
Mean bodyweight throughout the study initiation was 11.3 kg, with a range of 9.6-13.7 kg.
Food consumption/Compound intake:
Food consumption and compound intake were observed by eye only. For that reason no further specification is available.
Clinical Chemistry:
The mean steady-state serum bromide concentration was 245 mg/dL. Steady-state bromide concentrations were widely variable between dogs, ranging from 178 to 269 mg/dL. From 60 days until the final sampling time during maintenance dosing (115 days), serum bromide concentrations increased very slowly. The subsequent dose adjustment successfully raised the serum bromide concentration of 400 mg/dL. The median serum bromide concentration post-dose adjustment was 397 mg/dL.
The mean median t1/2 using a one-compartment model was 15.2 days. A majority of dogs reached a serum bromide concentration that was 75% of the apparent steady-state concentration by 30 days and at least 90% of the apparent steady-state concentration by 60 days.
Urinalysis:
Median renal clearance was 8.2 mL/kg bw/day . Renal clearance varied between dogs (range 6.03-12.6 mL/kg bw/day) and was not correlated with total body clearance.
Other:
Electrodiagnostic tests revealed subtle changes over time. Spectrally analyzed EEG frequencies either remained unchanged or showed insignificant reductions in power at all frequencies with increasing bromide concentrations. Examination of individual trends revealed central latency shifts that either progressively increased over time or else coincided with the appearance of adverse side-effects. Cortical somatosensory potentials remained constant in latency and amplitude with no significant changes over time. The electromyography, motor nerve conduction velocity, repetitive nerve stimulation and characteristics of the compound muscle action potential after peripheral nerve stimulation were all normal at all time points and did not show significant differences over time. Muscle and nerve biopsies from all dogs were normal. Brain stem auditory evoked response latencies were prolonged with increasing serum bromide concentrations, then appeared to stabilize or even decrease slightly. After a dose increase, latencies again increased. - Conclusions:
- There were no adverse neurologic side-effects in any of the dogs treated with 30 mg/kg/day of sodium bromide. Neither the magnitude of nor the relative change in the serum bromide concentration was related to the development of neurologic deficits. The mean steady-state serum bromide concentration was 245 mg/dL and the mean median t1/2 was 15.2 days.
Only BAER latencies were prolonged with increasing serum bromide concentrations. These findings suggest that conduction along peripheral and central sensory pathways may be delayed when serum and CSF bromide concentrations are elevated. The BAER may be useful in documenting and assessing the severity of clinical bromism.
LOAEL: 400 mg bromide/dL (serum concentration)
NOAEL: 269 mg bromide/dL (serum concentration) - Executive summary:
Materials and Methods
The purpose of the study was to develop a multidose method of bromide administration that targets serum bromide concentrations in the range of 200-300 mg/dL and to examine the dynamics of bromide pharmacokinetics during the accumulation phase and at steady-state. Pharmacokinetic parameters from other studies have served as the basis for currently recommended KBr maintainance doses in the dog. Recommended doses of 30-40 mg/kg bw/day of potassium bromide (20-27 mg Bromide/kg bw) will hypothetically produce a steady-state bromide concentration of 100-200 mg/dL. Therefore, the selection of the initial maintenance dose was based on calculation of a dose required to maintain a steady-state serum concentration of 275 mg/dL. Previously reported bromide pharmacokinetic values were used to calculate the maintainance dose. Prior to administration of 30 mg/kg bw/day of potassium bromide (mixed with canned food over 12 hours) for a period of 115 days, baseline serum, urine and cerebrospinal fluid bromide concentrations were measured. Urine was collected over a 24-hour period and total volume was recorded.. The daily dose was divided in order to reduce the likelihood of gastrointestinal side-effects. After steady-state was attained, blood samples were collected every 2 hours over a 12-hour dosing interval in all dogs in order to determine if serum concentrations of bromide underwent significant fluctuations between doses. Cerebrospinal fluid samples were taken on Days 0, 9, 45 and 155. On the same days electrodiagnostic testing was performed. A complete neurologic examination was performed once per week and the degree of ataxia and/or paresis were scored. Bodyweights were determined weekly. Consumption of food was observed by eye to ensure entire uptake of the test substance. Subsequent to Day 115, an adjusted loading dose of potassium bromide was administered as divided doses over a period of 5 days to rapidly increase and maintain bromide serum concentrations at 400 mg/dL. Dose adjustment was designed to increase serum bromide to a new steady-state concentration rapidly in the event of inadequate seizure control but may also increase the risk of bromide toxicity. The loading dose required to achieve the new stead-state concentration was calculated using the following formula: Volume distribution at steady-state x (target serum concentration – observed serum concentration) with 400 mg/dL being the target serum concentration. After this 5-day-period, serum, urine and cerebrospinal fluid were collected and neurodiagnostic testing was performed. Following the supplemental loading dose, all dogs were administered a maintenance dose calculated to maintain a 400 mg/dL bromide serum concentration. Muscle and nerve biopsies were collected at termination of the study (quadriceps, cranial tibial, and triceps muscle and sciatic nerve were submitted for histochemical evaluation). Electrodiagnostic testing (electroencephalography (EEG), electromyography (EMG), motor nerve conduction velocity (MNCV), repetitive nerve stimulation (RNS), brain stem auditory evoked responses (BAERs), and cortical somatosensory evoked potentials (SSEPS)) was performed at Days 0, 9, 45 and 115.
Results and Discussion
The results of all pharmacological parameters determined are summarized in table A6.10/17-1. Steady-state bromide concentrations were variable (range 178-268 mg/dL) reflecting differences in clearance and/or bioavailability between dogs. The mean steady-state serum bromide concentration was 245 mg/dL. From 60 days until the final sampling time during maintenance dosing (115 days), serum bromide concentrations increased very slowly. The subsequent dose adjustment successfully raised the serum bromide concentration of 400 mg/dL. The median serum bromide concentration postdose adjustment was 397 mg/dL. The mean median t1/2 using a one-compartment model was 15.2 days. Median apparent total body clearance was 16.4 mL/kg/day and median apparent volume of distribution was 0.4 L/kg. Median renal clearance was 8.2 ml/kg/day, varied between dogs and was not correlated with total body clearance. The CSF to serum bromide concentration ratio was 0.63 at 9 days after initial dosing (see also table A6.10/17-2). After a significant increase in the ratio between 9 and 45 days, there was no significant change in this ratio between Day 45 and 115. The ratio increased significantly from days 115 to 121, coincident with the supplemental dose that was administered. The median CSF bromide concentration postdose adjustment was 353.3 mg/dL and the median CSF to serum ratio was 0.86. There were no adverse neurologic side-effects from Day 0 to 115 in any of the dogs. Following dose adjustment, two dogs exhibited caudal paresis and ataxia characterized by a wide-based and/or crouched pelvic limb stance, difficulty rising from a sitting position, and decreased hemistanding and flexor withdrawal reflexes in the pelvic limbs. Neither the magnitude of nor the relative change in the serum bromide concentration was related to the development of neurologic deficits. Electrodiagnostic tests revealed subtle changes over time. EEG frequencies and SSEPs either remained unchanged or showed insignificant reductions in power with increasing bromide concentrations. EMG, MNCV, RNS and characteristics of the compound muscle action potential after peripheral nerve stimulation were. Muscle and nerve biopsies from all dogs were normal. BAER latencies were prolonged with increasing serum bromide concentrations, than appeared to stabilize or even decrease slightly. After a dose increase, latencies again increased. These findings suggest that conduction along peripheral and central sensory pathways may be delayed when serum and CSF bromide concentrations are elevated. Slowed neuronal conduction may be related to the hyperpolarizing effect of bromide on neuronal membrane potential.
- Endpoint:
- specific investigations: other studies
- Type of information:
- other: review article on waivy ribs in the rat
- Adequacy of study:
- key study
- Study period:
- 1994
- Reliability:
- other: Not applicable (2 with focus on information provided in the review article)
- Qualifier:
- no guideline followed
- Deviations:
- not applicable
- Remarks:
- review article
- GLP compliance:
- no
- Type of method:
- in vivo
- Endpoint addressed:
- developmental toxicity / teratogenicity
- Species:
- other: rat, mouse and hamster
- Strain:
- not specified
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- No details given on test animals. - Route of administration:
- other: Not indicated
- Vehicle:
- not specified
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- Day 7-20 of gestation
- Frequency of treatment:
- Not indicated
- Post exposure period:
- Not indicated
- Remarks:
- Doses / Concentrations:
Not indicated
Basis:
no data - No. of animals per sex per dose:
- Not indicated
- Control animals:
- yes
- Details on study design:
- Occurrence of wavy ribs in fetuses following administration of different substances to pregnant animals of different species during organogenesis was reviewed.
- Examinations:
- General:
Litter Size, Nr. of dead Foetuses
Skeleton:
Alizarin staining of foetuses and X-Ray examinations at several time-points post partum were performed. - Details on results:
- Maternal toxic effects:
There were no maternal toxic effects described in the review, since it focuses on the wavy ribs occurring in fetuses following administration of different substances during the period of gestation
Teratogenic effects:
The review focuses on the appearance and disappearance of wavy ribs. Therefore, only these effects are described and summarized below (5.2). - Conclusions:
- It can be concluded from the literature that wavy ribs in foetuses of small rodents are caused by a large number of compounds of various chemical structures and biological activities and that these effects are secondary to maternal toxicity per se but also endocrine disturbances are amongst the main causative factors for the formation of wavy ribs and, thus, for an impairment of the development of the offspring. However, a uniform, generally accepted explanation for the etiology and pathogenesis is still missing. The wavy ribs develop during the perinatal period from Day 17 through Day 20 of gestation and are more or less completely reversible within three weeks after birth. Therefore, wavy ribs cannot be considered as true malformations, but are a reversible pathologic finding.
- Executive summary:
Materials and Methods
In a review on the occurrence of wavy ribs in fetuses of small rodents following administration of different substances during the period of gestation data of 74 scientific publications have been summarised. The data were collected in support of a computer-assisted search on MEDLINE, TOXLINE, BIOSIS and EXPERTA MEDICA systems and has been critically examined. Numerous compounds of a large variation in chemical structure and biological activity were given to pregnant rats during the later period of organogenesis.
Results and Discussion
In this publication, the occurrence of wavy ribs in fetuses following administration of different substances to pregnant animals of different species during organogenesis was reviewed. Synonyms used for waved or wavy ribs are: bent, undulated, nodumalted, bulbous, flexible, kinky, distored or misshapen ribs. The wavy ribs are often combined with limb bone flexures, like bent and short scapula, humerus, radius and ulna; and may also occur together with anasacra. Anatomy of wavy ribs: With respect to the anatomy of wavy ribs it was observed that, basically, the earliest appearance of skeletal cartilage in rats, as shown by stainability with toluidine blue, is in the 3rd and 9th ribs at Day 15 of gestation. Ossification of the ribs begins with ribs 3-9 on Day 15.5 and during the following 2 days spreads in both directions, slightly more rapidly caudal than rostral. Via electron-microscopy mineralisation defects were found in the region of hypertrophic cartilage and the perichondral bone sheath of the limb bones. In a different study, similar conditions in Syrian gold hamsters were studied by light- and electron-microscopy and a defect was founding the extracellular maturation of collagen. Another study showed wavy outlines of the cartilaginous matrix located within the bony sheath using alcian blue staining. Firstly, a delay of ossification was seen in the middle of the ribs on Day 17 of gestation. Thereafter, middle part of the ribs began to curve, and the curves were calcified until end of gestation. On Day 17, inhibition of endochondral ossification was observed. Causative factors: Wavy ribs are caused by numerous compounds of a large variation in chemical structure, chemical class and biological activity.
The following modes of etiopathology have been discussed:
1)Alkaline phosphatase: It has been postulated that the anomaly of wavy ribs results from delayed development of alkaline phosphatase (AP) activity and arrested calcium deposition in ribs. Ribs that lack rigidity may bent in response to muscular tension or other physical forces. It was found, that AP significantly decreased in fetal serum, when the dams were treated with fenoterol from Day 7-20 of gestation. Of these fetuses, 53.4% showed wavy ribs.
2)Dietary vitamins and minerals: A gross dietary deficiency of either calcium or phosphorus leads to rarefaction of bone. Deficiency of vitamins A, C and D also result in a diminuation in the rate of growth of the skeleton, disturbance of ossification and diminuation of the calcium content. Such bones are easily deformed.
3)Maternal toxicity: Numerous compounds including anthelmintics, herbicides, insecticides, pesticides, antibiotics, antidiabetics, antiallergics, antidepressives, antiepileptics and anxiolytics are causing wavy ribs if given at high enough dosages. Maternal toxicity seems to be an important factor.
4)Renal loop diuretics: Several researchers found wavy ribs after renal loop diuretics. In one of the publications the incidence of wavy ribs was reduced and the scapular and humeral bone flexures eliminated, both caused by a loop diuretic, by providing a 1% solution of potassium via the drinking water during the dosing period. Another group, studying the pathogenesis of furosemide-induced wavy ribs, described protective effects of sodium hydrocarbonate or ammonium chloride against maternal hypochloremic metabolic alkalosis and successfully prevented the fetal lesion.
5)Myometrial constriction: Mechanical actions like myometrial constriction or increase in uterine pressure and reductions in the amount of amniotic fluid may contribute to the deformities of wavy ribs. Hysterectomy prevented the formation of wavy ribs in those fetuses freed from the uterus whilst deformed fetuses were obtained in the closed horn of the same dam. According to a different publication, it is probable that wavy ribs are due to increased uterine pressure caused by impaired growth of the uterus due to oestrogen deficiency. Then, however, fenoterol, isoxsuprine or other tokolytics should protect against the lesion, but actually, those beta-adrenoceptor-stimulants are causing wavy ribs.
6)Endocrine disturbances: In another mode of action, endocrine effects upon bone have to be considered. The activity of the parathyroid glands appears to be regulated by a negative feedback mechanism in which the blood calcium-ion level itself exerts a direct effect upon parathyroid activity. Methallibure blocks the action of the pituitary thereby lowering gonadotropine and consequently reducing ovarian oestrogen secretion.
7)Beta-adrenoceptor stimulation and blocking (bone morphogenic proteins theory): According to literature data, treatment of pregnant rats with high doses of a beta-adrenoceptor stimulant during gestational Days 7-20 caused calcified and edematous placentar labyrinths indicating an impairment of the maternal site of placentar circulation and resulting in significant changes of fetal blood, namely a decrease of fetal total protein from 3.5 ± 0.7 g/dL in controls to 2.1 ± 0.1 g/dL in treated animals. In parallel, there was an as well significant decrease of alkaline phosphatase.
Based on these results, the following theory is added by the author of the review: it has been known for some time that the bone morphogenic proteins are responsible for the process of bone formation seen during embryonic long bone development. Cartilage is formed at earlier times and then replaced by bone at later times. The bone morphogenic proteins involved in the development of cartilage and bone during embryogenesis are due to a set of proteins which may be poor in foetal serum, when the dams are treated with beta-adrenoceptor stimulants, blockers or other compounds causing wavy ribs. In parallel to the retarded development of osteoblast progenitors at different stages of differentiation, alkaline phosphatase activity remains low. After delivery, when the neonate’s serum protein content normalizes, the bone morphogenic proteins activate cells that participate in bone repair until weaning. Beta-stimulation was also a factor found by two research groups. Both succeeded in reducing the incidence of wavy ribs by beta-blocking with bunitrolol or carazolol, respectively. On the contrary, another group reported the occurrence of 19% fetuses with wavy ribs after 300 mg/kg bunitrolol. Occurrence and repairability: Wavy ribs occur rarely in fetuses of untreated dams, but some investigators reported 0.4-6.1%. In groups treated with various compounds during the period of organogenesis, the incidence may be moderately increased or extremely high in a dose-dependent manner. According to one publication, the sensitive period for treatment is during Days 13-17 of gestation, corresponding to the time of appearance of chondrification and ossification centres. In control fetuses, ossification of ribs starts on Day 16 of gestation. One day later, ossification spreads towards both ends of the rib. On Day 18, the ribs are ossified. Pathologic ribs do not differ morphologically from controls on Day 16. On Day 17 and 18, however, ossification centre has separated to single, shortened ossification centres. Curving of insufficiently ossified ribs occurs on Day 19, and on Day 20 the curves become ossified. Following treatment with 1000 mg/kg fenoterol for instance, the rate of the total of “wavy ribs” observed by alizarin staining did not differ significantly between fetuses obtained by caesarean section on Day 21 of gestation, and various age groups of pups at 1, 3, 7 and 14 days post partum, but the severity decreased gradually from distinctly visible to hardly visible, and the incidence of hardly visible wavy ribs was finally reduced at weaning. X-Ray examinations of individual pups confirmed that the distortion of ribs which was distinctly visible on the first day post partum gradually disappeared during three-week lactation period and was gone or hardly visible at weaning.
The reversibility of wavy ribs was repeatedly confirmed by several investigations, the conclusions thereof are summarized below:
-Development of wavy ribs on Day 18 of gestation, disappearance on Day 10 post partum. A surface remodelling may straighten the bend in the subsequent bone growth.
-Alizarin staining showed 19% animals with wavy ribs at birth, but 0% at weaning.
-Ribs revert to normal within a few days after birth.
-Wavy ribs are temporary and disappear in adults.
-X-ray examination showed 18.5% animals with wavy ribs directly after birth, 2.7% on Day 11 and 0% on Day 21 post partum.
-On Day 33 post partum, this rib anomaly was no longer present, clearly showing the transient nature.
-Bending of the long bones disappears during the weaning period
Species-specificity:
Wavy ribs occurred in rats, mice, pigs and Golden Syrian hamsters. They were not described in rabbits. According to one publication from 1980, the condition seems not related to certain strains of rat, whereas another publication found that strains of Sprague-Dawley rats were relatively resistant. The present literature survey performed for this review did not indicate strain differences among both treated and control rats. The finding seems not to be relevant for man, a species with a long gestation period. Even in rat, the lesion disappears spontaneously after birth.
Evaluation:
Most authors consider wavy ribs as a malformation, but some also consider them as defects, mild forms as variants or non-specific skeletal variants. One author states, that little is known about the significance of bent or wavy ribs, and that they are foetal aberrations rather than malformations. A different publication considered wavy ribs as reversible pathologic finding but not as true malformation whereas one publication saw an embryotoxic effect.
- Endpoint:
- mechanistic studies
- Type of information:
- migrated information: read-across based on grouping of substances (category approach)
- Adequacy of study:
- supporting study
- Study period:
- 1997
- Reliability:
- 2 (reliable with restrictions)
- Qualifier:
- no guideline available
- Deviations:
- not applicable
- Remarks:
- no guideline available for the conduct of mechanistic studies
- Principles of method if other than guideline:
- study was performed according to good experimental practice
- GLP compliance:
- no
- Type of method:
- in vivo
- Endpoint addressed:
- not applicable
- Species:
- other: human and laboratory rat
- Strain:
- not specified
- Sex:
- not specified
- Route of administration:
- other: route of administration not stated within the review report.
- Details on results:
- Long-term perturbations of the pituitary-thyroid axis by various xenobiotics or physiological alterations (e.g. iodine deficiency, partial thyroidectomy, natural goitrogens in food) are more likely to predispose the laboratory rat to a higher incidence of proliferative lesions in response to chronic thyroid stimulating hormone (TSH) stimulation than in the human thyroid. The greater sensitivity of the rodent thyroid also is related to the shorter plasma half-life of thyroxine (T4) than in humans due to the considerable differences between species in the transport proteins for thyroid hormones.
The plasma T4 half-life in rats is considerably shorter (12-24 hours) than in humans (5-9 days). In human beings and monkeys, circulating T4 is bound primarily to thyroxine binding globulin (TBG), but this high-affinity binding protein is absent in rodents, birds, amphibians and fish.
Binding affinity of TBG for T4 is approximately 1000-times higher than for prealbumin. The percentage of unbound active T4 is lower in species with high levels of TBG than in animals in which T4 binding is limited to albumin and prealbumin. Therefore, a rat without a functional thyroid requires about 10-times more T4 (20 µg/kg bw) for full substitution than an adult human (2.2 µg/kg bw). Triioothyronine (T3) is transported bound to TBG and albumin in humans, monkey and dog but only to albumin in mouse, rat and chicken. In general, T3 is bound less avidly to transport proteins than T4, resulting in a faster turnover and shorter plasma half-life in most species. These differences in plasma half-life of thyroid hormones and binding to transport proteins between rats and humans may be one factor in the greater sensitivity of the rat thyroid to develop hyperplastic and/or neoplastic nodules in response to chronic TSH stimulation. A broad spectrum of marketed drugs (diverse product classes) results in a thyroid tumorigenic response. - Conclusions:
- The regulation of thyroid hormone synthesis as well as the possible mechanism for the induction of thyroid tumours in the rodent, mostly in the rat, were discussed. It was shown that thyroid cell proliferation is mostly a result of increased TSH levels as a consequence of reduced T3 levels in the plasma. This results in a sustained increase of circulating TSH levels causing hypertrophy in follicular cells. The mechanism for a decrease in T3 levels consists of an inhibition of 5´-deiodinase which biotransforms T4 to T3 or is mediated by an increased excretion of T4 as a consequence of hepatic enzyme induction and resulting enhanced enzymatic glucuronidation of T4. In the review it was stated that this occurs particularly with rodents, first because UDP-glucuronyl transferase can be easily induced in rodents, and second because thyroxine metabolism takes place very rapidly in rats in the absence of thyroxine binding globuline. In humans, a lowering of the circulating thyroxine level but no change in TSH and T3 levels has been observed but only at high doses of powerful enzyme-inducing compounds. In this context it was concluded that there is no convincing evidence that humans treated with drugs or exposed to chemicals that induce hepatic enzymes are at increased risk for the formation of thyroid tumours. An investigation with relative high dose of phenobarbital (100 mg for 14 days) demonstrated no influence on T3, T4 and TSH levels in humans and no hormonal imbalance was noted. Long-term administration of therapeutic doses of phenobarbital was not associated with a higher risk for the development of thyroid neoplasia.
In conclusion, rats show a greater sensitivity to derangement by drugs, certain chemicals and physiologic perturbations than humans regarding the thyroid gland. This is due to the absence of TBG in rodents, which is the major thyroxine and triiodothyronine binding protein in man with a much higher binding activity than albumin. Therefore, species that lack TBG, like the rat, are more sensitive to develop follicular cell hypertrophy and hyperplasia in response to chronic TSH stimulation. Another cause for the greater sensitivity of rodents is the shorter plasma half-life of T4 compared to man: 12-24 hours in rats compared to 5-9 days in humans. - Executive summary:
Please refer to section "remarks on results".
- Endpoint:
- endocrine system modulation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1990
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: No guideline is available for the investigation of effects related to the possible mechanism of action of a substance and to study effects on body specific substances.
- Qualifier:
- no guideline available
- Deviations:
- not applicable
- Remarks:
- no guideline available for this special mechanistic investigation
- Principles of method if other than guideline:
- Study is a publication and was performed according to good experimental practice.
- GLP compliance:
- no
- Type of method:
- in vivo
- Endpoint addressed:
- repeated dose toxicity: oral
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, Germany
- Age at study initiation: 11 weeks
- Weight at study initiation: not stated within the report - Route of administration:
- oral: feed
- Vehicle:
- other: No vehicle used, test substance was mixed with the diet
- Details on exposure:
- DIET PREPARATION
- Concentration of test substances in food: 4, 8 and 16 g/kg - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 4 weeks
- Frequency of treatment:
- daily
- Post exposure period:
- No postexposure period; animals were sacrificed at termination of the treatment period.
- Remarks:
- Doses / Concentrations:
4, 8 and 16 g/kg
Basis:
nominal in diet
corresponding to 200, 400 and 800 mg/kg bw/day with 1 ppm = 0.05 mg/kg bw/day for older rats - No. of animals per sex per dose:
- 12/sex/group
- Control animals:
- other: Two control groups receiving either plain diet or low iodine diet
- Details on study design:
- The animals underwent a 2-week pre-treatment period and a 4-week treatment period. During these periods animals were fed either a normal laboratory rodent diet or a low iodine diet with or without varying amounts of sodium bromide.
Total T4 and free T4 in serum were measured by radioimmunoassay. Thyroid stimulating hormone (TSH) in serum was measured by an immunoradiometric assay. - Examinations:
- Clinical signs: twice daily
Bodyweight: weekly
Food consumption: weekly
Clinical chemistry: blood samples were taken on Day 1, 14, 22, 29, 36 and 43 and analyzed for bromide and iodide content and T4 and TSH levels. No other clinical-chemical parameters were investigated.
Urinalysis: urine samples were taken on Day 1, 14, 22, 29, 36 and 43 and analyzed for iodide content.
Organ weights: Thyroid and parathyroid weights were taken only.
Determination of bromo- and iodosubstituted thyronines in the thyroid: bromo- and iodosubstituted thyronines were quantitatively released from thyroglobulin by digestion of the tissue with pronase-E and subsequently analyzed by HPLC combined with UV detection at 230 nm and with off-line radioimmunoassay detection.
Statistics: Statistics were performed with the Kruskal- Wallis test for differences between more than two groups of non-normally distributed data. - Details on results:
- Clinical signs:
For animals in group 5 (low-iodine diet, 16 g sodium bromide/kg) hypoactivity, ruffled fur and emaciation were noticed in Week 3. From Week 4, hypoactivity was found in group 4 (low-iodine diet, 8 g/kg of sodium bromide). No abnormal clinical signs were observed in groups 1-3.
Mortality:
All animals of the highest dosage group (16 g/kg) fed a low-iodine diet, were either found dead or had to be sacrificed because of poor general appearance. Most of the animals of this group were dead on Day 37 of the investigation. From the 8 g/kg group, one male was found dead on Day 33 and another male was dead by Day 42 of the study.
Bodyweight gain:
Analysis of bodyweight showed dramatically reduced bodyweights for the animals receiving 16 g sodium bromide/kg from Day 29 onwards. Bodyweight gain in group 4 (8 g/kg) was also reduced from Day 29 on. Animals receiving the lowest sodium bromide concentration used (4 g/kg) had bodyweights comparable to the control groups (normal diet and low-iodine diet).
Food consumption:
Food consumption was reduced in animals treated with 8 and 16 g/kg.
Clinical Chemistry:
For determination of bromide, total T4, free T4 and TSH in serum the samples of four animals of each cage were pooled. No differences in the results of both sexes could be detected, so the data were combined. With increasing serum bromide concentrations free T4 and total T4 levels decreased in all treatment groups. The higher the sodium bromide concentration in food, the higher was the observed serum bromide level and the lower the T4 levels. Group one, which was fed normal rodent diet, had higher serum bromide concentrations and higher T4 levels than the animals treated with low-iodine diet but without sodium bromide.
Urinalysis:
Urine was analyzed for iodide content. There were no differences found between male and female animals and for that reason, the data were combined for both sexes. The results show iodine deficiency for all animals receiving the iodine-poor diet. Between the groups of animals treated with different amounts of sodium bromide, there were no differences observed.
Organ weights:
Thyroid weights were increasing with increasing sodium bromide content in food with exception of the highest dosage group which showed organ weights being lower than the ones from the lowest treatment group. Thyroid weights of animals fed iodine-poor diet were higher than the ones from the animals receiving the normal rodent diet.
Thyroid hormones:
The concentrations of T4, T3 and 3,3´,5´-triiodothyronine (reverse T3) in the thyroid gland decreased with increasing bromide concentrations and were lower in animals fed the low-iodine diet compared to the animals receiving the normal rodent diet (control). T3, reverse T3 and T4 levels decreased from 19.6, 5.5 and 143 from control to <2 for all parameters in bromide treated animals respectively. Trisubstituted bromo/iodothyronines were detected in the thyroids of all groups treated with bromide, but not in groups one and two fed a normal or iodine-poor diet. The amount of these bromo/iodosubstituted thyronines was at least one order of magnitude lower than that of the thyroid hormone T3 in normal rats. - Conclusions:
- All animals fed with an iodine-poor diet were in a state of hypothyroidism (decrease of total and free T4 and increase of TSH in blood). This was further enhanced by intake of sodium bromide. Effects on thyroid hormone and bromide levels in serum followed a dose-response relationship. Based on the findings made, a NOAEL could not be derived in this study.
LOAEL: 4 g/kg diet corresponding to 200mg NaBr (156 mg bromide)/kg bw/day
NOAEL: < 4 g/kg diet (< 200 mg NaBr (156 mg bromide)/kg bw/day) day - Executive summary:
Materials and Methods
The investigation was performed to study the influence of bromide on the thyroid of rats, paying special attention to the possibility of biosynthesis of brominated thyroid hormone analogues. Bromide doses were chosen where effects on the thyroid were to be expected. Animals were treated with 4-16 g sodium bromide/kg diet for four weeks (corresponding to 200, 400 and 800 mg/kg bw/day). Rats were separated into two groups; one group receiving the normal rodent diet and the other group was fed a low iodine diet. Within these two groups animals were given varying amounts of sodium bromide within the diet. At Day 1, 14, 22, 29, 36 and 43 urine samples were collected and analyzed for iodide content. On the same days, blood samples were taken and analyzed for bromide content; in addition total and free T4 and TSH levels were determined in serum. At the end of the treatment period all rats were sacrificed, thyroid glands and parathyroid glands removed and organ weights taken. Bromo- and iodosubstituted thyronines were determined.
Results and Discussion
All animals of the highest dosage group (16 g/kg) fed a low-iodine diet, were either found dead or had to be sacrificed because of poor general appearance. Most of the animals of this group were dead on Day 37 of the investigation. From the 8 g/kg group, one male was found dead on Day 33 and another male was dead by Day 42 of the study. There were no premature decedents within the animals receiving either the standard diet or the low-iodine diet or the iodine-poor diet in combination with the lowest amount of sodium bromide used (4 g/kg). For animals in group 5 (low-iodine diet, 16 g sodium bromide/kg) hypoactivity, ruffled fur and emaciation were noticed in Week 3. From Week 4, hypoactivity was found in group 4 (low-iodine diet, 8 g/kg of sodium bromide). No abnormal clinical signs were observed in groups 1-3. Animals in the highest dosage group (16 g/kg) had dramatically reduced bodyweights from Day 29 onwards. Also the animals treated with 8 g/kg showed reduced bodyweight gain from Day 29 on. The bodyweights from the lowest treatment group were comparable to control groups. Food consumption was reduced in the 16 and 8 g/kg groups. The actual iodine intake was checked by determining the urinary iodide secretion. There were no differences for male and female animals; for this reason, the data of both sexes were combined. The results obtained show a iodine deficiency for all animals receiving the low-iodine diet. The values between the different sodium bromide concentrations did not show differences in iodide excretion. For determination of bromide, total T4, free T4 and TSH in serum, the samples of four animals of each cage were pooled. Since no differences in the results of both sexes could be detected, the data were combined. With increasing serum bromide concentrations free T4 and total T4 levels decreased in all treatment groups. The higher the sodium bromide concentration in food, the higher was the observed serum bromide level and the lower the T4 levels. Group one, which was fed normal rodent diet, had higher serum bromide concentrations and higher T4 levels than the animals treated with low-iodine diet but without sodium bromide. The differences in the concentrations of free T4 of the iodine-deficient groups were statistically highly significant (p < 0.001) on Day 22, 29, 36 and 43. The wet weights of the thyroid glands at the end of the study showed increasing absolute and relative organ weights with increasing sodium bromide concentrations in food. Exception of this rule were the animals receiving the highest sodium bromide amount; absolute and relative thyroid weights were lower than from the lowest treatment group (4 g/kg). Thyroid weights of animals fed iodine-poor diet were higher than the ones from the animals receiving the normal rodent diet. The concentrations of T4, T3 and 3,3´,5´-triiodothyronine (reverse T3) in the thyroid gland decreased with increasing bromide concentrations and were lower in animals fed the low-iodine diet compared to the animals receiving the normal rodent diet (control). Trisubstituted bromo/iodothyronines were detected in the thyroids of all groups treated with bromide, but not in groups one and two fed a normal or iodine-poor diet. The amount of these bromo/iodosubstituted thyronines was at least one order of magnitude lower than that of the thyroid hormone T3 in normal rats.
- Endpoint:
- endocrine system modulation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1983
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: no guideline available for mechanistic investigations
- Qualifier:
- no guideline available
- Deviations:
- not applicable
- Remarks:
- there is no guideline available for this special investigation
- Principles of method if other than guideline:
- Study was performed according to good experimental practice
- GLP compliance:
- no
- Type of method:
- in vivo
- Endpoint addressed:
- repeated dose toxicity: oral
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Not indicated
- Age at study initiation: 3 weeks
- Weight at study initiation: 60-100 g - Route of administration:
- oral: feed
- Vehicle:
- other: test substance was administered with the diet
- Details on exposure:
- Diets and water were available ad libitum.
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 4 or 12 weeks
- Frequency of treatment:
- daily
- Post exposure period:
- No postexposure period. Animals were decapitated and exsanguinated at the end of the 4- or 12-week treatment period.
- Remarks:
- Doses / Concentrations:
0, 20, 75, 300, 1200 and 19200 mg/kg diet
Basis:
nominal in diet
corresponding to 1.5, 5.6, 22.5, 90 and 1440 mg NaBr/kg bw/day - No. of animals per sex per dose:
- 10/group
- Control animals:
- yes, plain diet
- Examinations:
- Clinical Chemistry:
Serum hormone levels (thyroxine, testosterone, corticosterone, thyroid-stimulating hormone, follicle-stimulating hormone, adrenocorticotropic hormone, insulin and growth hormone) were established by radioimmunoassay.
Organ weights:
Weight was taken from pituitary gland, thyroid and testes only.
Gross and Histopathology:
Pituitary gland, thyroid and testes were examined by histopathological and immunocytochemical techniques.
Statistics:
Significance of differences in the radioimmunoassay between groups was analyzed by Student´s t test. - Details on results:
- Clinical Chemistry:
Serum hormone levels (thyroxine, testosterone, corticosterone, thyroid-stimulating hormone, follicle-stimulating hormone, adrenocorticotropic hormone, insulin and growth hormone) were established by radioimmunoassay.
There was a statistically significant decrease of T4 both after the 4- and the 12-week treatment with the 19200 mg NaBr/kg diet. Also in the 1200 mg/kg diet group, the T4 level was significantly reduced after the 4-week exposure period. On the other hand, TSH levels were significantly increased in the highest dose group. TRH had no effect on the T4 level, but as might be expected, it caused an increase in the TSH levels both in the control group and in the bromide group.
With regard to the gonadotropic hormones LH and FSH there are a number of striking observations. In both series of experiments FSH increased significantly after exposure to a high level of bromide. This did not hold for LH. After 4 weeks a statistically significant decrease of the LH level in the highest dose group was seen compared to untreated animals, but this effect had disappeared after 12 weeks. The level of both LH and FSH was higher in the 4-week groups treated with 20- 1200 mg NaBr/kg diet than in those killed at 12 weeks. Since all animals were 3 weeks old at the beginning of the experiment, the 4-week groups were younger when killed than were the 12-week groups. With regard to the LH and FSH findings in the TRH test, it is surprising that only LH rises both in the control and in the bromide animals.
A statistically significant decrease in testosterone was observed after treatment with the 19200 mg NaBr/kg diet for 4 and 12 weeks. The results of the GH assay are difficult to interpret. The large variation within each dosage group may have been due to rapid fluctuations in GH secretion. Episodic bursts of GH secretion are responsible for the 10-20-folg increase in the GH concentration in the serum within 15-30 minutes. Nevertheless, it appears that with the highest dietary level of 19200 mg/kg there was a significant decrease after 12 weeks. Insulin levels were significantly increased by bromide treatment only in the highest dosage group but after 4 as well as after 12 weeks. Corticosterone showed a tendency to decline in the sodium bromide-treated rats, particularly in the highest dosage group.
Organ weights:
Compared to control animals, the group on the 19200 mg NaBr/kg diet showed growth retardation, which was noticeable after 4 weeks and more pronounced after 12 weeks. There was a distinct increase in the thyroid weight in the 1200 mg/kg group after 4 weeks but not after 12 weeks. In the 19200 mg/kg group, however, a statistically significant increase was observed after both 4 and 12 weeks.
Gross and Histopathology:
Pituitary gland:
No histopathological changes could be detected in the haematoxylin/eosin-stained sections of the pituitary glands of rats exposed to sodium bromide for 4 or 12 weeks. Using the immunoperoxidase techniques, selective immunocytochemical staining could be localized in different types of cell. Immunoreactive GH cells were seen to be scattered throughout the anterior pituitary gland, frequently forming small clusters or strings. The cytoplasm was strongly stained and the cells had round to oval shapes. The thyrotropic cells revealed with the anti-TSH serum appeared to be polygonal, and were mainly located in the central part of the anterior pituitary gland. The staining reaction was again strong. Many stellate cells with cytoplasmic processes lying between other cells were positive for ACTH, and were preferentially localized in the lateral part of the anterior lobe. No immunocytochemical reaction was observed when the anti-hormone sera were substituted to normal sera. Immunostaining procedures for GH, TSH and ACTH in the different types of cell in the anterior pituitary gland of the control rats and of animals treated with sodium bromide yielded different results. The average number of size of the immunoreactive cells and the intensitiy of the staining reaction in each procedure were first ascertained to serve as a base-line against which any change could be judged. In the pituitary gland of rats treated with 19200 mg NaBr/kg diet for 4 and 12 weeks, only a slight tendency towards less GH immunoreactivity was observed in comparison with the control animals. On the other hand there was distinctly more immunoreactivity for TSH and ACTH, but only after 12 weeks.
Thyroid:
After exposure of rats to 19200 mg NaBr/kg diet for four weeks, remarkable histopathological changes in thyroid were observed, characterized by an increase of follicles and a decrease in their size. The follicular epithelium was greatly heightened while the colloid was decreased in amount and more granular in appearance. This was also seen after an exposure time of 12 weeks. No marked changes in the histological picture of the thyroid were detected in the lower dosage groups, irrespective of the exposure time. Using the PAP (peroxidase-antiperoxidase) method, T4 was generally detected particularly in the follicular colloid but also in the follicular epithelium of normal rat thyroid tissue, but its distribution within the follicular colloid and the intensity of the reaction between follicles varied. In comparison with the reaction in the control animals, the follicles of the rats treated with 19200 mg NaBr/kg diet for 4 or 12 weeks were less intensely stained. Moreover, in the latter groups there was less variation in the intensity of the reaction within the follicular colloid.
Testes:
A decreased spermatogenesis and a reduction of tubule diameter were observed in the rats of the highest dosage group after 12 weeks of treatment. No immunocytochemical staining for testosterone could be achieved by the PAP method whatever fixation procedure was carried out. The spontaneous loss of testosterone from the Leydig cells during the fixation and embedding procedures is well recognized. - Conclusions:
- In conclusion it may be postulated that sodium bromide acts directly on certain endocrine organs such as the thyroid, adrenals and testes, thereby inducing alterations in the pituitary gland by feedback mechanisms.
LOAEL:
1200 mg NaBr/kg diet, corresponding to 93.6 mg bromide/kg bw/day based on a distinct increase in thyroid weight after four weeks of treatment.
NOAEL:
300 mg NaBr/kg diet, corresponding to 22.5 mg bromide/kg bw/day. - Executive summary:
Materials and Methods
The present study was initiated to ascertain whether alterations detected during a semichronic feeding study in rats (increase of relative weights of thyroid and adrenals and decrease in relative prostate weights; activation of the thyroid; decreased spermatogenesis) could be detected in male rats after exposure to high dietary concentrations of sodium bromide, and whether histopathological and immunocytochemical findings could be correlated with serum-hormone levels. Furthermore, a range of lower dietary concentrations of sodium bromide was studied to investigate whether a previously observed decrease in the serum-thyroxine level could be confirmed. Inbred male Wistar rats within a weight range of 60-100 g were used throughout. Sodium bromide was mixed with the diet to give final concentrations of 0, 20, 75, 300, 1200 and 19200 mg/kg (corresponding to about 0, 1.5, 5.63, 22.5, 90 and 1440 mg/kg bw/day based on a default conversion of 1 ppm = 0.075 mg/kg bw/day (mean of 0.05 mg/kg bw/day and 0.1 mg/kg bw/day for older and young rats, respectively)). Separate controls were used for the group on the latter diet. Each test and control group for each exposure period consisted of 10 rats. Diets and tap-water were available ad libitum. After 4 or 12 weeks, animals were decapitated and exsanguinated. After macroscopic inspection, the pituitary gland, thyroid and testes were weighed. Pituitary gland was fixed in 8 % (w/v) formaldehyde solution containing 4.5 % (w/v) mercuric chloride (sublimate) and 0.5 % (w/v) sodium chloride. Thyroid and testes were fixed in 4 % (w/v) formaldehyde in 0.067 M-Sörensen buffer, pH 6.9, and in Bouin Holland´s solution, respectively. Embedding was performed in paraplast and 5 µm sections were prepared. The sublimate was removed by treatment with lugol and sodium thiosulphate. The sections were stained with haematoxylin and eosin. Two immunoperoxidase techniques were used for the localization of the hormones. For thyroid-stimulating hormone (TSH), growth hormone (GH) and adrenocorticotropic hormone (ACTH), immunocytochemical staining was carried out in accordance with the indirect peroxidise-labelled antibody method using 3,3´-diaminobenzidine and hydrogen peroxidise as substrates for peroxidase. The antisera used were the following: rabbit antiserum to rat TSH, specific anti-rat-GSH serum prepared in monkey and specific anti-pig-ACTH serum prepared in rabbit. Peroxidase-labelled secondary antibodies were used for the detection of bound specific primary immunoglobulins. Immunocytochemical controls were done with normal rabbit and monkey sera. All sera were diluted in phosphate-buffered saline (PBS), pH 7.2. For thyroxine (T4) and testosterone, the unlabelled antibody method of peroxidase-antiperoxidase (PAP) was applied, using Immulok Histoset Immunoperoxidase Staining Kits with 3-amino-9-ethylcarbazole in N,N-dimethylformamid and hydrogen peroxide as substartes for the peroxidise reaction. Counterstaining with haematoxylin was sometimes performed. The hormone concentrations in the sera were measured by radioimmunoassay. For TSH, GH, luteinising hormone (LH) and follicle-stimulating hormone (FSH) determinations, materials supplied by NIAMDD were used. Iodination was carried out using the Chloramine-T technique with slight modification. For TH, LH and FSH, separation of antibody-bound and free hormone was achieved by addition of sheep-anti-rabbit-γ-globulin coupled to a solid phase. For GH, liquid-phase goat-anti-monkey- γ-globulin was used. Counting data were evaluated using a computer programme. Testosterone and corticosterone were measured I ethylene glycol diethyl ether and ethylene glycol-toluene extracts, respectively. For T4 determinations the Corning Immophase T4 Kit was used, slightly modified for rat serum. In a separate 12-week experiment, five animals receiving 19200 mg NaBr/kg diet and live control animals were submitted to a release test using thyrotropin-releasing hormone (TRH) in a dose of 1 µg/kg bodyweight. Five minutes after intravenous injection of the TRH the rats were decapitated and exsanguinated. Following clotting of the trunk blood, the serum was harvested and kept at -20°C prior to use for the hormone assay.
Results and Discussion
Compared to control animals, the group on the 19200 mg NaBr/kg diet showed growth retardation, which was noticeable after 4 weeks and more pronounced after 12 weeks. There was a distinct increase in the thyroid weight in the 1200 mg/kg group after 4 weeks but not after 12 weeks. In the 19200 mg/kg group, however, a statistically significant increase was observed after both 4 and 12 weeks. No histopathological changes could be detected in the haematoxylin/eosin-stained sections of the pituitary glands of rats exposed to sodium bromide for 4 or 12 weeks. Using the immunoperoxidase techniques, selective immunocytochemical staining could be localized in different types of cell. Immunoreactive GH cells were seen to be scattered throughout the anterior pituitary gland, frequently forming small clusters or strings. The cytoplasm was strongly stained and the cells had round to oval shapes. The thyrotropic cells revealed with the anti-TSH serum appeared to be polygonal, and were mainly located in the central part of the anterior pituitary gland. The staining reaction was again strong. Many stellate cells with cytoplasmic processes lying between other cells were positive for ACTH, and were preferentially localized in the lateral part of the anterior lobe. No immunocytochemical reaction was observed when the anti-hormone sera were substituted to normal sera. Immunostaining procedures for GH, TSH and ACTH in the different types of cell in the anterior pituitary gland of the control rats and of animals treated with sodium bromide yielded different results. The average number of size of the immunoreactive cells and the intensitiy of the staining reaction in each procedure were first ascertained to serve as a base-line against which any change could be judged. In the pituitary gland of rats treated with 19200 mg NaBr/kg diet for 4 and 12 weeks, only a slight tendency towards less GH immunoreactivity was observed in comparison with the control animals. On the other hand there was distinctly more immunoreactivity for TSH and ACTH, but only after 12 weeks. After exposure of rats to 19200 mg NaBr/kg diet for four weeks (corresponding to 1440 mg/kg bw/day), remarkable histopathological changes in thyroid were observed, characterized by an increase of follicles and a decrease in their size. The follicular epithelium was greatly heightened while the colloid was decreased in amount and more granular in appearance. This was also seen after an exposure time of 12 weeks. No marked changes in the histological picture of the thyroid were detected in the lower dosage groups, irrespective of the exposure time. Using the PAP (peroxidase-antiperoxidase) method, T4 was generally detected particularly in the follicular colloid but also in the follicular epithelium of normal rat thyroid tissue, but its distribution within the follicular colloid and the intensity of the reaction between follicles varied. In comparison with the reaction in the control animals, the follicles of the rats treated with 19200 mg NaBr/kg diet for 4 or 12 weeks were less intensely stained. Moreover, in the latter groups there was less variation in the intensity of the reaction within the follicular colloid. A decreased spermatogenesis and a reduction of tubule diameter were observed in the rats of the highest dosage group after 12 weeks of treatment. No immunocytochemical staining for testosterone could be achieved by the PAP method whatever fixation procedure was carried out. The spontaneous loss of testosterone from the Leydig cells during the fixation and embedding procedures is well recognized. Nevertheless, further studies to demonstrate testosterone in the rat testis will be performed. There was a statistically significant decrease of T4 both after the 4- and the 12-week treatment with the 19200 mg NaBr/kg diet. Also in the 1200 mg/kg group, the T4 level was significantly reduced after the 4-week exposure period. On the other hand, TSH levels were significantly increased in the highest dose group. TRH had no effect on the T4 level, but as might be expected, it caused an increase in the TSH levels both in the control group and in the bromide group. With regard to the gonadotropic hormones LH and FSH there are a number of striking observations. In both series of experiments FSH increased significantly after exposure to a high level of bromide. This did not hold for LH. After 4 weeks a statistically significant decrease of the LH level in the highest dose group was seen compared to control animals, but this effect had disappeared after 12 weeks. The level of both LH and FSH was higher in the 4-week groups (20 -1200 mg NaBr/kg) than in those killed at 12 weeks. Since all animals were 3 weeks old at the beginning of the experiment, the 4-week groups were younger when killed than were the 12-week groups. Similar differences in LH and FSH levels in the sera of animals of these ages were observed before. With regard to the LH and FSH findings in the TRH test, it is surprising that only LH rises both in the control and in the bromide animals. Since FSH does not change, contamination of the TRH preparation with the LH/FSH-releasing hormone (LHRH) can be excluded. There may be two explanations for this phenomenon: (a) the observed rise of LH is due to cross-reacting TSH, although the antiserum is supposed to be specific for LH. Nevertheless, pituitary hormones have been shown to exist in different molecular forms and it is possible that the TSH form released by the pituitary gland after TRH treatment is different from that released after sodium bromide treatment. (b) extra handling stress is caused by the injection of TRH. It is well known that acute stress affects the level of a number of hormones, e.g. ACTH and GH. LH does not seem to be very sensitive in this respect, but this possibly cannot be excluded completely. In this respect the slight elevation of the corticosterone levels in both the control and the bromide groups after TSH injection should be noted. A statistically significant decrease in testosterone was observed after treatment with the 19200 mg NaBr/kg diet for 4 and 12 weeks. The results of the GH assay are difficult to interpret. The large variation within each dosage group may have been due to rapid fluctuations in GH secretion. Episodic bursts of GH secretion are responsible for the 10-20-fold increase in the GH concentration in the serum within 15-30 minutes. Nevertheless, it appears that with the highest dietary level of 19200 mg/kg there was a significant decrease after 12 weeks. Insulin levels were significantly increased by bromide treatment only in the highest dosage group but after 4 as well as after 12 weeks. Corticosterone showed a tendency to decline in the sodium bromide-treated rats, particularly in the highest dosage group. In the present study attention was focused mainly on the pituitary gland, the thyroid and the testes. Several sites of action of sodium bromide can be distinguished. One of them is a direct effect on the thyroid. After treatment with sodium bromide, T4 production was hampered, as evidenced by immunocytochemistry and radioimmunoassay. Due to feedback regulation the pituitary gland was stimulated to produce and release TSH. In the TRH experiment the increase of TSH in the control group and the bromide group were of similar magnitude. However, only for the control group was this increase statistically significant. From this it might be concluded that in bromide-treated animals the pituitary gland has little capacity for releasing even more TSH. Marked histopathological and immunocytochemical changes in the thyroid tissue were induced, indicative of a typical hypothyroidism. In a hitherto unresolved way, at least two other hormones are affected by changes in the T4 level, GH and insulin. The observation that a decrease in T4 concentration was accompanied by a decrease in GH concentration in the serum is in agreement with similar findings reported previously. Moreover, T4 was shown to exert a diabetogenic effect. It is, therefore, conceivable that a decrease in the T4 level results in an increase in the insulin level. The changes in serum levels of both GH and insulin are in accord with the observed growth retardation of the rats. Finally, since GH may act as a thymotropic hormone, a decrease in GH level may also be connected with the reduction in relative thymus weight found earlier. The effect of sodium bromide on the adrenals seems to parallel that on the thyroid. A decrease in the corticosterone level in the serum, as assessed by radioimmunoassay, results in an increase in the production of ACTH by the pituitary gland, as found by immunocytochemical staining. Also the testes are affected by sodium bromide. Histopathological findings now and in the past have shown an inhibition of spermatogenesis indicating a deterioration of the Sertoli-cell function. It has been shown that the Sertoli cell produces a substance called inhibin, which hampers FSH release by the pituitary gland. This may be an explanation for the rise in FSH level observed in the bromide-treated rats. In addition, the Leydig cells may be affected, resulting in a decreased production of testosterone and consequently in a loweing of the secretory activity of the prostate. An expected rise in LH level triggered by the decreased testosterone level could not be detected. Rather, after 4 weeks a slight fall was observed. It is conceivable that the damage caused in the Leydig cells is small compared to that in the Sertoli cells, thus giving rise to changes in FSH but not in the LH levels.
- Endpoint:
- endocrine system modulation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1982
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: there is no guideline available for this special mechanistic investigation
- Qualifier:
- no guideline available
- Deviations:
- not applicable
- Remarks:
- no guideline available for this special mechanistic investigation
- Principles of method if other than guideline:
- Study was performed according to good experimental practice.
- GLP compliance:
- no
- Type of method:
- in vivo
- Endpoint addressed:
- not applicable
- Species:
- human
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- 21 human volunteers were subdivided into weight classes of 10 kg. All persons from one weight class were administered the same daily amount of bromide (1 mg/kg) as NaBr in capsules for 8 weeks or two full cycles.
- Route of administration:
- oral: capsule
- Vehicle:
- other: test substances was administered as capsules.
- Details on exposure:
- DIET PREPARATION
- The experimental diets had a normal sodium chloride level, since plasma half-life of bromide is related to the chloride content of the food. - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 8 weeks or two full cycles
- Frequency of treatment:
- daily
- Post exposure period:
- No postexposure period.
- Remarks:
- Doses / Concentrations:
1mg/kg bw
Basis:
actual ingested - No. of animals per sex per dose:
- 21 volunteers were subdivided into weight classes of 10 kg. All persons from one weight class were administered the same daily amount of bromide (1 mg/kg) as NaBr in capsules.
- Control animals:
- no
- Examinations:
- Blood was analysed for haemoglobin concentration, haematocrtit, MCV (mean erythrocyte volume), MCH (mean corpuscular haemoglobin), MCHC (mean corpuscular haemoglobin concentration), leucocyte concentration, differential white cell count and thrombocyte concentration.
Furthermore, the following biochemical parameters were investigated: urea, creatinine, alkaline phosphatase, γ-glutamyl transpeptidase, glutamic oxalacetic and glutamic pyruvic transaminase, lactic dehydrogenase and protein. Urinalysis was performed for glucose, albumin and sediment. - Details on results:
- General aspect:
The medical histories and physical examinations revealed a few relevant findings. In one female a cyst had been removed from the right ovary two years before the investigation started. In two males a cryptochidism operation had been performed when they were 10 and 13 years of age respectively. In these three persons neither physical examination nor endocrine analyses disclosed abnormalities. Five females had used oral contraceptives during 3 month to 2 years in the past. The use of these medicines had been stopped at least 6 months before the start of the experiment. Seven persons had an atopic constitution, one had eczema. Acne vulgaris was detected in 3 volunteers.
Two persons out of the group of 8 with an atopic and allergic constitution developed dermal changes during the experiment. On the third day of the experiment one female developed itching erythematous spots and slightly elevated vesicles with a diameter of 1 mm on the face, neck, arms and upper half of the chest. She reported these changes 4 days after they were first noticed; by that time the lesions were already disappearing. After another 4 days no abnormalities were observable. A second female developed similar dermal abnormalities on the first day of NaBr ingestion. She preferred not to participate in the experiment further. In a few days the abnormalities disappeared. One other female withdrew because of reasons not related to the experiment.
The results of the medical histories and the physical examinations at the end of the experiment were identical with those at the beginning.
The results of all haematological, biochemical and urine analyses at the start and at the end of the experiment were within normal limits and no significant changes were observed.
The mean plasma bromide concentration at the start of the experiments was 0.08 ± 0.01 mmol/L in females and males. At the end of the experiment the mean plasma bromide concentration in females was 0.97 ± 0.18 mmol and in males 0.83 ± 0.09 mmol/L. The rise in the bromide concentration was not gradual in all individuals. In some individuals slight decreases were observed between the 4th and 6th or 6th and 8th week. During the experiment the total plasma concentration of halide ions remained constant (for details please refer to Table 6.10/03-1).
The mean bromide excretion at the start of the experiment was 9.8 ± 2.9 mg NaBr/24 hours/person in females and in males 9.5 ± 4.2 mg NaBr/24 hours/person. At the start of the experiment the mean chloride excretion in females was 9.8 ± 2.9 g NaCl/24 hours and in males 8.5 ± 2.0 g NaCl/24 hours. This corresponds well with the usual sodium chloride consumption in the Netherlands. The mean sodium chloride excretion in females and males at the end of the experiment did not differ from the excretion at the start, although large individual differences were measured. In these individuals only slight differences in the creatinine excretion in the urine of 24 hours were observed.
At the end of the experiment the mean bromide excretion in females was 90.6 ± 30.2 mg NaBr/24 hours. The mean daily dose administered was 85 ± 10 mg NaBr. In males the mean bromide excretion was lower than the daily administered dose: mean excretion was 79.7 ± 18.5 mg NaBr/24 hours, administered dose was 95 ± 9 mg NaBr. This may indicate that a steady state had not yet been reached in males (for details please refer to Table 6.10-03/2).
Endocrine system:
Concentrations of T4, fT4, TBG and T3 were determined in serum at the start and at the end of the experiment and no significant differences were observed between the values.
Furthermore, no differences were observed in serum concentrations of cortisol, testosterone, estradiol, progesterone, TSH, prolactin, LH, FSH when comparing the levels at the start and at the end of the experiment.
The response to administration of TRH and LHRH was within normal limits and no differences were observed at the start and at the end of bromide administration. - Conclusions:
- No biological effect could be established in any of the parameters investigated in human volunteers who ingested daily 1 mg bromide/kg bw during 8 weeks or 2 full cycles.
Presumably a difference exists between rat and man regarding the effects of and susceptibility to sodium bromide. Therefore the results from animal experiments in rodents may have limited relevance to man when used as a basis for an “acceptable daily intake” for bromide. - Executive summary:
Materials and Methods
Inorganic bromide-containing compounds have been widely used in medicine for more than 100 years as a hypnotic or sedative. The used decreased because of the availability of more selective and more effective medicines. Toxicity of bromide in therapeutic dosages is low.
The study was performed to determine whether ingestion of a dose of bromide equal to the acceptable daily intake might induce effects in humans. Bromide was administered at concentrations of 1 mg/kg as sodium bromide to 21 healthy volunteers (11 females not using oral contraceptives and not pregnant; 10 males) during 8 weeks or 2 full cycles. Special attention was paid to the endocrine system because endocrine changes were predominant in rats receiving sodium bromide in their diets. The experimental diets had a normal sodium chloride level, since plasma half-life of bromide is related to the chloride content of the food.
At the start and at the end of the investigation a full medical history was obtained and physical examination performed. In addition, venous blood samples were obtained for the haematological and clinical chemical analyses.
At start, every 14 days and at the end of the investigation, serum electrolytes and plasma bromide concentration were determined.
During physical examination special attention was paid to those endocrine organs accessible to this method of investigation. Serum concentration of the following hormones were determined at start and at the end of the study: thyroxine (T4), free thyroxine (fT4), thyroxine binding globulin (TBG), triiodothyronine (T3), cortisol, testosterone, estradiol, progesterone.
Radioimmunassay was performed at beginning and end of the investigation to determine prolactin, tyrotrophin, luteinising hormone and follicle stimulating hormone before and 20 and 60 minutes after i.v. administration of 200 µg thyrotrophin releasing hormoe and 100 µg luteinising hormone releasing hormone.
Statistical comparison on initial and final results was performed using Student`s t-test.
Results and Discussion
The purpose of the study was to determine whether administration of bromide in a dose equal to the acceptable daily intake allocated by the WHO (i.e. 1 mg/kg bw/day) might induce an effect in healthy human volunteers taking their normal diet. Special attention was paid to the endocrine system, because in rats, plasma bromide concentrations similar to the therapeutic range in man induced endocrine changes particularly in the thyroid gland. After daily ingestion of 1 mg bromide/kg during 8 weeks the mean plasma bromide concentration rose to 0.97 ± 0.18 mmol/L in females (corresponding to 4.6 mg/kg bw, with a mean bodyweight of 67.4 kg and 4 L of blood) and to 0.83 ± 0.09 mmol/L in males (corresponding to 3.9 mg/kg bw, with a mean bodyweight of 67.4 kg and 4 L of blood). These concentrations are about 10% of the therapeutic plasma concentration of 6-12 mmol/L and also about 10% of the mean plasma concentration in rats of 7.7 ± 1.1 mmol/L in which effects were observed.
If all persons consumed a daily diet containing a more or less constant amount of bromide and chloride it might be assumed that they were in a steady state as far as absorption and elimination of total halide ions are concerned. Based upon this premise, bromide excretion at the start of the experiment reflects the average daily bromide intake. This corresponds well with the mean bromide content measured in 24-h duplicate meals which was 9.75 mg bromide. The mean bromide excretion at the end of the experiment did not differ significantly from the administered dose.
Plasma half-life of bromide in man is about 12 days. It was, therefore, to be expected that at the end of the experiment the plasma bromide concentration would have reached a steady state during only the last week. The course of the plasma bromide concentration suggests that this might have happened. On the other hand it may well be possible that, particularly in males, no steady state had been reached yet.
The mean chloride excretion in the urine indicates that the consumption of sodium chloride on average was constant during the experiment. However, rather large individual differences were observed. Possibly, changes in individual daily sodium chloride consumption may explain the decrease in plasma bromide concentration that occurred in some persons in the course of the experiment. Another explanation is that the capsules had not been taken regularly. Since the experiment was not performed under metabolic balance circumstances the results are not suitable for further kinetic interpretations.
The results of the medical histories, the physical examinations and the haematological, biochemical and urine analyses after completion of the experiment showed no abnormalities when compared to pre-experimental values. In two females a short lasting itching dermatosis with small vesicles was observed at the beginning of the experiment which was shown to be reversible. A relation between these dermal changes and bromide ingestion seems highly improbable.
Daily ingestion of 1 mg bromide/kg bw did not change the serum concentration of any of the hormones measured, produced by thyroid, adrenals, gonads and pituitary gland. The response of the pituitary upon administration of TRH and LHRH was not influenced during the experiment.
The results of the investigation, no influence of bromide on the endocrine system in humans, fit well with other investigations. Bromide concentrations comparable to the ones used in diverse rat studies were used for investigations in dogs. Like in humans, no influence on thyroid morphology could be detected and changes in thyroid hormone concentrations appeared in the control animals as well. In addition sodium bromide is used as anticonvulsant in dogs with serum concentrations of 10-20 mmol bromide/L. Regarding the thyroid gland, diverse functional and morphological differences exist between humans and rodents. The most important finding is that rodents lack the thyroid binding globulin (TGB) which is the predominant plasma protein that binds and transports thyroid hormone in the blood in humans. Thyroxine (T4), therefore, is mainly protein bound in humans and free in the blood in rodents. For dogs the same is true as stated for man. This might be one of the reasons why rodents respond in a more sensitive manner to bromide ions compared to dogs. Another point is the half-life of T4, which is 12 hours in the rat compared to 5-9 days in humans; serum TSH is 25-times higher in the rodent as compared to man. These findings indicate a much higher functional activity in the rodent thyroid gland as compared to the primate, a conclusion also supported by the histological appearance of the thyroid. In primates, the follicles are uniformly large with abundant colloid. In contrast, the rodent thyroid has large follicles only in the periphery of the gland; the interior is composed of comparatively small follicles with small amounts of colloid. These species differences make it difficult to calculate a possible risk for humans from animal data.
In addition to this, the present investigation showed that a daily intake of 1 mg bromide/kg bodyweight did not influence the endocrine system in humans nor resulted in other effects regarding haematological, biochemical and urine analyses or physical examinations.
- Endpoint:
- endocrine system modulation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1993
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: The publication investigated mechanistic properties of the test substance and no guideline exists for the conduction of such studies
- Qualifier:
- no guideline available
- Deviations:
- not applicable
- Remarks:
- no guideline available for this special investigation
- Principles of method if other than guideline:
- study was performed according to good experimental practice
- GLP compliance:
- no
- Type of method:
- in vivo
- Endpoint addressed:
- repeated dose toxicity: oral
- Species:
- human
- Sex:
- female
- Details on test animals or test system and environmental conditions:
- HUMAN VOLUNTEERS
The study was performed on 48 healthy female volunteers aged 20-28 years. Because 3 volunteers withdrew, only 45 subjects completed the study: 15 subjects in every dosage group - Route of administration:
- oral: capsule
- Vehicle:
- other: test material was applied as capsules
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- The experiment covered six menstrual cycles. During the first three cycles females were treated at 0, 4 or 9 mg/kg bw/day with sodium bromide capsules and during the remaining cycles no sodium bromide was administered.
- Frequency of treatment:
- daily
- Post exposure period:
- 3 cycles
- Remarks:
- Doses / Concentrations:
0, 4, 9 mg/kg bw/day
Basis:
actual ingested - No. of animals per sex per dose:
- 15 females per dose group
- Control animals:
- yes, concurrent no treatment
- Examinations:
- Routine blood tests and a full physical examination were performed before and after bromide administration. Concentration of bromide was determined once each cycle. Thyroxine, free thyroxine, thyroxine-binding globulin, triiodothyronine and thyrotropin were determined before and at the end of the study. The activity of the central nervous system was evaluated before, in the middle and at the end of the study by physical examination and the recording of a quantitative EEG using three different montages (monopolar fronto-occipital, bipolar fronto occipital and bipolar transversal). Besides, the Visual Evoked Response (VER) was recorded using standard clinical procedures.
- Details on results:
- General:
Three volunteers withdrew from the study. Two of them before the start, one due to unrelated medical problems. The third female withdrew after 21 days of ingestion of the capsules because of skin effects not related to the experiment or bromide in particular. Another volunteer developed skin effects after 5 days of ingestion, continued the participation. The skin effects disappeared spontaneously within a couple of days and were apparently not related to bromide. Thus, 45 subjects completed the study: 15 subjects in every dosage group.
The medical history and physical examination before and after the experiment showed no irregularities besides complaints of nausea shortly after the ingestion of the capsules. In none of the volunteers of the control group nausea in relation to ingestion of the capsules was observed. Three volunteers of the 4 mg bromide/kg bw/day group and 11 of the 9 mg/kg bw/day group complained of nausea, which is a known effect of bromide. Statistical analysis reveals that both at 4 mg/kg and at 9 mg/kg nausea is likely to have been caused by the administration of bromide (P<0.0001 for all groups). After three cycles one subject of each of the groups complained about drowsiness and vertigo. Another three of each group complained about sleepiness.
The routine blood tests before and after the study showed no abnormalities.
Bromide:
The plasma bromide concentration did not differ between the three groups before the start of the experiment. In the control group the plasma bromide concentration did not change during the experiment. In the group treated at 4 mg/kg bw/day, the bromide concentration rose to 3.22 ± 0.93 mmol/L and in the 9 mg/kg bw/day group to 7.99 ± 1.89 mmol/L after the administration of bromide for 3 full cycles. At the end of the experiment the bromide concentration did not differ between the three groups.
Thyroid:
The concentrations of T4, fT4, TBG, T3 and TSH were within the normal limits of the used detection methods at each point of time (start of experiment, after 3 and 6 cycles). For none of the variables significant differences have been found between the dosage groups.
Central nervous system:
No neurological differences were found on clinical neurological examination at three test moments.
The interindividual variability of the quantitative EEG parameters was within normal limits for healthy subjects. The individual variability was rather small when care was taken to minimize attention deficits during recording and the subject was at all test moments recorded at the same time of day to minimize diurnal variation.
On visual inspection of the EEG, no effect was visible. The non-systemic differences found could all be related to variability of attention during recording.
Quantitative analysis of the spectral values showed that the significant decrease in activity in the δ-band, found in the previous study, did not attain the required level of significance in the current investigation. However, a significant effect was found in the α1-band in the majority of the channels, albeit with a weak level of significance (P<0.1). This, however, persisted when comparing the first test moment to the second as well as when comparing the third moment to the second. The same results were found for the β-bands. These effects were observed both at doses of 4 and 9 mg/kg bw/day.
Correlation analysis indicated a relation between blood bromide levels and the significant EEG-parameters, however, also with rather weak levels of significance (P<0.09 to P<0.01).
The VER (visual evoked response) did not display significant effects of sodium bromide. - Conclusions:
- This investigation was performed to confirm the results obtained in a previous study with healthy male and female volunteers. The results of this study demonstrated that females represented a more sensitive subpopulation.
Based on the results obtained in the present investigation, bromide was found to be the cause of the nausea in both dose groups (4 and 9 mg).
No effects on clinical pathology and on the function of endocrine glands, especially the thyroid function were observed. There was an effect in the quantitative EEG the significance of which was considered to be borderline. Most interestingly, the direction and type of changes was opposite to those observed in the previous investigation. The most plausible explanation is that the changes in the quantitative EEG caused by sodium bromide are very small in both dosages used in this study and therefore easily obscured by the individual variability. It presumably indicates that the borderline effects of sodium bromide on the central nervous system are buffered between relatively broad limits where the upper level may be over 9 mg/kg and the lower at approximately 4 mg/kg.
The ADI for sodium bromide, as determined in 1988, is based on recent animal as well as human experiments. In the establishment of this ADI the data of healthy volunteer experiments were decisive. It can therefore be concluded that solid data from healthy volunteer experiments influence the setting of an ADI to a great extent.
Based on the results of obtained in this present investigation and considering the borderline relevance and not consistent change in neurophysiological parameters, the NOEL and the NOAEL of this investigation is derived at 4 and 9 mg/kg bw/day, respectively. The NOAEL of 9 mg/kg bw/day serves as the point of departure for the deduction of the ADI which is derived at about 1 mg/kg bw/day applying an assessment factor of 10 to the NOAEL. Since the NOAEL is based on human data and derived from a more sensitive subpopulation, the assessment factor of 10 is considered to be appropriate and to provide a sufficient level of safety. - Executive summary:
Materials and Methods
The investigation was performed to study if the findings made in female volunteers during a previous investigation on the effect of bromide on humans could be reproduced. From this study a NOAEL of 4 mg/kg bw/day was established with minor changes in the thyroid hormone concentration in females treated at 9 mg/kg bw/day and some effects on the central nervous system in females and males taking the same daily dose. The effects seen at 9 mg/kg bw/day were within the normal variance but differed from the results of the examination at study start.
In the present investigation female volunteers were observed for changes in thyroid hormone concentration over 6 menstrual cycles, where during the first three cycles bromide was administered in doses of 0, 4 or 9 mg/kg bw/day as sodium bromide capsules. Volunteers were divided in groups according to their bodyweight; within one group each subject ingested the same amount of sodium bromide.
Bromide concentration in plasma was determined once each cycle.
Before and at the end of the experiment, thyroxine (T4), free thyroxine (fT4), thyroxine-binding globulin (TBG), triiodothyronine (T3) and thyrotropin (TSH) were determined.
The activity of the central nervous system was evaluated before, in the middle and at the end of the study by physical examination and the recording of a quantitative EEG, the latter by using 3 different montages (monopolar fronto-occipital, bipolar fronto-occipital and bipolar transversal), each consisting of 12 channels.
For the blood sample parameters, an analysis of variance was performed on the variables recorded. Further statistic analysis was performed with one-sided analysis of variance of the logartithms of the variables. For the quantitative EEG parameters a multivariante analysis was performed on the power values of the Matousek bands for each montage over the channels; all of these tests were two-sided.
Results and Discussion
The rationale to perform the present experiment was to investigate whether the results of the previously performed investigation with sodium bromide in human volunteers could be confirmed. The present experiment with sodium bromide showed effects on the physiological condition of the volunteers and on their EEG. Female volunteers were observed for changes in thyroid hormone concentration over 6 menstrual cycles, where during the first three cycles bromide was administered in doses of 0, 4 or 9 mg/kg bw/day as sodium bromide capsules. The nausea can be considered as an effect of the relatively large amount of bromide administered in one capsule to the stomach and is certainly not a systemic effect. When exposure to bromide is caused by food products, the bromide will be consumed during the day in small amounts and it is unlikely that nausea will occur.
In the previous study a significant decrease was found in the power of both δ- bands (δ1 and δ2) with an increase in the activity in the β-band. In that study changes in the α1-band were found only in the 4 mg/kg bw/day group, not attaining detection levels in the 9 mg/kg bw/day group. In the current study, the situation was reversed: the activity in the δ- bands did not reach detection levels while changes in the α1-band were significant in both groups. The most plausible explanation is that the changes in the quantitative EEG caused by sodium bromide are very small in both dosages used and therefore easily obscured by the individual variability. It presumably indicates that the borderline effects of sodium bromide on the central nervous system are buffered between relatively broad limits where the upper level may be over 9 mg/kg and the lower at approximately 4 mg/kg. It is therefore better to refer to borderline effect levels instead of no-effect-levels, due to the sensitive nature of these detection methods.
In the present study an increase in the concentration of T4 and T3, observed in the previous experiment, could not be confirmed. The former study had shown significant increases in T4 and T3, although all changes remained within normal limits.
From the results from the experiments with sodium bromide at dose levels of 0, 4 and 9 mg bromide/kg bw/day in healthy volunteers, it can be concluded that bromide can be considered the cause of the nausea in both dose groups (4 and 9 mg). Sodium bromide does not have effects on the function of endocrine glands, especially the thyroid function in man. Quantitative analysis of the EEGs shows differences at doses of 4 and 9 mg/kg in the present experiment. In the previous experiment significant differences were only observed at a dose of 9 mg sodium bromide/kg. For the reasons given above, the effects observed at a dose level of 4 mg/kg can be considered to be coincidental.
The aim of the series of studies was to determine the no-effect-level of sodium bromide in healthy subjects. Based on the results obtained during the present investigation and the earlier studies, it can be concluded that the only systemic changes found in the variables analysed were observed in the quantitative EEG. With treatment at 4 as well as 9 mg/kg bw/day, group reversible changes have been demonstrated, albeit at a weak level of significance. As has been stated, due to the sensitive nature of the method it is better to observe borderline effects instead of no-effect-levels. The borderline being defined as those levels at which the effects are equal and rather weak. Therefore it can be concluded that based on the EEG parameters, 9 mg/kg bw/day is within this borderline effect level and can be taken as a save dose for the calculation of ADI values.
The previous study gave, with regards to the thyroid function, an indication of increased activity, which, however, could not be confirmed in the present investigation. This affirms the conclusion that in practical terms 4 mg/kg bw/day can be considered as a no-effect level, taking into account the considerations mentioned above. This is in agreement with the conclusions from the Joint Meeting WHO/FAO on Pesticides Residues in 1988 where a new ADI (acceptable daily intake) was determined based on both animal and human experiments. This new ADI of 1 mg/kg bw/day was based on a no-observed adverse effect level for rats of 12 mg/kg bw (240 ppm) and on a NOAEL for humans of 9 mg/kg bw, based on the bromide studies in healthy volunteers as described here.
A NOAEL is used to establish an ADI. In the extrapolation from a NOAEL determined by animal experimentation, a factor of 100 is used, consisting of two (sub)factors: a factor of 10 for extrapolation from animal to human (interspecies variation correction factor) and another factor of 10 to account for intraspecies variability in man. In the case of a NOAEL obtained from healthy volunteer research, it seems warranted to use a safety factor of ten in the assessment of an ADI. This would be in agreement with the WHO-FAO conclusions, where a factor of 9 was used probably because it seemed most practical to set the ADI at 1 mg/kg bw/day. Moreover, a factor of nine seems adequate, considering the fact that the experiment described here was performed in healthy female volunteers who were more sensitive to bromide than males. Because the NOAEL has been determined in a more sensitive subpopulation the extrapolation factor could have been smaller, for example a factor of five for the extrapolation from a sub-chronic experiment to life-time exposition. The ADI would then have been 9/5 = 1.8 mg/kg bw/day. However, applying the precautionary principle, an assessment factor of 10 seems appropriate resulting in a tolerable daily intake of about 1 mg/kg bw/day which is consistent with the ADI derived by WHO-FAO.
- Endpoint:
- specific investigations: other studies
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 4 (not assignable)
- Principles of method if other than guideline:
- other
- GLP compliance:
- no
- Species:
- mouse
- Strain:
- Swiss Webster
- Sex:
- male
- Route of administration:
- intraperitoneal
- Duration of treatment / exposure:
- Exposure period: 30 hour(s)
- Frequency of treatment:
- single dose
- Remarks:
- Doses / Concentrations:
0, 0.2, 0.5, 1.0 and 2.0 g/kg
Basis: - No. of animals per sex per dose:
- 120
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- Type: other
Observation period: hourly for the first 14 hours, then every 2 hour for a further 16 hours - Details on results:
- Administration of NaBr 0.2 to 2.0 g/kg i.p. suppressed the convulsions in a dose-related fashion. Bromide was effective at subsedative doses. However, bromide increased the postwithdrawal mortality.
- Endpoint:
- specific investigations: other studies
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 4 (not assignable)
- Principles of method if other than guideline:
- other
- GLP compliance:
- no
- Species:
- dog
- Strain:
- other: mongrel dogs
- Sex:
- male
- Route of administration:
- other: oral
- Vehicle:
- other: sodium bromide was dosed in gelatin capsules
- Duration of treatment / exposure:
- Exposure period: 5 day(s)
- Frequency of treatment:
- daily
- Remarks:
- Doses / Concentrations:
1.13 g/day
Basis: - No. of animals per sex per dose:
- 4
- Details on study design:
- Type: other
Observation period: 5 days - Details on results:
- Two days after dosing the mean (± SE) serum level of bromide was 28.0 ± 4.0 mEq/L and the serum chloride level had decreased from a pre-treatment value of 112.5 ± 1.0 mEq/L to 86.5 ± 3.7 mEq/L.
In the basal prostatic secretion, the mean prostastic fluid to secretion (PF/S) ratios for bromide and chloride were 0.56 ± 0.15 and 0.53 ± 0.11, respectively, and were not different (P > 0.05).
At higher rates of secretion provoked by intravenous pilocarpine the corresponding PF/S ratios of 1.48 ± 0.04 and 1.32 ± 0.01 were significantly different (P < 0.05).
It is conclulded that the processes involved in forming the basal and pilocarpine induced prostatic secretions must differ and that the ability of the chloride-transporting system to transport bromide is slightly greater than that for chloride. Because it may impair sperm motility, bromide secreted in prostatic fluid potentially could adversely affect reproduction.
- Endpoint:
- endocrine system modulation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1983
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: there is no guideline available for this special mechanistic investigation
- Qualifier:
- no guideline available
- Deviations:
- not applicable
- Remarks:
- no guideline available for this special mechanistic investigation
- Principles of method if other than guideline:
- study was performed according to good experimental practice
- GLP compliance:
- no
- Type of method:
- in vivo
- Endpoint addressed:
- not applicable
- Species:
- human
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- HUMAN VOLUNTEERS
- All volunteers were divided into weight classes of 10 kg. Females and males were randomly divided into groups. A quantitative analysis of the EEG and the evoked response at the start and end of the investigation was performed - Route of administration:
- oral: capsule
- Vehicle:
- other: test substance was administered as capsules
- Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- three month or three full cycles
- Frequency of treatment:
- daily
- Post exposure period:
- no postexposure period
- Remarks:
- Doses / Concentrations:
0, 4 and 9 mg bromide/kg bw/day
Basis:
actual ingested - No. of animals per sex per dose:
- Volunteers were divided into weight classes of 10 kg. Females and males were randomly divided into groups.
- Control animals:
- yes, concurrent no treatment
- Examinations:
- A quantitative analysis of the EEG and the evoked response at the start and end of the investigation was performed to obtain information on the functioning of the CNS. For the spontaneous EEG three different montages were used, each consisting of 12 channels. For each montage, 150 seconds were recorded. The visual evoked response (VER) was recorded with eight channels. From the same signal were calculated the Hjorth parameters (time-domain) indicating mean power, mean frequency and band width.
Haematological and biochemical analyses were performed at the beginning and at the end of the investigtation. Haemoglobin concentration, haematocrit, MCV, MCH, MCHC, leucocte concentration, differential white cell count, thrombocyte concentration (in blood), urea, creatinine, sodium, potassium, chloride, alkaline phosphatase, γ-glutamyl transpeptidase, glutamic-oxalacetic and glutamic-pyruvic transaminases, lactic dehydrogenases, protein (in serum), glucose and albumin (in urine) were determined. - Details on results:
- General aspects:
The medical histories obtained and the physical examinations made during the selection revealed no findings relevant for the investigation. Eight females had used oral contraceptives in the past, bit this use had been stopped at least 6 months before the start of the study.
One female volunteer dropped out, because of an intercurrent illness while another appeared in the postovulatory phase of her cycle due to a misunderstanding. Results of two other female volunteers were excluded because of the development of intercurrent gynaecological pathology not related to the investigation. Therefore, the results of only four females in the group receiving 4 mg bromide/kg bw/day and only 6 in the group receiving 9 mg bromide/kg bw/day have been evaluated.
The results of the medical histories and physical examinations at the end of the investigation were identical with those at the time of the study initiation except for the occurrence of nausea and mention of a decrease in concentration and an increased need of sleep. Two subjects receiving 4 mg bromide/kg bw/day noticed some nausea on several consecutive days after ingestion of the capsules. The symptom disappeared when the capsules were taken during the meal. This occurred as well in five persons in the group ingesting 9 mg bromide/kg bw/day. A decreased ability to concentrate and an increase in sleepiness was mentioned by five male volunteers receiving 4 mg bromide/kg bw/day and by one female and one male volunteer taking 9 mg/kg bw/day.
In contrast to the examination at the start of the experiment, one male volunteer showed a granulocytopenia at the end of the investigation. The performance of several additional measurements suggested that he had a cyclic neutropenia. No changes in the haematological variables were demonstrated during the investigation in the other volunteers.
The results of the serum biochemical and urine analyses at the start and at the end of the study were within normal limits and no significant changes were observed except that three subjects showed an increase in the concentraton of γ-glutamyl transpeptidase. In two of them, this appeared to be related to the consumption of ethanol-containing beverages.
Bromide:
The mean plasma bromide concentration in all subjects ranged between 0.06 ± 0.01 and 0.08 ± 0.01 mmol/L at the start of the investigation. At the end the levels were 0.07 ± 0.01 mmol/L for the females and 0.08 ± 0.02 mmol/L for the males in the control group. No changes occurred in the mean plasma bromide concentration of the control groups during the investigation. The two groups receiving sodium bromide showed bromide concentrations that gradually increased during the first six weeks of the experiment and then remained stable except in the males receiving 9 mg/kg bw/day. Here, plasma concentration increased until week 8 after which a non-significant decrease was observed. No significant differences were found between females and males at either dose level. The mean plasma bromide concentrations for the two dose levels of bromide differed significantly from each other, both for the females (0.02>P> 0.01) and for the males (P<0.001), showing 3.05 ± 0.82 and 4.93 ± 1.07 mmol/L for the females and 2.14 ± 0.72 and 4.3 ± 0.71 mmol/L for the males for 4 and 9 mg bromide/kg bw/day, respectively.
Considering the fact that a substance, when regularly administered, usually reaches a steady state in plasma after four times the plasma half-life and that in this study a steady state for bromide was reached after 6 weeks, it may be concluded that in these volunteers the plasma half-life of bromide was about 10 days.
Analysis of the 24-hour urine samples revealed that the mean bromide excretion at the start ranged from 0.06 ± 0.03 to 0.09 ± 0.03 mmol/24h. Assuming that the bromide excretion in a 24-hour urine sample reflects the average daily bromide consumption there is no difference from the mean bromide content of 0.1 mmol bromide in a 24-hour duplicate meals.
In the control group the mean bromide excretion did not change. At the end of the investigation, bromide excretion in females receiving 4 or 9 mg/kg bw/day was 2.61 ± 0.45 and 2.69 ± 1.61 mmol/24 h, respectively, whereas the daily administered dose of bromide was 1.76 ± 0.14 and 3.66 ± 0.5 mmol, respectively. The mean excretion was higher (0.02>P>0.01) in the 4 mg group and lower (but not significantly) in the 9 mg group than the daily administered dose of bromide. The mean bromide excretion in the two groups did not differ significantly.
In the male groups receiving 4 and 9 mg bromide/kg bw/day the mean bromide excretion was 2.41 ± 1.62 and 6.07 ± 3.31 mmol/24h, respectively. The mean dose administered daily was 1.88 ± 0.14 and 4.31 ± 0.39 mmol, respectively. In neither group was the mean excretion significantly higher than the daily administered dose.
The mean chloride excretion in the 24-hour urines of the different groups did not differ significantly and reflected a sodium chloride consumption ranging from 4.9 to 10.2 g, which is normal for the Netherlands. However, large individual differences were observed in the chloride excretion at the start and end of the investigation. The same applies to the individual creatinine excretion which under physiological circumstances is relatively stable. No differences were found in the mean creatinine excretion of the different groups at the start and end of the investigation.
Endocrine system:
Evaluation of thyroid parameters (T4, fT4, TBG and T3) revealed that the log (Ce/Cs) for each of the control groups did not differ significantly (at P<0.01) from zero. The log (Ce/Cs) of T4 and T3 in the female volunteers receiving 9 mg bromide/kg bw/day differed significantly from zero (0.01>P>0.01). In these six females, the change in T4 and T3 concentration was on average +14 and +20% (for T4 the 99% confidence interval for the mean percentage increase includes values in the range of 1-28%; for the T3 the interval include values ranging from 3 to 38%). No significant differences (at the 0.01 significance level) were recorded for fT4 and TGB concentrations. No significant differences in any of the four variables were found in the females receiving 4 mg bromide/kg bw/day or in either group of male volunteers receiving bromide.
For none of the variables related to the function of the gonades and adrenals (estradiol, progesterone, testosterone and cortisol) did log (Ce/Cs) differ significantly from zero in any of the groups. Similarly, no significant values for log (Ce/Cs) wre recorded in respect of the TSH, prolactin, LH and FSH values in either sex in any of the three groups before administration of TRH and LHRH. The changes in these four variables after TRH/LHRH administration to each individual were within physiological limits; no significant differences from zero were demonstrated in the 40-minute concentration values expressed as log (Me/Ms), quantifying the response to bromide after TRH/LHRH administration in terms of multiples of the concentration before this treatment.
Central nervous system:
Since no differences between men and women were found regarding the investigations on the central nervous system, the results of both sexes were combined for each dose group. In addition, testing was done on 5% and 10% level of significance (two-sided) because little or no effect was expected and there was only a small number of subjects per group examined. Visual inspection of the EEG records did not reveal overt differences caused by bromide. A slight increase in sleep patterns was detectable in the second recording for volunteers treated with bromide. Examination of the inspected parameters revealed that in the temporo-occipital and parieto-occipital areas practically no effect was visible.
Regarding the total power, a moderate effect was detectable in the frontal areas. In other parts only a trend in that direction could be seen. For the three dosage groups alike, a slight decrease in overall power indicated a general effect attributable to small attenuation differences. The most important changes were found in the δ-bands, the α2-bands and the β-bands for individuals treated at 9 mg/kg bw/day. Regarding scalp distribution, these effects were most pronounced over the temporal and central areas. The occipital and parietal areas showed practically no significantly changes.
The observed effects were symmetrical. Apparent asymmetries in significance were due to the rather small changes and the relatively small number of subjects. Although in the normal EEG asymmetrics between the left and right hemispheres exist because of the differences in hemispheric dominance, these are irrelevant here as only the differences between the start and the end of the investigation were analysed.
Correlation analysis shows that the effects were generalized. This means that all the different areas displayed the same direction of change due to bromide for the different parameters of the EEG. Only the magnitudes differed. For one cortical area, a decrease in power is visible (δ1-band course). The mean decrease induced by bromide as 3dB for the fronto-central and fronto-temporal areas and 2dB for the temporal and central parts. In contrast the β-bands showed an increase in power amounting to about 3dB. Although visible in the graph, the effect is much better observed, when the relative instead of the absolute power is used, beacausse of the statistically better proportions of the former.
A remarkable course is seen for the α1-band. All areas showed a significant decrease with the lower dose group but, in some areas, the effect disappeared with the higher dose. The significance of this finding is not clear. It may have been fortuitous or the expression of the start of a shift in power between frequency bands.
The mobility parameter clearly indicated a general increase in the mean frequency, which varied between 1-3 Hz for the fronto-central and 3-4 Hz for the temporal areas. It was calculated directly from the initial EEG in quite a different way and confirmed the shift in power from the lower frequency bands to the higher already demonstrated in the individual bands of the power spectrum.
The VER`s were analysed in exactly the same way for the differences between the three groups. Here the latencies and amplitudes of the six main components were used. The only effect visible was a relatively small decrease in the latency of wave IV and a slight increase in the amplitude of waves II and IV in the channels that referred to temporal or frontal electrodes, in the group on the highest bromide dosage. As the VER is generated in the occipital lobes and the spontaneous EEG analysis showed that the effects were located mainly in the more anterior cortical areas, these rather moderate changes were to be expected. - Conclusions:
- The results of this study demonstrated a half-life of bromide of 10 days in man which is in good agreement with the half-lives reported for bromide in man elsewhere.
At 9 mg/kg bw/day, a slight but significant increase in T4 and T3 was observed although the individual concentrations of T4 and T3 in this group were within normal limits at the start and the end of the investigation. The effect found on the female thyroid gland parameters is only minor in degree and opposite to the effects seen in investigations performed in the rat. The rat showed a severe decrease in T4 and T3 levels, whereas in humans only minor increase in T4 and T3 was recognized with levels being within the normal range of variability. In addition, no changes in TSH were observed and, thus, a direct effect on thyroid morphology by an initiation of the TRH-TSH induced proliferation of the thyroid as seen in the rat seems unlikely in humans. a direct effect An alteration in neurophysiological parameters was evident at 9 mg/kg bw/day and was characterised by a decrease in the δ-activity, a decrease in the β-activity and an increase in mean frequency, expressed in the mobility. No changes on haematology and clinical chemistry was evident when comparing the pre-experimental values with those determined at the end of the study.
Based on the results obtained in this 12-week study in humans, the NOEL of this study is derived at 4 mg/kg bw/day while the NOAEL can be set at 9 mg/kg bw/day as the effects seen in females at this dose level are only slight in degree and of borderline nature, not seen in males and are, thus, not considered to be toxicologically adverse. - Executive summary:
Materials and Methods
The effect of inorganic bromide in the rat had been extensively studied and shown dose dependent changes in all endocrine organs at serum concentrations comparable to therapeutic levels in man. The most sensitive organ had been the thyroid gland in female animals; increased activity of the thyroid was demonstrated after bromide administration. Because of the apparent differences between man and rat in the effect of bromide, it was decided that the obtained results from rat studies might have only a limited relevance when establishing an “acceptable daily intake” for bromide. Sodium bromide was administered orally in capsules to healthy volunteers (7 males and 7 non-pregnant females, not using oral contraceptives) in doses of 0, 4 and 9 mg/kg/day for 12 weeks or 3 full cycles. Special attention was paid to the endocrine and central nervous systems. At the start and at the end of the study, a full medical history (physical examination, haematology, clinical chemistry, urine analysis) was recorded for each individual. In addition, a quantitative analysis of the EEG and the evoked response at the start and end of the investigation was performed to obtain information on the functioning of the CNS. Haematological and biochemical analyses were performed at the beginning and at the end of the investigtation. At the start, after 14 days and at the end of the experiment, serum-electrolyte and plasma-bromide concentrations were determined, as well as sodium, chloride, bromide and creatinine concentration in a 24-hour urine sample. At the start and at the end of the study the following hormones were determined by radioimmunoassay (RIA): thyroxine (T4), free thyroxine (fT4), thyroxine-binding globulin (TBG), triiodothyronine (T3), cortisol, oestradiol, progesterone, testosterone, thyrotropin, prolactin, luteinising hormone (LH) and follicle stimulating hormone (FSH). TSH, prolactin, LH and FSH were determined as well. Statistical analyses with respect to the separate endocrine variable were performed by application of Student`s t-test in each group to the logarithm of the individual ratios: Concentration in the end/concentration at start (Ce/Cs).
Results and Discussion
The purpose of the study was to determine whether administration of 4 or 9 mg bromide/kg bw/day for 3 months in males or for 3 full cycles in females might induce any effect on healthy human volunteers taking their normal diet. Special attention was paid to the endocrine system, because endocrine effects were induced in rats by the administration of sodium bromide; and the central nervous system because the therapeutic use of sodium bromide suggested that an effect might be expected. The plasma- bromide concentration at the start of the investigation in all volunteers was the same magnitude as in draftees in whom a mean concentration of 0.06 ± 0.02 mmol/L was measured. In the control group of volunteers the plasma-bromide level did not change during the investigation. The therapeutic plasma-bromide concentration ranges from 6 to 12 mmol/L. In rats an effect was measured at plasma concentrations of 7.7 ± 1.1 mmol/L and higher. At the end of our investigation the mean plasma concentration in females and males receiving 9 mg bromide/kg bw/day was therefore 66% of the lower therapeutic plasma concentration and over 50% of the plasma concentration at which effects were observed in rats (4.3 and 4.93 mmol/L in males and females, respectively). In the four groups receiving sodium bromide, the plasma-bromide concentration reached a steady state after 6 weeks. The estimated plasma half-life of bromide of about 10 days corresponds very well with the literature data. In the male volunteers taking 9 mg bromide/kg bw/day, the plasma bromide concentration decreased slightly from week 8 to 12. Whether this decrease was induced by changes in chloride intake, by physiological variation or by the inaccurate taking of the capsules could not be established. The mean bromide and chloride excretion measured in urine collected at the start over 24 hours for all subjects reflected the usual bromide and chloride content of the daily diet in the Netherlands. The individual creatinine excretion figures at the start and end of the investigation led to the conclusion that unfortunately not all subjects collected urine accurately. Therefore, the results of the urine analyses were not used for further kinetic interpretation. In 38 subjects remaining on the study, gastric discomfort (nausea) associated with the ingestion of bromide-containing capsules was recorded. In subjects taking sodium bromide a relation between the dose administered and the incidence of nausea was present, but this was absent in subjects taking placebos. Gastric discomfort is a known side effect of sodium bromide. Since the investigation was performed to establish a no-effect-level in man with respect to food while the bromide dose was ingested once a day, this phenomenon may not be considered to be relevant to the study. Several subjects mentioned a decrease in mental concentration and an increase in sleepiness, a known possible effect. However, on consideration of the relation between mentioning of this symptom and the dose administered, association with the bromide ingested seemed unlikely. The results of the medical histories, the physical examinations and the haematological and biochemical analyses revealed no other effects attributable to bromide. In the female subjects taking 9 mg bromide/kg bw/day, a significant increase in T4 and T3 was observed (P < 0.01), although the individual concentrations of T4 and T3 in this group were within normal limits at the start and the end of the investigation. fT4 and TBG concentrations did not change. The slight increase in T4 and T3 did not induce a measurable decrease or an increase of TSH. Thus, a direct effect on thyroid morphology by an initiation of the TRH-TSH induced proliferation of the thyroid as seen in the rat seems unlikely in humans. The reaction of the pituitary to the administration of THRH did not change either. No such changes in the endocrine parameters relating to the function of the thyroid were observed in male subjects receiving the same dosage of sodium bromide or in males or females taking 4 mg bromide/kg bw/day. No changes were observed in measurements of hormones produced by the adrenals, the gonads and the pituitary gland. It is striking that in man an effect is found in the female thyroid, because the female thyroid is also the most sensitive endocrine organ in rats. However, in contrast to man, rats showed a decrease in thyroid function. When bromide replaces iodide in the thyroid gland, this interferes with the production of thyroidal hormones leading to decreased T4 and T3 levels. In response to decreased circulating thyroxine levels the pituitary induces the production and release of TSH which in turn leads to growth stimulation in the thyroid gland to compensate for the decreased thyroid hormone level. Thyroid hormones are not bound to proteins in rats, in contrast to man, where thyroxine is found mainly bound to proteins like TBG, pre-albumin and albumin (McClain, 1995). Therefore it seems likely, that in rats where, in addition, the half-life of T4 is about 12 hours (in man: 5-9 days), thyroid hormones are more susceptible to changes in iodide concentration. This fits with the results obtained in this investigation, showing rather increased than decreased levels of T4 ad T3. It is therefore questionable, if the effects of bromide administration seen in rats are conferrable to humans. Statistically significant changes were found in the EEG. These were apparent as shifts in power in spectral bands and a shift in mean frequency. These shifts never exceeded normal limits but reflected a shift in background EEG activity. The results show a decrease in the δ-activity, a decrease in the β-activity and an increase in mean frequency, expressed in the mobility. The rather conspicuous course of the α1-band may have been fortuitous and due to the small number of subjects and the small shift in power. It may, however, also have reflected the fact that the arbitrary choice of limits of the power bands may obscure effects. If the effect of bromide at low doses consists of a slight decrease in power in the middle frequency range, this will be visible. When, however, with plasma concentrations, the power distribution is shifted to higher frequencies, the former effect disappears. Such a differential effect of drugs in low doses is not uncommon. Another result is that not all cortical areas are involved to the same extent. The occipital and parietal areas show only minor changes while the changes are most pronounced in the fronto-temporal and central areas. The effects are strikingly symmetrical. The asymmetrical occurrence of significant variables apparent may only reflect the small differences between the two hemispheres that may be expected in a small number of subjects. Neurophysiological variables differ from biochemical entities in that they express dynamic rather than static values. Differences in patterns and different cortical areas, particularly between right and left, form the basis of the evaluation of EEG parameters. Provisionally the effects found in the group receiving 4 mg bromide/kg bw/day must be considered to be fortuitous. However, before regarding the changes in the higher dose group as an effect and 4 mg bromide/kg bw/day as a no-effect-level in man, it will be necessary to perform another study confirming the observed endocrine and neurophysiological results.
- Endpoint:
- endocrine system modulation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Study period:
- 1988
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: no guideline available for this special investigation
- Qualifier:
- no guideline available
- Deviations:
- not applicable
- Remarks:
- no guideline available for this special investigation
- Principles of method if other than guideline:
- Study is a publication and was performed according to good experimental practice.
- GLP compliance:
- no
- Type of method:
- in vivo
- Endpoint addressed:
- repeated dose toxicity: oral
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Not applicable
- Age at study initiation: Age not indicated, based on the weight animals should have been about 8-10 weeks of age
- Weight at study initiation: 200-300 g - Route of administration:
- oral: feed
- Vehicle:
- other: No vehicle used; test substance was mixed with diet
- Details on exposure:
- DIET PREPARATION
- Concentration of test substance in food: 19 g NaBr/kg - Analytical verification of doses or concentrations:
- not specified
- Duration of treatment / exposure:
- 14 days
- Frequency of treatment:
- daily
- Post exposure period:
- No postexposure period; animals were sacrificed at the end of the treatment period.
- Remarks:
- Doses / Concentrations:
19 g NaBr/kg
Basis:
nominal in diet
corresponding to 950 mg/kg bw/day using a default conversion of 1 ppm = 0.05 mg/kg bw for older rats - No. of animals per sex per dose:
- 8/group
- Control animals:
- other: Two control groups included in the investigation: Plain diet and 11 g NaCl/kg in diet
- Details on study design:
Animals held on the same regimen were intubated after two weeks with 0.25 ml phosphate buffered saline (0.1 mM, pH 7.2) containing 28 µg NaI/mL and 2.4 µCi 125-I/mL. After 24 hours the animals were desanguinated and the thyroid glands were excised for the determination of 125-I with a gamma-scintillation counter.- Examinations:
- Bodyweight: at the and of the investigation
Clinical chemistry: T4 and TSH levels were determined in serum; no other clinical chemical parameters were investigated
Organ weights: thyroid weights were taken only
Thyroid peroxidase (TPO) activities: Thyroids were homogenized in 50 mM phosphate buffer, pH 7.4 (4 ml/g tissue). Homogenates were centrifuged (15 min, 1000 g) and the supernatant diluted 10-times in buffer. TPO activity was determined using iodide as substrate. The incubation mixture consisted of 50 mM phosphate buffer, pH 7.4; 0.135 mM H2O2, 5 mM KI and 50 µl diluted thyroid homogenate in a total volume of 1 ml. tHe reatio was followed spectrophotometrically at 353 nm.
Peroxidise activity: guaiacol as used as substrate. Final concentrations in the incubation mixture were: 50 mM phosphate buffer, pH 7.4, 0.27 mM H2O2, 30 mM guaiacol and 50 µl diluted homogenate in a total volume of 1 ml. The oxidation of guaiaol was followed at 470 nm.
NADH and NADPH activity: 80 mM phosphate buffer, pH 7.6, 6.07 mM sodium azide, 50 µl diluted homogenate and 100 nM NADH/25 nM cytochrome c or 100 nM NADPH/100 nM cytochrome c. Cytochrome c reduction was monitored at 550 nm. - Details on results:
- Mortality:
There were no premature decedents during the treatment period
Clinical signs:
No clinical signs were reported within this investigation.
Bodyweight gain:
Bodyweight of animals treated with sodium bromide (19 g/kg = 950 mg/kg bw/day) was lower than in the controls.
Clinical chemistry:
In the serum of NaBr-treated rats the concentration of T4 was lower and that of TSH higher than in the serum of control animals. No other clinical chemical parameters were examined in this investigation.
Organ weights:
The absolute and relative weight of the thyroid gland was significantly higher in animals treated with sodium bromide compared to controls. No further organ weight determinations were performed.
Other:
I-TPO activity was strongly decreased in NaBr-treated rats compared to control animals, but also the guaiacol-TPO activity was significantly lower.
Hardly any effect was observed in the activity of NADPH cytochrome c reductase. However, both NaCl and NaBr induced an increase in activity of NADH cytochrome c reductase, with NaBr showing the strongest effect. - Conclusions:
- Bromide inhibits the uptake of iodide in the thyroid gland. Bromide inhibits the oxidation of iodide to iodine and thus incorporation of iodine in thyrosine residues. Bromide inhibits the coupling of tyrosine residues to thyronine. Bromide causes an increase in NADH cytochrome c reductase activity, probably as the result of an increased TSH stimulus.
- Executive summary:
Materials and Methods
The investigation was performed to clarify the mechanism of action of bromide ion on the thyroid gland. Furthermore, the effects of bromide on active uptake of iodide by the thyroid gland, oxidation of iodide by the thyroid peroxidase enzyme and bromide effects on thyroid hormone biosynthesis were investigated. Male rats were fed a diet containing 19 g NaBr/kg (corresponding to about 950 mg/kg bw/day based on a default conversion of 1 ppm = 0.05 mg/kg bw/day for older rats) or 11 g NaCl/kg for two weeks. Animals were sacrificed after the treatment period and blood was colleted for determination of T4 and TSH levels. Thyroid glands were weighed and homogenized. Thyroid peroxidise (TPO) activities, peroxidise activity and activity of NADH and NADPH were determined. Additionally, animals held on the same regimen were intubated after two weeks with phosphate buffered saline containing 28 µg NaI/mL and 2.4 µCi 125-I/mL. After 24 hours the animals were desanguinated and the thyroid glands were excised for the determination of 125-I with a gamma-scintillation counter.
Results and Discussion
After two weeks feeding of a NaBr-containing diet, bodyweight of animals was lower than in the controls, and the absolute and relative weight of the thyroid gland was significantly higher. In the serum of these rats the concentration of T4 was lower and that of TSH higher than in the serum of control animals. These findings are in accordance with the earlier findings pointing to a disturbance of thyroid hormone biosynthesis, consequently followed by a compensatory increase in TSH. In addition to this, in NaBr-treated animals the uptake of 125-I by the thyroid gland was significantly lower than in control animals. Surprisingly, the animals fed an equimolar dose of NaCl also showed an increase in absolute and relative thyroid weight. However, no alterations were found in the concentration of T4 and TSH. So far, the origin of this latter effect is unknown. NaBr caused a marked effect on the activity of thyroid peroxidase. Particularly the I-TPO activity was strongly decreased compared to control animals, but also the guaiacol-TPO activity was significantly lower. NaCl treatment did not cause any effect on peroxidise activity. I-TPO activity might reflect the oxidase potential of thyroid peroxidise and the ability to incorporate iodine in tyrosine residues, whereas the guaiacol-TPO reaction reflects the activity of the coupling reaction of tyrosine residues in thyroglobulin by peroxidise. Therefore, it can be hypothesized that bromide strongly inhibits the oxidation of iodide into iodine by H2O2, and to a lesser extent inhibits the coupling of iodinated tyrosine residues to thyronines. This inhibition of peroxidise activity, combined with the earlier mentioned reduction in iodide uptake by the thyroid gland, will result in a decrease of T3 and T4 into the circulation. With respect to the enzymes involved in the production of H2O2 in the thyroid gland, hardly any effect was observed in the activity of NADPH cytochrome c reductase. However, both NaCl and NaBr induced an increase in activity of NADH cytochrome c reductase, with NaBr showing the strongest effect. This increase in activity after NaCl and NaBr treatment in vivo is remarkable, especially if one realizes that upon incubation of thyroid homogenate with either of the compounds in vitro, inhibition of NADH cytochrome c reductase activity was found. At least for NaBr the increase in NADH cytochrome c reductase activity in vivo can be explained as a result of a compensatory stimulation of the thyroid gland by TSH, in an attempt to compensate for the decreased thyroid hormone biosynthesis through an increased H2O2 production. However this cannot be the case for NaCl, since for the animals in this group no increase in TSH was found. Further investigations to clarify this problem are necessary.
- Endpoint:
- mechanistic studies
- Type of information:
- other: review on species differences in thyroid gland biochemistry and physiology (rodent/human) and the mechanisms for altered thyroid function
- Adequacy of study:
- supporting study
- Study period:
- 1995
- Reliability:
- 2 (reliable with restrictions)
- Qualifier:
- no guideline available
- Deviations:
- not applicable
- Remarks:
- no guideline available for the conduct of mechanistic studies
- Principles of method if other than guideline:
- study was performed according to good experimental practice
- GLP compliance:
- no
- Type of method:
- other: no method applicable; review on species differences
- Endpoint addressed:
- not applicable
- Species:
- other: a comparison was made regarding function and morphology of the thyroid gland between rodents, non-human primates and humans.
- Strain:
- not specified
- Sex:
- not specified
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
No information on test animals available in the review. - Route of administration:
- other: not applicable; review of several mechanistic studies
- Vehicle:
- not specified
- Examinations:
- regulation of thyroid function, the biosynthesis of thyroid hormones,species differences in thyroid gland biochemistry and physiology and the mechanisms for altered thyroid function
- Details on results:
- There are important species differences in thyroid gland physiology between rodents and humans that may account for a marked species difference in the inherent species susceptibility for neoplasia to hormone imbalance. Thyroid gland neoplasia is mediated by thyroid stimulating hormone (TSH) in response to altered thyroid gland function. It is known that certain chemicals can modulate TSH response for autoregulation of follicular cell function in the thyroid gland and thereby increase or decrease the response of the follicular cell to TSH. It is thus important to consider mechanisms for the evaluation of potential cancer risks.
Many chemicals stimulate thyroid gland growth as a compensatory response to altered thyroid function. This response is mediated via TSH released by the pituitary in response to decreased circulating levels of thyroid hormone. The plasma membrane surface of the follicular cells has receptors for TSH. Small organic molecules are not known to be direct TSH receptor agonists or antagonists so far; however, various antibodies found in autoimmune disease can directly stimulate or inhibit the TSH receptor. Chemicals can, however, modulate the TSH response by effects on components of the TSH receptor responsible for autoregulation of follicular cell function. - Conclusions:
- The regulation of thyroid hormone synthesis, the binding of thyroid hormones to plasma protein and their half life in the blood have been demonstrated to be different in rodents and primates. The rodent thyroid reveals a much higher functional activity and operates at a considerably higher level with respect to thyroid hormone turnover as compared to the primate, a conclusion also supported by the histological appearance of the thyroid. The species differences between rodents and primates in thyroid gland physiology, the spontaneous incidents of thyroid gland neoplasia and the apparent susceptibility to neoplasia secondary to simple hypothyroidism support the conclusion that there is a marked species difference in thyroid gland neoplasia secondary to hormone imbalance. The rodent will exhibit an increase in thyroid gland neoplasia in the presence of mild to moderate increase in TSH. In contrast, no clear etiologic role for hypothyroidism in human thyroid cancer has been established even though chronic hypothyroidism, in the moderate to severe range, has occurred in humans in areas of endemic goitre. This again demonstrates that the regulation of thyroid hormones reveals a profound difference in rodents and humans and rodents appear to more sensitive to the effect of chemicals on the thyroid functions compared to rodents.
- Executive summary:
Please refer to section "remarks on results".
Referenceopen allclose all
Table A6.10/08-1: Development of wavy ribs in fetuses of dams dosed with 1000 mg/kg fenoterol on Days 7-17 of gestation
Day of gestation |
Number of litters |
Fetal death ([%])* |
Number of live fetuses |
Number of fetuses with pathologic ribs ([%])* |
|||
Stage I |
Stage II |
Stage III |
Total |
||||
16 |
5 |
2 (3.1) |
68 |
0 |
0 |
0 |
0 |
17 |
5 |
6 (11.3) |
57 |
26 (47.4) |
0 |
0 |
26 (47.4) |
18 |
5 |
9 (12.8) |
62 |
25 (41.2) |
0 |
0 |
25 (41.2) |
19 |
5 |
7 (10.4) |
59 |
1 (1.7) |
27 (45.2) |
0 |
28 (46.8) |
20 |
5 |
20 (29.7) |
51 |
0 |
10 (22.2) |
17 (27.1) |
27 (49.4) |
* %-values were averaged from litter values
Stage I: Insufficiently ossified (cartilage straight)
Stage II: Insufficiently ossified (cartilage curved)
Stage III:Ossified wavy ribs
Table A6.10/08-2: Repairability of wavy ribs in the offspring of dams dosed with 1000 mg/kg fenoterol by gavage on Days 7-17 of gestation
(alizarin staining method)
Group |
Day of observation |
Number of litters |
Mortality of pups ([%])* |
Number of viables |
Number of viables with wavy ribs ([%])* |
|||
Degree |
||||||||
++ |
+ |
(+) |
Total |
|||||
I |
Day 21 of gestation |
5 |
- |
59 fetuses |
25 (43.0) |
13 (23.4) |
3 (4.6) |
41 (71.0) |
II |
Day 1 p.p. |
5 |
2 (2.6) |
59 pups |
18 (27.2) |
5 (9.3) |
9 (17.2) |
32 (53.7) |
III |
Day 3 p.p. |
6 |
13 (14.5) |
45 pups |
6 (12.7) |
10 (24.7) |
3 (5.8) |
19 (43.2) |
IV |
Day 7 p.p. |
7 |
28 (36.4) |
50 pups |
0 |
18 (34.7) |
14 (21.7) |
32 (56.4) |
V |
Day 14 p.p. |
6 |
15 (25.5) |
42 pups |
0 |
2 (6.7) |
14 (35.8) |
16 (41.3) |
VI |
Day 21 p.p. |
6 |
12 (19.7) |
46 pups |
0 |
0 |
12 (32.4) |
12 (32.4) |
* % averaded from litter values
++ distinctly visible
+ slightly visible
(+) hardly visible
p.p. post partum
Table A6.10/08-3: Repairability of wavy ribs in individual pups after birth from dams dosed with 1000 mg/kg fenoterol on Days 7-17 of gestation
(X-ray examination method)
Pup number |
Degree of wavy ribs |
||||
Day 1 |
Day 3 |
Day 7 |
Day 14 |
Day 21 |
|
1751-1 |
+ |
(+) |
8+ |
- |
- |
1751-2 |
8+) |
- |
- |
- |
- |
1751-3 |
- |
- |
- |
- |
- |
1751-4 |
- |
- |
- |
- |
- |
1751-5 |
++ |
++ |
Died |
- |
- |
1751-6 |
- |
Died |
- |
- |
- |
1751-7 |
(+) |
(+) |
- |
- |
- |
1751-8 |
+ |
Died |
- |
- |
- |
1752-1 |
++ |
++ |
+ |
(+) |
- |
1752-2 |
++ |
++ |
+ |
(+) |
(+) |
1752-3 |
++ |
Died |
- |
- |
- |
1752-4 |
++ |
++ |
++ |
(+) |
- |
1752-5 |
(+) |
(+) |
- |
- |
(+) |
1752-6 |
++ |
++ |
+ |
+ |
- |
1752-7 |
(+) |
(+) |
- |
- |
+ |
1752-8 |
++ |
++ |
++ |
+ |
- |
1752-9 |
- |
- |
- |
- |
- |
1752-10 |
- |
- |
- |
- |
|
1753-1 |
++ |
Died |
- |
|
|
1753-2 |
++ |
Died |
- |
|
|
1753-3 |
- |
Died |
- |
|
|
1753-4 |
+ |
Died |
- |
|
|
1753-5 |
+ |
+ |
Died |
|
|
1753-6 |
- |
- |
Died |
|
|
1753-7 |
- |
Died |
- |
|
|
1753-8 |
++ |
Died |
- |
|
|
1754-1 |
++ |
+ |
+ |
(+) |
- |
1753-2 |
++ |
++ |
+ |
(+) |
- |
1753-3 |
+ |
+ |
Died |
|
|
1753-4 |
++ |
++ |
++ |
+ |
(+) |
1753-5 |
(+) |
(+) |
- |
- |
- |
1753-6 |
++ |
++ |
+ |
(+) |
- |
1753-7 |
- |
- |
- |
- |
- |
1753-8 |
+ |
+ |
Died |
|
|
1753-9 |
++ |
+ |
Died |
|
|
1753-10 |
- |
- |
- |
- |
- |
1753-11 |
++ |
++ |
++ |
+ |
+ |
1753-12 |
+ |
+ |
(+) |
- |
- |
1753-13 |
++ |
++ |
+ |
(+) |
(+) |
1753-14 |
- |
- |
- |
- |
- |
++ distinctly visible
+ slightly visible
(+) hardly visible
- not visible
Table A6.10/18-1:Comparison of Anticanine Thyroglobulin Antibody (TgA) Optical Density (OD), Body Weight, Weight Gain and Serum Free Thyroxine (fT4) Hormone Concentrations between Potassium Bromide (KBr)-Treated and Control Dogs
Parameter |
TgA (OD) |
Body Weight (kg) |
Weight Gain (kg) |
fT4 (pmol/L) |
||
Day |
0 |
0 |
177 |
177 |
0 |
177 |
KBr-treated Group |
||||||
Dog 1 |
0.039 |
24 |
31.8 |
6.8 |
26 |
19 |
Dog 2 |
0.024 |
24.5 |
28.2 |
3.7 |
17 |
12 |
Dog 3 |
0.072 |
19.5 |
22.3 |
2.8 |
27 |
10 |
Dog 4 |
0.133 |
23.2 |
29.1 |
5.8 |
27 |
6 |
Dog 5 |
0.044 |
18.6 |
25.4 |
6.8 |
27 |
13 |
Mean ±SD |
- |
22.2 ±2.9 |
27.4 ±3.6 |
5.2 ±1.8 |
25 ±4.0 |
12 ± 5.0 |
Control Group |
||||||
Dog 6 |
0.034 |
22.3 |
25.9 |
3.6 |
26 |
12 |
Dog 7 |
0.153 |
23.6 |
25.4 |
1.8 |
23 |
12 |
Dog 8 |
0.021 |
20 |
21.8 |
1.8 |
12 |
10 |
Dog 9 |
0.02 |
24.4 |
26.4 |
1 |
28 |
13 |
Dog 10 |
0.009 |
20.4 |
23.2 |
2.8 |
27 |
13 |
Mean ±SD |
|
22.3 ±2.2 |
24.5 ±2.0 |
2.2 ±1.0 |
23 ±6.0 |
12 ± 1.0 |
Reference |
< 0.124 |
- |
- |
- |
9-40 |
|
Table A6.10/18-2:Thyroid Weight and Histopathology Scoring in Potassium Bromide (KBr)-Treated and Control Dogs
|
Thyroid Weight |
Scored Thyroid Histopathology Criteria |
||
|
Day 177 |
MFD |
IFC |
V |
KBr- treated Group Dog 1 |
0.065 |
3 |
2 |
1.5 |
Dog 2 |
0.04. |
2 |
1 |
1.2 |
Dog 3 |
0.028 |
3 |
2 |
1.6 |
Dog 4 |
0.028 |
2 |
2 |
1.5 |
Dog 5 |
0.024 |
2 |
3 |
1.4 |
Mean ±SD |
0.037 ±0.017 |
1.4 ±0.2 |
2.4 ±0.5 |
2.0 ±0.7 |
Control Group Dog 6 |
0.023 |
4 |
4 |
1.5 |
Dog 7 |
0.063 |
4 |
4 |
1.3 |
Dog 8 |
0.046 |
1 |
1 |
1.5 |
Dog 9 |
0.039 |
2 |
2 |
1.4 |
Dog 10 |
0.052 |
2 |
2 |
1.4 |
Mean ± SD |
0.045 ±0.015 |
1.4 ±0.1 |
2.6 ±1.3 |
2.6 ± 1.3 |
MFD microfollicular development
IFC intrafollicular colloid
V vascularity
Table A6.10/18-3:TRH Stimulation Test Results in Potassium Bromide (KBr)-Treated and Control Dogs on day 0 and 177
TRH Stimulation |
Day 0 |
Day 177 |
||||||||
TT4 [µg/dL] |
TSH [ng/mL] |
% change |
TT4 [µg/dL] |
TSH [ng/mL] |
% change |
|||||
preTRH* |
postTRH§ |
preTRH** |
postTRH# |
preTRH |
postTRH |
preTRH |
post TRH |
|||
Dog 1 |
2.8 |
3.9 |
0.05 |
0.04 |
-20 |
2.2 |
2.6 |
0.03. |
0.15 |
400 |
Dog 2 |
1.8 |
1.7 |
0.13 |
0.1 |
-23 |
1.1 |
2.0 |
0.1 |
0.56 |
460 |
Dog 3 |
2.0 |
1.6 |
0.07 |
0.05 |
-28 |
1.1 |
1.3 |
0.08 |
0.27 |
238 |
Dog 4 |
2.9 |
2.9 |
0.15 |
0.13 |
-13 |
1.0 |
1.9 |
0.11 |
1.2 |
991 |
Dog 5 |
4.3 |
4.4 |
0.06 |
0.06 |
0 |
1.7 |
2.6 |
0.06 |
0.26 |
333 |
Mean ±SD |
2.8 ±1.0 |
2.6 ±1.3 |
0.1 ±0.04 |
0.09 ±0.04 |
- |
1.4 ±0.5 |
2.1 ±0.5 |
0.08 ±0.03 |
0.49 ±0.42 |
- |
Dog 6 |
2.7 |
1.5 |
0.22 |
0.22 |
-9 |
1.4 |
1.3 |
0.16 |
0.29 |
81 |
Dog 7 |
2.8 |
2.8 |
0.12 |
0.12 |
-16 |
1.5 |
2.1 |
0.03 |
0.15 |
400 |
Dog 8 |
1.1 |
1.7 |
0.05 |
0.05 |
0 |
1.2 |
2.0 |
0.07 |
0.43 |
514 |
Dog 9 |
2.8 |
3.1 |
0.08 |
0.08 |
-38 |
1.8 |
3.7 |
0.06 |
0.28 |
366 |
Dog 10 |
2.4 |
3.5 |
0.09 |
0.09 |
0 |
1.0 |
3.2 |
0.22 |
0.98 |
345 |
Mean ± SD |
2.4 ± |
2.5 ±0.9 |
0.11 ±0.06 |
0.11 ±0.06 |
- |
1.4 ±0.3 |
2.5 ±1.0 |
0.11 ±0.08 |
0.43 ±0.32 |
- |
TT4 total thyroxine
TSH tyroid-stimulating hormone
* reference range: 1.3-4 µg/dL
§ reference range: > 1.5 µg/dL
** reference range:0-0.65 ng/mL
# a percentage change in postTRH stimulation TSH>100% was considered supportive of euthyroidism
Table A6.10/11-1: Content of bromine and iodine and concentration ratio I/Br in the rat thyroid gland
Group |
Bromide concentration in drinking water [mg/L] |
Duration in days |
Concentration in the thyroid gland |
Concentration ratio I/Br |
||
Br [mg/kg dw] |
I [mg/kg dw] |
Weight [g] |
Molar* |
|||
1 |
0 |
16 |
49 |
888 |
18.1 |
11.4 |
2 |
10 |
16 |
102 |
639 |
6.3 |
3.9 |
3 |
50 |
16 |
369 |
736 |
2.0 |
1.3 |
4 |
100 |
16 |
615 |
653 |
1.1 |
0.67 |
5 |
0 |
66 |
44 |
914 |
21.4 |
13.5 |
6 |
10 |
66 |
158 |
1076 |
6.8 |
4.3 |
7 |
50 |
66 |
530 |
829 |
1.6 |
1.0 |
8 |
100 |
66 |
1375 |
805 |
0.59 |
0.37 |
dw: dry weight
*Molar ratio I/Br: weight ratio I/Br multiplied by a factor of 0.6296 (atomic mass Br/I = 79.904/126.9045)
Table A6.10/13-1: Bromine and iodine content and the concentration ratio of I/Br in the thyroid gland of rats after bromide treatment for 133 days.
Group |
Bromine concentration in drinking water [mg/L] |
Concentration in the thyroid gland [mg/kg dw] |
Concentration ratio I/Br |
||
Br |
I |
Weight |
Molar* |
||
1 |
0 |
51.8 |
1472.9 |
28.43 |
17.9 |
2 |
100 |
1013.0 |
1213.6 |
1.20 |
0.75 |
3 |
200 |
1181.6 |
1137.4 |
0.96 |
0.61 |
4 |
400 |
1926.3 |
1204.1 |
0.63 |
0.39 |
dw: dry weight
*Molar ratio I/Br: weight ratio I/Br multiplied by a factor of 0.6296 (atomic mass Br/I = 79.904/126.9045)
Table A6.10/17-1:Individual and Mean Pharmacokinetic Parameters in six Dogs following 115 Days of Oral Bromide administration
Model parameter |
Dog 1 |
Dog 2 |
Dog 3 |
Dog 4 |
Dog 5 |
Dog 6 |
Median |
Range |
weight |
11.3 |
10.5 |
13.7 |
12.5 |
10 |
9.6 |
11.3 |
9.6-13.7 |
T1/2 (days) |
17 |
12.9 |
19.1 |
20.3 |
12.2 |
13.4 |
15.2 |
12.2-20.3 |
Css (mg/dL) |
246 |
204 |
269 |
256 |
178 |
244 |
245 |
178-269 |
Cl/F (mL/kg/day) |
16.4 |
19.6 |
14.9 |
15.6 |
22.5 |
16.4 |
16.4 |
14.9-22.5 |
Clc (mL/kg/day) |
8.5 |
12.6 |
6.7 |
6 |
10 |
7.9 |
8.2 |
6-12.6 |
Vd/F (L/kg) |
0.4 |
0.37 |
0.41 |
0.46 |
0.4 |
0.32 |
0.4 |
0.32-0.46 |
t1/2 estimated elimination half-life
Css steady-state serum concentration
Cl/F total body clearance/bioavailability
Clc renal clearance
Vd/F apparent volume distribution/bioavailability
Table A6.10/17-2:Individual and Median Cerebrospinal fluid to Serum Bromide Concentration Ratios
|
Dog 1 |
Dog 2 |
Dog 3 |
Dog 4 |
Dog 5 |
Dog 6 |
Median |
Range |
Day 9 |
0.63 |
0.59 |
0.69 |
0.63 |
0.64 |
0.63 |
0.63 |
0.59-0.69 |
Day 45 |
0.75 |
0.77 |
0.82 |
0.77 |
0.79 |
0.73 |
0.77* |
0.73-0.82 |
Day 115 |
0.74 |
0.75 |
0.79 |
0.83 |
0.83 |
0.75 |
0.77* |
0.74-0.83 |
Day 121 |
0.84 |
0.86 |
0.86 |
0.94 |
0.99 |
0.82 |
0.86** |
0.82-0.99 |
* Significantly different from Day 9 median ratio (P< 0.05)
** Significantly different from Day 9, Day 45 and Day 115 median ratios (P< 0.05)
Mechanisms of toxicity of thyroid follicular cells:
Numerous studies have reported that chronic treatment of rodents with goitrogenic compounds results in the development of follicular cell adenomas. Thiouracil and its derivatives have this effect in rats and mice. This phenomenon also has been observed in rats that consumed brassica seeds, erythrosine, sulfonamides and many other compounds.
The pathogenic mechanism of this phenomenon has been understood for some time and is widely accepted. These goitrogenic agents either directly interfere with thyroid hormone synthesis or secretion in the thyroid gland, increase thyroid hormoneexcretion into the bile, or disrupt the peripheral conversion of T4 to T3. The ensuing decrease in circulating thyroid hormone levels results in a compensatory increased secretion of pituitary TSH. The receptor-mediated TSH stimulation of the thyroid gland leads to proliferative changes of follicular cells that include hypertrophy, hyperplasia, and ultimately, neoplasia in rodents. In the multistage model of carcinogenesis, proliferative lesions often begin with hyperplasia, may proceed to the development of benign tumours (adenoma), and infrequently develop into a malignant tumour. Although these lesions are usually classified as discrete entities, it is important to emphasize that they represent a morphologic continuum with imprecise criteria to separate borderline proliferative lesions.
Excessive secretion of TSH alone also has been reported to produce a high incidence of thyroid tumours in rodents. This has been observed in rats fed an iodine-deficient diet and in mice that received TSH-secreting pituitary tumour transplants. The pathogenic mechanism of thyroid follicular cell tumour development in rodents involves a sustained excessive stimulation of the thyroid gland by TSH. In addition, iodine deficiency is an important promoter of the development of thyroid tumours in rodents induced by intravenous injection of N-methyl-N-nitrosurea.
Inhibition of 5´-Deiodinase: FD&C Red No. 3 (erythrosine) is an example of a well-characterized xenobiotic that results in perturbation of thyroid function in rodents and in long-term studies is associated with an increased incidence of benign thyroid tumours. Red No. 3 is a widely used colour additive in food, cosmetics, and pharmaceuticals. A chronic toxicity/carcinogenicity study revealed that male Sprague-Dawley rats fed a 4% dietary concentration of Red No. 3 beginningin uteroand extending over their life-time developed a 22% incidence of well-differentiated thyroid adenomas derived from follicular cells compared to 1.5% in control rats and a historical incidence of 1.8% for this strain.
The thyroid adenomas were well demarcated from the normal gland by a thin layer of fibrous connective tissue, and adjacent follicles were slightly compressed. The neoplastic cells were more hyperchromatic than the surrounding normal follicular cells and either formed variability sized colloid-containing follicles, lined large cystic spaces, or formed solid sheets. Some neoplatic cells formed papillary structures extending into cystic spaces. There was no evidence of vascular invasion. In addition, male rats developed a 23% incidence of follicular cystic hyperplasia. These focal lesions were formed by the coalescence of adjacent colloid-distended follicles. The follicular wall was lined by 1 or 2 layers of low cuboidal epithelium with hyperchromatic nuclei that occasionally formed papillary projections. There was not a significant increase in follicular cell adenomas in the lower dose groups of male rats or an increase of malignant thyroid follicular cell tumours. Female rats fed similar amounts of the colour did not develop a significant increase in either benign or malignant thyroid tumours. Feeding of the colour at the high dose level provided male rats with 2.464 mg/kg of Red No. 3 daily; by comparison, human consumption in theis estimated to be 0.023 mg/kg/day.
The results of mechanistic studies have suggested that a primary (direct) action of FD&C Red No. 3 on the thyroid is unlikely due to (a) failure of the colour to accumulate in the gland, (b) negative genotoxicity and mutagenicity assays, (c) lack of an oncogenic response in mice and gerbils, (d) a failure to result in thyroid tumour development at dietary concentrations of 1% or less in male and female rats, and (e) a lack of increased tumour development in other organs. Investigations with radiolabelled compound have demonstrated that the colour does not accumulate in the thyroid glands of rats following the feeding of either 0.5 or 4% FD&C Red No. 3 for one week prior to the oral dose of14C-labeled material.
Subsequent mechanistic investigations included a 60-day study in male Sprague-Dawley rats fed either 4% or 0.25% FD&C Red No. 3 compared to controls in order to determine the effects of the colour on thyroid hormone economy and morphometric changes in follicular cells. The experimental design of the study was to terminate groups of rats fed Red No. 3 and controls after 0, 3, 7, 10, 14, 21, 30 and 60 days.
A consistent effect of Red No. 3 on thyroid hormone economy was the striking increase in serum reverse T3. In the high-dose group, reverse T3 as increased at all intervals compared to controls and at 10, 14 and 21 days in the low-dosed animals. The mechanisms responsible for the increased serum reverse T3 appear to be, first, substrate (T4) accumulation due to 5´- deiodinase inhibition with subsequent conversion to reverse T3 rather than active T3 and, second, reverse T3 accumulation due to 5´-deiodinase inhibition resulting in an inability to degrade reverse T3 further to diiodothyronine (T2).
Serum triiodothyronine (T3) was decreased significantly at all intervals in rats of the high-dose group compare to interval controls. The mechanism responsible for the reduced serum T3 following feeding of Red No. 3 was decreased monodeiodination of T4 due to an inhibition of the 5´-deiodinase by the colour.
Serum TSH was increased significantly at all intervals in rats of the high-dose group compared to controls. Rats fed 0.25% Red No. 3 had increased serum TSH only at Days 21, 30 and 60. The mechanism responsible for the increased serum TSH following ingestion of Red No. 3 was a compensatory response by the pituitary gland to the low circulating levels of T3 that resulted from an inhibition of the 5´-deiodinase.
Serum T4 also was increased significantly at all intervals in rats fed 4% Red No. 3 compared o controls. The mechanism responsible for this was, first, accumulation due to an inability to monodeiodinate T4 to T3 in the liver and kidney from the inhibition of 5´-deiodinase by the colour and, second, TSH stimulation of increased T4 production by the thyroid gland.
125I-labeled T4 metabolism was significantly altered in liver homogenates prepared from rats fed 5% FD&C Red No. 3. Degradation of labelled T4 was decreased to approximately 40% of values in control homogenates. This was associated with a 75% decrease in percentage of generation of125I and an approximately 80% decrease in percentage of generation of125I-labeled T3 from radiolabelled T4 substrate. These mechanistic investigations suggested that the colour results in a perturbation of thyroid hormone economy in rodents by inhibiting the 5´-deiodinase in the liver, resulting in long-term stimulation of follicular cells by TSH, which over their lifetime predisposed to an increased incidence of thyroid tumours. The colour was negative in standard genotoxic and mutagenic assays and did not increase the incidence of tumours in other organs.
Morphometric evaluation was performed on thyroid glands from all rats at each interval during the 60-day study. Four levels of thyroid were evaluated with 25 measurements from each rat using a Zeiss interactive digital analysis system. Thyroid follicular diameter was decreased significantly in both low- and high-dose groups at 3, 7, 10 and 14 days compared to interval controls. The area of follicular colloid generally reflected the decrease in thyroid follicular diameter and was decreased significantly at Days 3 and 10 in high-dose rats and Days 7 and 10 in the low-dose group compared to interval controls. These reductions in thyroid follicle diameter and colloid area were consistent with morphologic changes expected from an increased serum TSH concentration.
Thyroid follicular height was increased significantly only after feeding FD&C Red No. 3 for 60 days in both the high- and low-dose groups compared to interval controls. The absence of morphometric evidence of follicular cell hypertrophy at the earlier intervals was consistent with the modest increase in thyroid gland: bodyweight ratio after this relatively short exposure to the colour. The lack of follicular cell hypertrophy at the earlier intervals of feeding Red No. 3 I rats with several-fold elevations in serum TSH levels may be related, in part, to the high thyroid iodine content interfering with the receptor-mediated response to TSH. The thyroid responsiveness to TSH is known to vary inversely with iodine content. Thyroid glands of rats fed FD&C Red No. 3 would be exposed to an increased iodine primarily from sodium iodine contamination of the colour and, to a lesser extent, from metabolism of the compound and release of iodine.
Induction of hepatic microsomal enzymes: Hepatic microsomal enzymes play an important role in thyroid hormone economy because glucuronidation is the rate-limiting step in the biliary excretion of T4 and sulfation by phenol sulfotransferase for the excretion of T3. Longterm exposure of rats to a wide variety of different chemicals may induce these enzyme pathways and result in chronic stimulation of the thyroid by disrupting the hypothalamic-pituitary-thyroid axis. The resulting chronic stimulation of the thyroid by increased circulating levels of TSH often results in a greater risk of developing tumours derived from follicular cells in 2-year or lifetime studies with these compounds in rats.
Xenobiotics that induce liver microsomal enzymes and disrupt thyroid function in rats include central nervous system-acting drugs, calcium-channel-blockers, steroids, retinoids, chlorinated hydrocarbons, and polyhalogented biphenyls among others. Most of the hepatic microsomal enzyme inducers have no apparent intrinsic carcinogenic activity and produce little or no mutagenicity or DNA damage. Their promoting effect o thyroid tumours usually is greater in rats than in mice, with males more often developing a higher incidence of tumours than females. In certain strains of mice, these compounds alter liver cell turnover and promote the development of hepatic tumours from spontaneously initiated hepatocytes.
Phenobarbital has been studied extensively as a prototype for hepatic microsomal inducers that increase a spectrum of cytochrome P-450 isoenzymes. It had been reported tat the activity of uridine diphosphate (UDP)-gucuronyltransferase, the rate-imiting enzyme in T4 metabolism, is increased in purified hepatic microsomes of male rats when expressed as picomoles/min/mg microsomal protein or as total hepatic cumulative biliary excretion of125I-T4 and bile flow than in controls.
Phenobarbital-treated rats develop a characteristic pattern of changes in circulating thyroid hormone levels. Plasma T3 and T4 are markedly decreased after one week and remain decreased for 4 weeks. By Week 8, T3 levels return to near normal due to compensation by the hypothalamic-pituitary-thyroid-axis. Serum TSH values are elevated significantly throughout the first month but often decline after a new steady state is attained. Thyroid weights increase significantly after 2-4 weeks of phenobarbital, reach a maximum increase of 40-50% by Week 8, and remain elevated throughout the period of treatment.
In a series of experiments it was shown that supplemental administration of thyroxine blocked the thyroid tumour-promoting effects of phenobarbital and that the promoting effects were directly proportional to the level of plasma TSH in rats. The sustained increase in circulating TSH levels results initially in hypertrophy of follicular cells, followed by hyperplasia, and ultimately places the rat thyroid at greater risk to develop an increased incidence of benign tumours.
Phenobarbital has been reported to be a thyroid gland tumour promoter in a rat initiation-promotion model. Treatment with a nitrosamine followed by phenobarbital has been shown to increase serum TSH concentrations, thyroid gland weights, and the incidence of follicular cell tumours in the thyroid gland. These effects could be decreased in a dose-related manner by simultaneous treatment with increasing doses of exogenous thyroxine.
It was demonstrated that rats treated with phenobarbital have a significantly higher cumulative biliary excretion of125I-thyroxine than controls. Most of the increase in biliary excretion was accounted for an increase in T4-glucuronide due to an increased metabolism of thyroxine in phenobarbital-treated rats. This is consistent with enzymatic activity measurements that result in increased hepatic T4-UDP-glucuronyl transferase activity in phenobarbital treated rats. Results of this experiment are consistent with the hypothesis that promotion of thyroid tumours in rats was not a direct effect of phenobarbital on the thyroid gland but, rather an indirect effect mediated by TSH secretion from the pituitary secondary to the hepatic microsomal enzyme-induced increase of T4 excretion in the bile.
The activation of the thyroid gland during the treatment of rodents with substances that stimulate thyroxine catabolism is a well-known phenomenon and has been extensively investigated with phenobarbital and other compounds. It occurs particularly with rodents, first because UDP-glucuronyl transferase can easily be induced in rodent species, and second because thyroxine metabolism takes place very rapidly in rats in the absence of thyroxine binding globulin. In humans, a lowering of the circulating thyroxine level but no change in TSH and T3 concentrations has been observed only with high doses of very powerful enzyme-inducing compounds such as rifampicin, both with and without atipyrine.
There is no convincing evidence that humans treated with drugs or exposed to chemicals that induce hepatic microsomal enzymes are at increased risk for the development of thyroid cancer. In a study of the effects of microsomal enzyme-inducing compounds on thyroid hormone metabolism in normal, healthy adults, phenobarbital did not affect the serum T4, T3 or TSH levels. A decrease in serum T4 levels was observed after treatment with either a combination of phenobarbital/rifampicin or a combination of phenobarbital/antipyrine; however, these treatments had no effect on serum T3 or TSH levels. Epidemiologic studies of patients treated with therapeutic doses of phenobarbital have reported no increase in risk for the development of thyroid neoplasia. Highly sensitive assays for thyroid and pituitary hormones are readily available clinically to monitor circulating hormone levels in patients who are exposed to chemicals that could potentially disrupt homeostasis of the pituitary-thyroid axis.
Direct effects of xenobiotic chemicals on the thyroid gland
Inhibition of thyroid hormone synthesis:
(1) Blockage of iodine uptake by thyroid: The initial step in the biosynthesis of thyroid hormones is the uptake of iodide from the circulation and transport across a gradient of follicular cells to the lumen of the follicle. A number of anions act as competitive inhibitors of iodide transport in the thyroid gland including perchlorate, thiocyanate and pertechnetate. Thiocyanate is a potent inhibitor of iodide transport and is a competitive substrate for the thyroid peroxidise but it does not appear to be concentrated in the thyroid. Blockage of the iodide trapping mechanism has a similar disruptive effect on the thyroid-pituitary-axis to iodide deficiency. The blood levels of T4 and T3 decrease resulting in a compensatory increase in the secretion of TSH by the pituitary gland. The hypertrophy and hyperplasia of follicular cells following sustained exposure results in an increased thyroid weight and the development of goitre.
(2) Organification defect due to inhibition of thyroperoxidase: A wide variety of chemicals, drug and other xenobiotics affect the second step in thyroid hormone biosynthesis. The step-wise binding of iodide to the tyrosyl residues in thyroglobulin requires oxidation of inorganic iodide (I-) to molecular (reactive) iodide (I2) by the thyroid peroxidise present in the luminal aspect (microvillar membranes) of follicular cells and adjacent colloid. Classes of chemicals that inhibit the organification by thyroglobulin include the thionamides (such as thiourea, thiouracil, propylthiouracil, methimazole, carbimazole and goitrin), aniline derivatives and related compounds (e.g. sulfonamides, para-aminobenzoic acid, para-aminosalicylic acid and amphenone), and substituted phenols (such as resorcinol, phloroglucinol and 2,4-dihydroxybenzooic acid) and miscellaneous inhibitors (e.g. aminotriazole, tricyanoaminopyrene, antipyrine and its iodinated derivative).
Many of these chemicals exert their action by inhibiting the thyroid peroxidase. This results in a disruption of both the iodination of tyrosyl residues in thyroglobulin and also the coupling reaction of iodotyronines (e.g. monoiodotyrosine (MIT) and diiodotyrosine (DIT) to form iodothyronines (T3 and T4)). In rats, propylthiouracil (PTU) has been shown to affect each step in thyroid hormone synthesis beyond iodide transport. The order of susceptibility to the inhibition by PTU is the coupling reaction, iodination of MIT to form DIT, and iodination of tyrosyl residues to form MIT. Thiourea differs from PTU and other thioamides in that it does not inhibit guaiacol oxidation and does not inactivate the thyroid peroxidise in the absence of iodide. Its ability to inhibit organic iodinations is due to the reversible reduction of active I2to 2 I-.
The goitrogenic effects of sulfonamides have been known for approximately 50 years, since the reports of the action of sulfaguanidine on the rat thyroid. Sulfamethoyazole and trimetroprim exert a potent goitrogenic effect in rats, resulting in marked decreases in circulating T3 and T4, a substantial compensatory increase in TSH and increased thyroid weights due to follicular cell hyperplasia. The dog also is a sensitive species to the effects of sulfonamides, with markedly decreased T4 and T3 levels, hyperplasia of thyrotropic basophils, and increased thyroid weights.
By comparison, the thyroids of monkeys and humans are resistant to the development of changes that sulfonamides produce in rodents (rats and mice) and the dog. Rhesus monkeys treated for 52 weeks with sulfamethoxazole (doses up to 300 mg/kg/day) with and without trimethroprim had no changes in thyroid weights and thyroid histology was normal. In one publication the effects of PTU and a goitrogenic sulfonaide are compared on the activity of thyroid peroxidise in the rat and monkey using guaiacol peroxidation assay. The concentration required for a 50% inhibition of the peroxidise enzyme was designated as the IC50. When the IC50for PTU was set at x 1 for rats, it took 50-times the concentration of PTU to produce a comparable inhibition in the monkey. Sulfomonomethoxine was almost as potent as PTU in inhibiting the peroxidase in rats with a factor of x 2.5. However, it required about 500-times the concentration of sulphonamide to inhibit the peroxidase in the monkey compared to the rat.
Studies such as these with sulfonamides demonstrate distinct species differences between rodents and primates in the response of the thyroid to chemical inhibition of hormone synthesis. It is not surprising that the sensitive species (e.g. rat, mouse, dog) are much more likely to follicular cell hyperplasia and thyroid nodules after long-term exposure to sulfonamides than the resistant species (e.g. subhuman primate, humans, guinea pig, chicken).
A contemporary example of a chemical acting as a thyroperoxidase inhibitor is sulfamethazine. This is a widely used antibacterial compound in food-producing animals with a current permissible tissue residue level of 100 ppb. Recently completed carcinogenicity studies at NCTR reported a significant increase of thyroid tumours in male Fischer-344 rats administered the high dose of sulfamethazine (2400 ppm). The incidence of thyroid tumours was increased in both male and female B6C1F3mice after 2 years in the high dose group (4800 ppm), but not in the lower dose group. Quantitative risk assessment based on these new carcinogenicity findings, using low-dose linear extrapolation, yielded a 1 x 106lifetime risk of 90 ppb in female rats and 40 ppb in male rats. A consideration of the ratio of intact drug to metabolites further reduced the tissue residue level to 0.4 ppb, which would be unachievable in practice.
In collaboration with other research groups, a number of mechanistic studies were performed with the objective of developing a database that would support the hypothesis that the thyroid tumours observed in rats and mice from chronic studies were secondary to hormonal imbalances following the administration of high doses of sulfamethazine. In a 4-week mechanistic study, the effects of 10 dose levels of sulfamthazine, spanning the range that induced thyroid tumours in rodents, were evaluated on thyroid hormone economy in Sprague-Dawley rats. There was a characteristic log-dose response relationship in all parameters of thyroid function evaluated. There were no significant changes at the six lower doses ( 20 > 800 ppm) of sulfamethazine, followed by sharp relatively linear changes at the 4 higher dose levels (1600 > 12000 ppm) in percentage of decrease of serum T3 and T4, increase in serum TSH, and increase in thyroid weight. A similar, non-linear dose-response was present in the morphologic changes of thyroid follicular cells following the feeding of varying levels of sulfamethazine. Follicular cell hypertrophy was observed at lower doses of sulfamethazine than hyperplasia, which was increased only at doses levels of 3300 ppm and above. Other mode-of-action studies have demonstrated sulfamethazine to be a potent inhibitor of thyroperoxidase in rodents with an IC50of 1.2 x 10-6M. The morphologic effects on the thyroid were reversible after withdrawal of compound, and supplemental T4 in the diet inhibited the development of the functional and morphologic changes in the thyroid follicular cells. Hypophysectomized rats (with no TSH) administered sulfamethazine did not develop morphologic changes in the thyroid. Sulfamethazine did not increase thyroid cell proliferationin vitroin the absence of TSH, and there was no effect on thyroid structure/function in cynomolgus monkeys administered sulfamethyzine. Non-human primates and humans are known to be more resistant than rodents to the inhibition of thyroperoxidase.
Inhibition of thyroid hormone secretion: (1) Blockage of thyroid hormone release by excess iodide or lithium: Relatively few chemicals selectively inhibit the secretion of thyroid hormone from the thyroid gland. An excess of iodide has been known for years to inhibit the secretion of thyroid hormone and occasionally can result in goitre and hypothyroidism in animals and human patients. High doses of iodide have been used therapeutically in the treatment of patients with Graves Disease and hypothyroidism to lower circulating levels of thyroid hormones. Several mechanisms have been suggested for this effect of high iodide levels on the thyroid hormone secretion including a decrease in lysosomal protease activity (human glands), inhibition of colloid droplet formation (mice and rats), and inhibition of TSH-mediated increase in cAMP (dog thyroid slices). Rats fed an iodine-excess diet had a hypertrophy of the cytoplasmic area of follicular cells with an accumulation of numerous colloid droplets and lysosomal bodies. However, there was limited evidence ultrastructurally of fusion of the membranes of these organelles and degradation of the colloidnecessary for the release of T4 and T3 from the thyroglobulin. Circulating levelsof T3, T4 and rT3 are all decreased by an iodide excess.
Lithium also has been reported to have a striking inhibitory effect on thyroid hormone release. The widespread use of lithium carbonate in the treatment of manic states occasionally results in the development of goitre with either euthyroidism or occasionally hypothyroidism in human patients. Lithium inhibits colloid droplet formation stimulated by cAMPin vitroand inhibits the release of thyroid hormones.
(2) Xenobiotic-induced thyroid pigmentation or alterations in colloid: The antibiotic minocycline produces a stiking black discoloration of the thyroid lobes in laboratory animals and humans with the formation of brown pigment granules within follicular cells. The pigment granules stain similarly to melanin and are best visualized on thyroid sections stained with the Fontana-Masson procedure. Electron-dense material first accumulates in lysosomes-like granules and in the rough endoplasmic reticulum. The pigment appears to be a metabolic derivative of minocycline, and the administration of the antibiotic at high dose rats for extended periods may result in a disruption of thyroid function and the development of goitre. The release of T4 from perfused thyroids of minocycline-treated rats was significantly decreased, but the follicular cells retained the ability to phagocytose colloid in response to TSH and had numerous colloid droplets in their cytoplasm.
Other xenobiotics selectively localize in the thyroid colloid of rodents, resulting in abnormal clumping and increased basophilia to the colloid. Brown to black pigment granules may be present in follicular cells, colloid, and macrophages in the interthyroidal tissues resulting in a macroscopic darkening of both thyroid lobes. The physicochemically altered colloid in the lumen of thyroid follicles appears to be less able than normal colloid of either reacting with organic iodine in a step-wise manner to result in the orderly synthesis of iodothyronines or being phagocytised by follicular cells and enzymatically processed to release active thyroid hormones into the circulation. Serum T3 and T4 are decreased, serum TSH levels are increased by an expanded population of pituitary thyrotrophs, and thyroid follicular cells undergo hypertrophy and hyperplasia. As would be expected, the incidence of thyroid follicular cell tumours in 2-year carcinogenicity studies is significantly increased at the higher dose levels, usually with a greater effect in males than in females. Autoradiographic studies often demonstrate tritiated material to be preferentially localized in the colloid and not within follicular cells. Tissue distribution studies with 14C-labeled compound may reveal preferential uptake and persistence in the thyroid gland compared to other tissues. However, thyroperoxidase activity is normal, and the thyroids ability to take up radioactive iodine often is increased compared to controls in response to the greater circulating levels of TSH. Similar thyroid changes and/or functional alterations usually do not occur in dogs, monkeys or humans.
Secondary mechanisms of thyroid oncogenesis
Understanding the mechanism of action of xenobiotics on the thyroid gland provides a more rational basis to extrapolate findings from long-term rodent studies to safety assessment of a particular compound for humans. Many chemicals and drugs disrupt one or more steps in the synthesis and secretion of thyroid hormones, resulting in subnormal levels of T4 and T3 associated with a compensatory increased secretion of pituitary TSH. When tested in highly sensitive species, such as rats and mice, these compounds result early in follicular cell hypertrophy/hyperplasia and increased thyroid weights and in long-term studies an increased incidence of thyroid tumours by a secondary (indirect) mechanism.
In the secondary mechanism of thyroid oncogenesis in rodents, the specific xenobiotic chemical or physiologic perturbation evokes another stimulus (e.g. chronic hypersecretion of TSH) that promotes the development of nodular proliferative lesions (initially hypertrophy, followed by hyperplasia, subsequently adenomas, infrequently carcinomas) derived from follicular cells. Thresholds for a no-effect on the thyroid gland can be established by determining the dose of xenobiotic that fails to elicit an elevation in the circulating level of TSH. Compounds acting by this indirect (secondary) mechanism with hormonal imbalance usually have little or no evidence for mutagenicity or for producing DNA damage.
In human patients with markedly altered changes in thyroid function and elevated TSH levels, as in areas with a high incidence of endemic goitre due to iodine deficiency, there is little if any increase in the incidence of thyroid cancer. The relative resistance to the development of thyroid cancer in humans with elevated plasma TSH levels is in marked contrast to the response of the thyroid gland to chronic TSH stimulation in rats and mice. The human thyroid is much less sensitive to this pathogenic phenomenon than rodents.
Human patients with congenital defects in thyroid hormone synthesis (dyshormonogenetic goitre) and markedly increased circulating TSH levels have been reported to have an increased evidence of thyroid carcinomas. Likewise, thyrotoxic patients with Graves Disease where follicular cells are autonomously stimulated by an immunoglobulin (long-acting thyroid stimulator, LATS) also appear to be at greater risk to develop thyroid tumours. In summary, the literature suggests that prolonged stimulation of the thyroid by TSH will induce neoplasia only in exceptional circumstances and possibly acting together with some other metabolic or immunologic abnormality.
Table 6.10/02-1: Thyroxine (T4) binding to serum proteins in selected vertebrate species
Species |
T4-binding globulin |
Postalbumin |
Albumin |
Prealbumin |
Human |
++ |
- |
++ |
+ |
Monkey |
++ |
- |
++ |
+ |
Dog |
+ |
- |
++ |
- |
Mouse |
- |
++ |
++ |
- |
Rat |
- |
+ |
++ |
+ |
Chicken |
- |
- |
++ |
- |
+ or ++ degree of T4-binding to serum protein
- absence of T4 binding to serum protein
Table 6.10/02-2: Triiodothyronine (T3) binding to serum proteins in selected vertebrate species
Species |
T4-binding globulin |
Postalbumin |
Albumin |
Prealbumin |
Human |
+ |
- |
+ |
- |
Monkey |
+ |
- |
+ |
- |
Dog |
+ |
- |
+ |
- |
Mouse |
- |
+ |
+ |
- |
Rat |
- |
- |
+ |
- |
Chicken |
- |
- |
+ |
- |
+ or ++ degree of T4-binding to serum protein
- absence of T4 binding to serum protein
Table A6.10/14-1: Mean daily food consumption
Group |
Food consumption [g] |
|||||||
Sodium bromide [g/kg bw] |
Type of diet |
Sex |
Week 1 |
Week 2 |
Week 3 |
Week 4 |
Week 5 |
Week 6 |
pretreatment period |
treatment period |
|||||||
0 |
Normal |
male |
20.4 |
26.5 |
26.4 |
25.1 |
23.8 |
17.3 |
female |
17.5 |
18.2 |
19.3 |
18.6 |
17.7 |
14.4 |
||
0 |
Iodine-poor |
male |
19.2 |
23.2 |
23.2 |
20.2 |
20.7 |
19.3 |
female |
16.8 |
17.2 |
25.4 |
14.3 |
15.5 |
15.5 |
||
4 |
Iodine-poor |
male |
20.0 |
24.8 |
21.9 |
16.6 |
18.1 |
16.0 |
female |
16.5 |
15.7 |
15.6 |
17.0 |
18.4 |
14.4 |
||
8 |
Iodine-poor |
male |
19.8 |
24.3 |
19.7 |
13.3 |
15.7 |
13.9 |
female |
16.7 |
15.9 |
15.2 |
13.6 |
15.2 |
12.2 |
||
16 |
Iodine-poor |
male |
19.5 |
24.8 |
12.9 |
7.9 |
10.7 |
- |
female |
16.9 |
17.3 |
9.1 |
7.7 |
7.2 |
- |
||
Table A6.10/14-2: Medians and range values of urinary iodide excretion [µg/L]
group |
|
day of study |
||||||
Sodium bromide [g/kg bw] |
Type of diet |
1 |
14 |
22 |
29 |
36 |
43 |
|
0 |
Normal |
mean |
252 |
150 |
345 |
224 |
231 |
347 |
range |
98-421 |
54-406 |
267-497 |
164-462 |
217-564 |
264-466 |
||
0 |
Iodine-poor |
mean |
166 |
13 |
8 |
7 |
13 |
9 |
range |
77-362 |
9-23 |
5-17 |
3-28 |
7-17 |
6-13 |
||
4 |
Iodine-poor |
mean |
170 |
18 |
12 |
11 |
16 |
14 |
range |
86-448 |
11-25 |
9-18 |
7-45 |
5-22 |
12-18 |
||
8 |
Iodine-poor |
mean |
112 |
19 |
12 |
16 |
14 |
17 |
range |
46-386 |
1-29 |
10-18 |
8-18 |
10-24 |
6-22 |
||
16 |
Iodine-poor |
mean |
333 |
16 |
18 |
15 |
17 |
- |
range |
272-455 |
6-24 |
12-52 |
13-36 |
8-26 |
- |
||
Table A6.10 /14-3: Medians and range values of the concentrations of T3, reverse T3 and T4 in the thyroid
Group |
mg/kg wet weight |
||||
Sodium bromide [g/kg bw] |
Type of diet |
||||
T3 |
Reverse T3 |
T4 |
|
||
0 |
Normal |
19.6 (12.7-23.7) |
5.5 (4.5-8.1) |
143 (101-182) |
|
0 |
Iodine-poor |
7.1 (5.3-21.7) |
<2 |
28.0 (17.1-47.0) |
|
4-16 |
Iodine-poor |
<2 |
<2 |
<2 |
|
Table A6.10/09-1: Body Weight and Relative Organ Weights of male Rats Fed Sodium Bromide in the Diet for 4 or 12 weeks
Dietary concentration of NaBr [mg/kg] |
Bodyweight [g] |
Relative weight [g/100 g bodyweight] of: |
||
Pituitary gland |
Thyroid |
Testes |
||
4-week exposure |
||||
0 |
183 ± 25 |
0.0044 ± 0.0007 |
0.0065 ± 0.0015 |
0.967 ± 0.162 |
20 |
187 ± 22 |
0.0043 ± 0.0011 |
0.0075 ± 0.0011 |
0.989 ± 0.110 |
75 |
190 ± 22 |
0.0042 ± 0.0009 |
0.0074 ± 0.0014 |
0.989 ± 0.091 |
300 |
186 ± 17 |
0.0042 ± 0.0008 |
0.0070 ± 0.0009 |
0.983 ± 0.069 |
1200 |
189 ± 19 |
0.0042 ± 0.0007 |
0.0090 ± 0.0013 |
0.968 ± 0.082 |
0 |
243 ± 16 |
0.0038 ± 0.0010 |
0.0086 ± 0.0018 |
0.921 ± 0.066 |
19200 |
220 ± 21 |
0.0044 ± 0.0013 |
0.0111 ± 0.0021 |
0.886 ± 0.182 |
12-week exposure |
||||
0 |
314 ± 25 |
0.0035 ± 0.0003 |
0.0064 ± 0.0014 |
0.799 ± 0.089 |
20 |
309 ± 25 |
0.0026 ± 0.0006 |
0.0071 ± 0.0014 |
0.835 ± 0.031 |
75 |
314 ± 30 |
0.0035 ± 0.0006 |
0.0061 ± 0.0019 |
0.818 ± 0.068 |
300 |
314 ± 20 |
0.0032 ± 0.0004 |
0.0067 ± 0.0019 |
0.824 ± 0.069 |
1200 |
309 ± 10 |
0.0036 ± 0.0004 |
0.0071 ± 0.0017 |
0.786 ± 0.087 |
0 |
322 ± 26 |
0.0033 ± 0.0005 |
0.0071 ± 0.0016 |
0.772 ± 0.094 |
19200 |
254 ± 28 |
0.0036 ± 0.0005 |
0.0129 ± 0.0023 |
0.681 ± 0.130 |
Table A6.10/09-2: Immunocytochemical findings in the pituitary gland of male Rats Fed Sodium Bromide in the Diet for 4 or 12 weeks
Immuncytochemical staining |
Findings after exposure for: |
|||
4 weeks |
12 weeks |
|||
0 mg NaBr/kg diet |
19200 mg NaBr/kg diet |
0 mg NaBr/kg diet |
19200 mg NaBr/kg diet |
|
Immunoreactive GH cells |
||||
Decrease |
4 |
6 |
4 |
6 |
Normal |
6 |
4 |
6 |
4 |
Immunoreactive TSH cells |
||||
Decrease |
3 |
3 |
3 |
1 |
Normal |
3 |
4 |
6 |
3 |
Slight increase |
4 |
3 |
0 |
0 |
Strong increase |
0 |
0 |
0 |
5 |
Immunoreactive ACTH cells |
||||
Decrease |
2 |
3 |
4 |
1 |
Normal |
5 |
4 |
5 |
3 |
Slight increase |
3 |
3 |
1 |
4 |
Strong increase |
0 |
0 |
0 |
2 |
Table A6.10/09-3: Levels of Thyroxine (T4) and Thyroid-Stimulating Hormone (TSH) in the Serum of male Rats Fed Sodium Bromide in the Diet for 4 or 12 weeks
Dietary concentration of NaBr [mg/kg] |
Serum concentration after exposure for: |
|||
4 weeks |
12 weeks |
|||
T4 [mmol/L] |
TSH [µg/L] |
T4 [mmol/L] |
TSH [µg/L] |
|
0 |
141 ± 21 |
142 ± 47 |
103 ± 12 |
186 ± 64 |
20 |
133 ± 24 |
120 ± 41 |
116 ± 23 |
176 ± 55 |
75 |
143 ± 18 |
155 ± 141 |
111 ± 9 |
196 ± 65 |
300 |
125 ± 14 |
100 ± 44 |
125 ± 18 |
265 ± 128 |
1200 |
109 ± 22 |
165 ± 79 |
114 ± 20 |
259 ± 189 |
0 |
128 ± 8 |
193 ± 113 |
111 ± 14 |
194 ± 70 |
19200 |
54 ± 9 |
420 ± 205 |
42 ± 9 |
935 ± 348 |
TRH (thyrotropin-releasing hormone) test |
||||
0 |
- |
- |
98 ± 23 |
442 ± 170 |
19200 |
- |
- |
45 ± 14 |
1148 ± 201 |
Table A6.10/09-4: Levels of Follicle-Stimulating Hormone (FSH), Luteinizing Hormone (LH) and Testosterone (T) in the Serum of male Rats Fed Sodium Bromide in the Diet for 4 or 12 weeks
Dietary concentration of NaBr [mg/kg] |
Serum concentration after exposure for: |
|||||
4 weeks |
12 weeks |
|||||
FSH [µg/L] |
LH [µg/L] |
T [nmol/L] |
FSH [µg/L] |
LH [µg/L] |
T [nmol/L] |
|
0 |
455 ± 80 |
42 ± 33 |
ND |
228 ± 44 |
24 ± 8 |
ND |
20 |
384 ± 72 |
37 ± 19 |
ND |
266 ± 38 |
18 ± 7 |
ND |
75 |
427 ± 60 |
32 ± 13 |
ND |
256 ± 47 |
20 ± 6 |
ND |
300 |
419 ± 107 |
37 ± 32 |
ND |
250 ± 48 |
24 ± 10 |
ND |
1200 |
438 ± 115 |
30 ± 22 |
ND |
276 ± 46 |
24 ± 6 |
ND |
0 |
540 ± 60 |
32 ± 16 |
15 ± 7.1 |
255 ± 46 |
27 ± 7 |
10.8 ± 5.8 |
19200 |
630 ± 100 |
17 ± 11 |
7.1 ± 3.4 |
482 ± 117 |
27 ± 12 |
5 ± 2.9 |
TRH (thyrotropin-releasing hormone) test |
||||||
0 |
- |
- |
- |
248 ± 31 |
47 ± 23 |
6.9 ± 3.0 |
19200 |
- |
- |
- |
469 ± 137 |
56 ± 13 |
3.1 ± 1.2 |
ND not determined
Table A6.10/09-5: Histopathological and immunocytochemical findings in the thyroid and testes of male Rats Fed Sodium Bromide in the Diet for 4 or 12 weeks
Criterion |
Findings after exposure for: |
||||||||||||||
|
4 weeks |
12 weeks |
|||||||||||||
NaBr [mg/kg diet] |
0 |
20 |
75 |
300 |
1200 |
19200 |
0 |
20 |
75 |
300 |
1200 |
19200 |
|||
THYROID |
|||||||||||||||
Haematoxylin-Eosin staining: degree of activation |
No. of examined |
20 |
10 |
10 |
10 |
10 |
10 |
20 |
10 |
10 |
10 |
10 |
10 |
||
- slight |
|
2 |
1 |
1 |
0 |
0 |
1 |
2 |
2 |
3 |
0 |
2 |
0 |
||
- moderate |
|
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
0 |
2 |
||
- strong |
|
0 |
0 |
0 |
0 |
0 |
9 |
0 |
0 |
0 |
0 |
0 |
8 |
||
Immunocytochemical staining: Intensity of T4 staining |
No. of examined |
20 |
0 |
0 |
0 |
10 |
10 |
19 |
0 |
0 |
0 |
10 |
10 |
||
-decrease |
|
1 |
- |
- |
- |
|
9 |
2 |
- |
- |
- |
0 |
7 |
||
- normal |
|
19 |
- |
- |
- |
10 |
1 |
17 |
- |
- |
- |
10 |
3 |
||
TESTES |
|||||||||||||||
Haematoxylin-Eosin staining: seminiferous tubules |
No. of examined |
20 |
10 |
10 |
10 |
10 |
10 |
20 |
10 |
10 |
10 |
10 |
10 |
||
- some atrophied |
|
0 |
0 |
0 |
0 |
0 |
1 |
0 |
0 |
0 |
0 |
0 |
4 |
||
- several atrophied |
|
0 |
0 |
0 |
0 |
0 |
0 |
0 |
0 |
1 |
1 |
0 |
3 |
||
Table A6.10/09-6: Levels of Growth Hormone (GH), Insulin and Corticosterone (C) in the Serum of male Rats Fed Sodium Bromide in the Diet for 4 or 12 weeks
Dietary concentration of NaBr [mg/kg] |
Serum concentration after exposure for: |
|||||
4 weeks |
12 weeks |
|||||
GH [µg/L] |
Insulin [mlU/L] |
C [nmol/L] |
GH [µg/L] |
Insulin [mlU/L] |
C [nmol/L] |
|
0 |
557 ± 868 |
ND |
119 ± 44 |
474 ± 314 |
11 ± 12 |
163 ± 135 |
20 |
445 ± 595 |
ND |
102 ± 15 |
364 ± 405 |
14 ± 13 |
244 ± 278 |
75 |
596 ± 822 |
ND |
95 ± 21 |
363 ± 436 |
10 ± 6 |
137 ± 99 |
300 |
673 ± 528 |
ND |
112 ± 31 |
203 ± 162 |
12 ± 11 |
121 ± 75 |
1200 |
476 ± 545 |
ND |
118 ± 26 |
603 ± 794 |
8 ± 4 |
105 ± 56 |
0 |
200 ± 276 |
21 ± 17 |
208 ± 152 |
525 ± 645 |
25 ± 13 |
299 ± 334 |
19200 |
368 ± 233 |
51 ± 15 |
89 ± 32 |
56 ± 62 |
41 ± 9 |
149 ± 187 |
TRH (thyrotropin-releasing hormone) test |
||||||
0 |
- |
- |
- |
536 ± 487 |
16 ± 9 |
780 ± 211 |
19200 |
- |
- |
- |
33 ± 22 |
36 ± 9 |
530 ± 262 |
ND not determined
Table 6.10/03-1: Plasma bromide concentration in human volunteers ingesting daily 1 mg bromide/kg bw as NaBr capsules
Week: |
Bromide concentration [mmol/L plasma] |
||||
0 |
2 |
4 |
6 |
8 |
|
Females |
0.08± 0.01 |
0.39± 0.1 |
0.68± 0.07 |
0.78± 0.17 |
0.97± 0.18 |
Males |
0.08± 0.01 |
0.5± 0.06 |
0.74± 0.1 |
0.89± 0.11 |
0.83± 0.09 |
Table 6.10/03-2: Bromide, chloride and creatinine excretion in urine of human volunteers before and after ingestion of a daily dose of 1 mg bromide/kg bw as NaBr capsules during 8 weeks
Week: |
Bromide excretion [mg NaBr/24h] |
Chloride excretion [g NaCl/24 h] |
Creatinine excretion [mmol creatinine/24h] |
|||
start |
end |
start |
end |
start |
end |
|
Females |
9.8± 2.9 |
90.6± 30.2 |
8.9± 2.2 |
8,7± 3.1 |
11.4± 1.9 |
11.3± 2.2 |
Males |
9.5± 4.2 |
79.7± 18.5 |
8.5± 2.6 |
8.6± 2.2 |
16.2± 2.4 |
15.1± 2.3 |
Table A6.10/05-1:Bromide concentration in plasma of healthy volunteers before, directly after and 3 full cycles after administration of bromide for 3 full cycles
Bromide Dosage [mg bromide/kg bw/day] |
Amount of bromide [mmol/L] |
||
Cycle 0 |
Cycle 3 |
Cycle 6 |
|
0 |
0.06±0.01 |
0.07±0.02 |
0.09±0.02 |
4 |
0.05±0.01 |
3.22±0.93 |
0.11±0.05 |
9 |
0.05±0.01 |
7.99±1.89 |
0.1±0.02 |
Table A6.10/05-2:T4 concentration in serum of healthy volunteers before, directly after and 3 full cycles after administration of bromide for 3 full cycles
Bromide Dosage [mg bromide/kg bw/day] |
Amount of T4 [nmol/L] |
||
Cycle 0 |
Cycle 3 |
Cycle 6 |
|
0 |
129±12 |
132±18 |
132±18 |
4 |
128±21 |
127±24 |
129±21 |
9 |
146±18 |
143±23 |
139±21 |
Table A6.10/05-3:T3 concentration in serum of healthy volunteers before, directly after and 3 full cycles after administration of bromide for 3 full cycles
Bromide Dosage [mg bromide/kg bw/day] |
Amount of T3 [nmol/L] |
||
Cycle 0 |
Cycle 3 |
Cycle 6 |
|
0 |
1.8±0.2 |
1.9±0.3 |
2.0±0.3 |
4 |
1.9±0.2 |
1.9±0.3 |
1.9±0.2 |
9 |
2.2±0.5 |
2.0±0.4 |
2.1±0.7 |
Table A6.10/04-1:Mean plasma bromide levels in volunteers given capsules containing 0, 4 or 9 mg bromide/kg/day as sodium bromide for 12 weeks or over 3 full menstrual cycles
Time [week] |
Dose [mg/kg bw/day] |
Bromide concentration [mmol/L plasma] |
|||||
Males |
Females |
||||||
0 |
4 |
9 |
0 |
4 |
9 |
||
No./group |
7 |
7 |
7 |
7 |
4 |
6 |
|
0 |
|
0.07±0.01 |
0.07±0.01 |
0.08±0.01 |
0.06±0.01 |
0.07±0.02 |
0.07±0.01 |
2 |
0.07±0.01 |
1.43±0.2 |
2.88±0.56 |
0.07±0.01 |
1.51±0.31 |
3.02±0.71 |
|
4 |
0.06±0.02 |
1.6±0.44 |
3.98±0.32 |
0.07±0.02 |
1.97±0.31 |
3.87±0.72 |
|
6 |
0.08±0.02 |
2.22±0.78 |
4.39±0.4 |
0.07±0.02 |
3.04±0.38 |
4.68±0.8 |
|
8 |
0.07±0.02 |
2.06±0.43 |
4.99±0.77 |
0.07±0.02 |
2.72±0.41 |
4.7±0.94 |
|
10 |
0.07±0.02 |
2.16±0.36 |
4.89±0.64 |
0.06±0.01 |
2.95±0.42 |
4.94±1.68 |
|
12 |
0.08±0.02 |
2.14±0.72 |
4.3±0.71 |
0.07±0.01 |
3.05±0.82 |
4.93±1.07 |
|
Table A6.10/04-2: Mean bromide, chloride and creatinine excretion in 24-hour urine samples from volunteers before and after ingestion of capsules containing 0, 4 or 9 mg bromide/kg bw/day for 12 weeks or over 3 full menstrual cycles
Bromide dose [mg/kg bw/day] |
No./group |
Excretion [mmol/24 hours] |
|||||
Start of test |
End of test |
||||||
Bromide |
Chloride |
Creatinine |
Bromide |
Chloride |
Creatinine |
||
MALES |
|||||||
0 |
7 |
0.07±0.03 |
152±91 |
14.9±5.5 |
0.11±0.04 |
202±75 |
13.3±3.4 |
4 |
7 |
0.07±0.04 |
126±68 |
15.2±4.6 |
2.41±1.62 |
177±81 |
13.8±2.1 |
9 |
7 |
0.09±0.03 |
176±71 |
15.3±1.6 |
6.17±3.31 |
208±82 |
14.8±1.0 |
FEMALES |
|||||||
0 |
7 |
0.06±0.03 |
85±35 |
9.6±2.3 |
0.06±0.03 |
118±46 |
10.0±5.0 |
4 |
4 |
0.08±0.04 |
145±54 |
13.8±4.3 |
2.61±0.45 |
109±33 |
10.8±4.0 |
9 |
6 |
0.08±0.03 |
141±53 |
11.6±2.4 |
2.69±1.61 |
103±27 |
9.6±2.8 |
Table A6.10/04-3: Serum concentration of thyroxine (T4) and triiodothyronine (T3) in volunteers before and after ingestion of 0, 4 or 9 mg bromide/kg bw/day for 12 weeks or 3 full menstrual cycles
Determination |
Bromide dose [mg/kg bw/day] |
Serum concentrations |
|||||
Males |
Females |
||||||
Start of test |
End of test |
Log (end/strt) |
Start of test |
End of test |
Log (end/start) |
||
T4 [nmol/L] |
0 |
104±24 |
11±15 |
0.035±0.072 |
119±25 |
117±22 |
-0.005V0.043 |
4 |
114±14 |
120±20 |
0.018±0.057 |
123±14 |
122±12 |
-0.002±0.027 |
|
9 |
108±16 |
108±12 |
0.002±0.036 |
115±11 |
131±15 |
0.056±0.046* |
|
T3 [nmol/L] |
0 |
1.7±0.3 |
1.9±0.2 |
0.049±0.06 |
1.6±0.2 |
1.8±0.3 |
0.048±0.056 |
4 |
1.9±0.4 |
1.9±0.4 |
-0.003±0.087 |
1.8±0.2 |
1.8±0.2 |
0.001±0.075 |
|
9 |
1.8±0.2 |
1.9±0.2 |
0.028±0.055 |
1.8±0.2 |
2.1±0.2 |
0.078±0.021* |
|
* 0.001< two sided P < 0.01
Table A6.10/04-4: Significant variables of different cortical areas with respect to the differences in the absolute powers of montage 2
Parameter |
Area… Hemisphere… |
Significant Variables |
|||||||||||
Frontal-Central |
Frontal-Temporal |
Central-Parietal |
Temporal-Temporalparietal |
Parietal-Occipital |
Temporalparietal-Occipital |
||||||||
Left |
Right |
Left |
Right |
Left |
Right |
Left |
Right |
Left |
Right |
Left |
Right |
||
Log power δ1-band |
|
0.04 |
0.02 |
0.06 |
0.08 |
0.06 |
0.07 |
0.1 |
- |
- |
- |
- |
- |
Log power δ2–band |
- |
0.05 |
0.04 |
- |
0.02 |
0.09 |
0.07 |
- |
- |
- |
- |
- |
|
Log power θ–band |
- |
- |
- |
- |
0.04 |
0.1 |
0.07 |
- |
- |
- |
- |
- |
|
Log power α1–band |
- |
0.03 |
0.02 |
0.09 |
0.04 |
0.01 |
0.07 |
0.02 |
0.09 |
- |
0.09 |
0.03 |
|
Log power β2–band |
0.04 |
0.08 |
0.07 |
0.01 |
- |
- |
- |
0.06 |
- |
- |
- |
- |
|
Log power β3–band |
- |
- |
0.07 |
0.02 |
- |
- |
- |
0.08 |
- |
- |
- |
- |
|
Log total power |
- |
0.01 |
- |
0.1 |
- |
- |
- |
- |
- |
- |
- |
- |
|
Quotient θ/α |
- |
0.07 |
0.1 |
- |
0.04 |
0.02 |
- |
0.04 |
- |
- |
0.04 |
0.07 |
|
Activity |
0.1 |
0.01 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
Mobility |
0.03 |
0.02 |
0.08 |
0.02 |
0.02 |
- |
- |
0.02 |
- |
- |
- |
- |
|
Complexity |
- |
- |
0.1 |
- |
- |
- |
- |
- |
- |
- |
- |
- |
|
Table A6.10/04-5: Means of variables for each cortical area of montage 2 for groups of 14, 13 and 11 volunteers taking 0, 4 or 9 mg bromide/kg bw/day for 12 weeks (males) or during three full cycles
Parameter |
Bromide dose [mg/kg bw/day] |
Mean Values ± SD for areas: |
|||||||
Frontal-Central |
Frontal-Temporal |
Temporal-Temporalparietal |
Central-Parietal |
||||||
Left |
Right |
Left |
Right |
Left |
Right |
Left |
Right |
||
Log power δ1-band |
0 |
0.078 ± 0.1.05 |
0.083 ± 0.131 |
0.18 ± 0.152 |
0.113 ± 0.161 |
0.154 ± 0.16 |
0.1 ± 0.107 |
0.058 ± 0.09 |
0.09 ± 0.079 |
4 |
0.11 ± 0.162 |
0.106 ± 0.154 |
0.225 ± 0.211 |
0.131 ± 0.33 |
0.092 ± 0.132 |
0.105 ± 0.112 |
0.086 ± 0.159 |
0.094 ± 0.12 |
|
9 |
0.268 ± 0.24 |
0.266 ± 0.266 |
0.363 ± 0.266 |
0.398 ± 0.436 |
0.223 ± 0.216 |
0.228 ± 0.246 |
0.162 ± 0.097 |
0.182 ± 0.121 |
|
Log power δ2–band |
0 |
0.047 ± 0.082 |
0.048 ± 0.14 |
0.097 ± 0.161 |
-0.004 ± 0.182 |
0.001 ± 0.129 |
0.015 ± 0.105 |
0.057 ± 0.081 |
0.06 ± 0.061 |
4 |
0.057 ± 0.128 |
0.073 ± 0.062 |
0.093 ± 0.108 |
0.051 ± 0.114 |
0.104 ± 0.118 |
0.094 ± 0.051 |
0.076 ± 0.145 |
0.087 ± 0.094 |
|
9 |
0.145 ± 0.117 |
0.144 ± 0.143 |
0.176 ± 0.111 |
0.16 ± 0.173 |
0.146 ± 0.149 |
0.071 ± 0.094 |
0.15 ± 0.105 |
0.155 ± 0.095 |
|
Log power α1–band |
0 |
0.027 ± 0.139 |
0.026 ± 0.16 |
-0.036 ± 0.213 |
-0.006 ± 0.218 |
-0.013 ± 0.267 |
0.014 ± 0.281 |
0.011 ± 0.208 |
0.035 ± 0.248 |
4 |
0.228 ± 0.244 |
0.17 ± 0.216 |
0.17 ± 0.274 |
0.21 ± 0.246 |
0.287 ± 0.27 |
0.25 ± 0.26 |
0.293 ± 0.256 |
0.273 ± 0.245 |
|
9 |
0.16 ± 0.161 |
0.088 ± 0.118 |
0.072 ± 0.205 |
0.023 ± 0.117 |
0.167 ± 0.225 |
0.121 ± 0.211 |
0.177 ± 0.216 |
0.174 ± 0.199 |
|
Log power β2-band |
0 |
0.034 ± 0.11 |
0.018 ± 0.08 |
-0.035 ± 0.197 |
0.074 ± 0.29 |
0.092 ± 0.22 |
0.133 ± 0.302 |
0.008 ± 0.094 |
0.047 ± 0.077 |
4 |
0.113 ± 0.156 |
0.063 ± 0.141 |
0.159 ± 0.388 |
0.223 ± 0.458 |
0.164 ± 0.357 |
0.155 ± 0.313 |
0.079 ± 0.161 |
0.071 ± 0.139 |
|
9 |
0.001 ± 0.108 |
0.049 ± 0.096 |
-0.215 ± 0.289 |
-0.132 ± 0.279 |
-0.098 ± 0.242 |
-0.066 ± 0.3349 |
0.019 ± 0.098 |
-0.01 ± 0.099 |
|
Mobility |
0 |
-0.158 ± 1.074 |
-0.537 ± 1.031 |
-0-847 ± 2.457 |
-0.222 ± 2.934 |
0.342 ± 2.442 |
0.338 ± 2.442 |
-0.057 ± 0.679 |
-0.168 ± 0.524 |
4 |
0.342 ± 2.064 |
-0.297 ± 0.984 |
00.515 ± 5.193 |
0.706 ± 5.678 |
0.290 ± 3.445 |
0.202 ± 3.689 |
-0.133 ± 1.21 |
0.069 ± 0.828 |
|
9 |
-1.455 ± 1.45 |
-1.513 ± 1.62 |
-5.063 ± 4.67 |
-3.352 ± 6.431 |
-2.435 ± 2.285 |
-1.273 ± 4.162 |
-0.665 ± 0.631 |
-0.795 ± 0.55 |
|
Altered thyroid gland function and thyroid neoplasia:
Two basic mechanisms are involved in thyroid carcinogenesis, the first of which involves chemicals that exert a direct carcinogenic effect on the thyroid gland (e.g. polycylic hydrocarbons, 2-acetylaminofluorene, dichlorbenzidine and a variety of nitrosamines). The second basic mechanism is the production of thyroid tumors by a variety of regimes that result in a hormone imbalance. Numerous studies performed in rats have shown that treatment with anti-thyroid substances (thiourea, thiouracil and their derivatives, 3-amino-1, 2, 4-triazole) will result in a high incidence of thyroid tumours in rats. Anti-thyroid drugs initially produce a hormonal imbalance by interfering with thyroid hormone production. As a result, a sustained increase in the synthesis and secretion of TSH occurs via the negative feedback system of the pituitary gland to stimulate thyroid function. Increased TSH stimulation produces a variety of morphological and functional changes in the follicular cell including follicular cell hypertrophy, hyperplasia and neoplasia. The sustained excessive level of TSH is considered to be the pathogenic factor responsible for thyroid tumour formation.
That excessive secretion of endogenous TSH alone will produce a high incidence of thyroid tumours has been clearly established by experiments in which rats were fed diets deficient in iodine or in which TSH-producing tumours were transplanted into mice with normal thyroids. Iodine deficient diets are goitrogenic and result in an increased TSH secretion with concomitant high incidence of thyroid tumours. These effects can be reversed by iodine supplementation, thyroid hormone replacement or hypophysectomy. Goitrogenic substances or regimes are also powerful promoters of thyroid gland neoplasia after administration of direct acting carcinogenic substances.
Thyroid hormone synthesis:
The functional unit of the thyroid gland is the follicle which consists of an area of colloid, the storage form of thyroid hormone, surrounded by a single layer of follicular epithelium. Hormone synthesis and storage are extracellular processes that occur at the spinal surface of the cell membrane. Thyroid hormone synthesis involves the active transport of iodine into the cell and a peroxidise-mediated iodination and coupling of thyrosine residues on thyroglobulin to form thyroid hormone which is stored as a colloid until released into the circulation. Thyroid hormone synthesis involves reactive biochemical processes, and the organisation of thyroid follicle structure and function serves to protect the cell from accidental iodination and cytotoxicity from oxidation products.
Thyroid hormone release involves pinocytosis of colloid by microvilli and digestion by lysosomes to release T4 and T3 which are secreted into plasma and bound to plasma proteins for transport to peripheral tissues. The most important aspect of the metabolism of the thyroid hormone is monodeiodination to form T3, the physiologically active form of thyroid hormone. T4 is also metabolized by glucuronidation and T3is predominantly sulphated and both are excreted in urine and bile.
Regulation of thyroid function:
Thyroid gland function is controlled by the hypothalamus and pituitary and is regulated via a negative feedback process in which thyrotropin-releasing factor (TRH) from the hypothalamus and TSH from the pituitary are released in response to decreased circulating levels of thyroid hormone. Another important aspect in the control of thyroid function is autoregulationviaa modulation of the receptor response to TSH. The TSH receptor has an extracellular component which undergoes a conformational change after binding to TSH. The catalytic unit of the receptor (adenylcyclase) produces cAMP which functions as a second messenger and mediates all the intracellular effects of TSH including the various differentiated functions of the follicular cell and cell proliferation. The adenylcyclase activity of the TSH receptor is subject to autoregulation and is controlled by a class of iodolipids that attenuates the formation of cAMP. When the iodolipids are low as a result of either iodine deficiency or an inhibition of the organification of iodine, the attenuation of the receptor response is removed and the response to TSH is enhanced. The physiologic role of this autoregulatory process is most likely to modulate the response of the thyroid to TSH depending upon the supply of iodine. Since autoregulation requires the enzymatic iodination of lipids, a process similar to thyroid hormone synthesis, inhibitors of iodination will decrease the iodolipid content and thereby enhance the TSH response. Iodine deficiency and chemicals such as propylthiouracil and methamidazole thus enhance the TSH response beyond that due to increased TSH alone. In contrast, excess iodine or chemicals that can either supply iodine or perhaps be recognized as a modulator similar to the endogenous iodolipids can attenuate the TSH response by inhibiting the formation of cAMP.
Species-differences in thyroid gland biochemistry and physiology:
There are marked species differences in thyroid gland physiology that must be taken into account in an evaluation of species differences in the induction of thyroid gland neoplasia secondary to hormone imbalance. The most obvious species difference between rodents and primates is the lack of thyroid binding globulin (TGB) in the rodent and other species including birds and reptiles. TGB is the predominant plasma protein that binds and transports thyroid hormone in the blood. In man, thyroxine binds to three plasma proteins, TGB, pre-albumin and albumin, with binding constants of 10-10, 10-7and 10-5, respectively. In rodents, the lack of TGB with a binding affinity of 3 and 5 orders of magnitude more than albumin and pre-albumin may be a factor responsible for species differences in thyroid gland function.
The half-life of thyroxine (T4) is 12 hours in the rat compared to 5-9 days in humans; serum TSH is 25-times higher in the rodent as compared to man. These findings indicate a much higher functional activity in the rodent thyroid gland as compared to the primate; a conclusion also supported by the histological appearance of the thyroid. For example, in the cynomolgus monkey, the follicles are uniformely large with abundant colloid and are lined by relatively flattened follicular epithelial cells. In contrast the rodent thyroid has large follicles only in the periphery of the gland. The interior is comprosed of comparatively small follicles with small amounts of colloid surrounded by a more cuboidal follicular epithelium. Interestingly, when rats are supplemented with exogenous thyroid hormone, the follicles accumulate colloid and increase in size. The epithelial cells assume a more flattened appearance, and the overall histological appearance resembles that of the monkey. Thus, both the physiologic parameters and the histologic appearance indicate that the rodent thyroid gland is markedly more active and operates at a considerably higher level with respect to thyroid hormone turnover as compared to the primate.
The incidence of “spontaneous” thyroid follicular cell neoplasia is also markedly different between rats and humans. The Fischer-344 male rat exhibits about a 2% incidence of thyroid follicular cell neoplasia (0.8% carcinoma and 1% adenoma) as compared to average incidence of approximately 0.004% with a range of 0.001% to 0.016% of carcinoma in humans. The prevalence of occult human carcinoma at autopsy has been high in some studies, all or most tumors being papillary carcinoma, a form rarely observed in the rat.
The relative susceptibility of rodents and humans to thyroid neoplasia secondary to hormone imbalance or simple hypothyroidism can be assessed by comparing humans in iodine deficient areas of endemic goitre to rats in these areas or rats treated with iodine deficient diets. Over the years there has been a slight, disputed association between endemic goitre and human thyroid cancer. Relatively extensive epidemiologic studies in areas of endemic goitre have not shown a clear etiologic role. In contrast, rodents in areas of endemic goitre or those treated with iodine deficient diets exhibit a high incidence of thyroid gland neoplasia.
The species differences between rodents and primates in thyroid gland physiology, the spontaneous incidents of thyroid gland neoplasia and the apparent susceptibility to neoplasia secondary to simple hypothyroidism support the conclusion that there is a marked species difference in thyroid gland neoplasia secondary to hormone imbalance. The rodent will exhibit an increase in thyroid gland neoplasia in the presence of mild to moderate increase in TSH. In contrast, no clear etiologic role for hypothyroidism in human thyroid cancer has been established even though chronic hypothyroidism, in the moderate to severe range, has occurred in humans in areas of endemic goitre. Thus, the contribution of endemic goitre to human thyroid cancer is small at most.
Mechanisms for altered thyroid function:
Thyroid hormone synthesis, release, transport cellular uptake, conversion of T4 to T3, hormone metabolism and the regulation of these processes by the hypothalamic-pituitary-thyroid axis and autoregulatory processes in the thyroid gland itself is a complex process and provides many ways in which chemicals can interfere with thyroid gland function. Regardless of mechanism, the response to hypothyroidism is similar. The pituitary will release TSH as a compensatory response to stimulate the thyroid to produce more hormone. Chronic stimulation of the thyroid gland by TSH in the rodent leads to the progression of follicular cell hypertrophy, hyperplasia and eventually neoplasia. Mechanisms for the action of chemicals can either be intra- or extracellular or both. Intrathyroidal mechanisms can involve iodine uptake or hormone synthesis, and extrathyroidal mechanisms can involve effects on hormone metabolism or disposition. A few important examples are discussed in the following.
Extrathyroidal mechanisms:
The conversion of thyroxine (T4) into the more active hormone triiodothyronine (T3) occurs in many peripheral tissues and is mediated bya microsomal 5´- monodeiodonase that removes one iodine at the 5´position in thyroxine. Various iodinated organic compounds such as tetraiodofluoresceine, amiodarone, and various iodinated radio-contrast media, will inhibit the 5´-monodeiodinase and disrupt the conversion of T4 to T3. The decrease in serum T3 values results in a compensatory increase in pituitary TSH and prolonged treatment with high dosages of tretraiodofluoresceine will result in a moderate increase in thyroid follicular neoplasia in rodents. PTU (Propylthiouracil)and methimidazole, in addition to inhibiting TPO (thyroperoxidase), will inhibit the monodeiodinases.
The effect of chemicals on various aspects of thyroid hormone metabolism have an important impact on thyroid hormone economy in the rodent. The monodeiodinases are quantitatively the most important path in the disposition of thyroxine. In addition, thyroxine is glucuronidated and T3 is sulphated and subsequently excreted in bile. Deamination, decarboxylation and cleavage of the ether link occur but are of quantitatively lesser importance. Many chemicals will induce hepatic microsomal enzymes at high dosages and alter thyroid function in rodents by increasing the hepatic disposition of thyroid hormone. Decreased serum thyroid hormone results in a compensatory increase in pituitary TSH which can exert a tumour promoting effect in initiation-promoting models or an increase in thyroid gland neoplasia in 2-year carcinogenicity studies.
Studies have shown that small amounts of thyroxine will block the tumour promoting effect of a microsomal enzyme inducer such as phenobarbital, thus this effect and presumably those observed in 2-year studies are considered to be secondary to hormone imbalance as opposed to a direct tumour promoting or direct carcinogenic effect in the thyroid gland.
With respect to the chemicals mentioned, tetraiodofluoresceine produced only mild effects on thyroid function in humans at very large multiples of the allowable daily intake. Chronic exposure to anticonvulsant drugs, many of which are enzyme-inducing at therapeutic dosages, result in only mild changes in thyroid function; these changes are not clinically significant.
Intrathyroidal mechanisms:
Two classes of chemicals known to inhibit thyroid hormone synthesis are the thioureylenes (thiourea, propylthiouracil, methimidazole) and the sulfonamides (sulfadiazine, sulfamethazine). The thioureylenes are considerably more potent in inhibiting thyroid hormone synthesis than the sulfonamides: however, sufficiently high dosages of many sulfonamides are goitrogenic in rodents. Although the goitrogenicity of the sulfonamides has been known since 1941, only a few have been tested for carcinogenicity. Sulfamethoxazole at dosages of 50 mg/kg/day or more is goitrogenic and produced thyroid neoplasia in rats within 50 weeks of treatment. Sulfamethazine produced goitrogenic effects and thyroid neoplasia in rats at doses of 600 ppm or more and in mice at doses of 4800 ppm. Sulfisoxazole which is only weakly goitrogenic in rodents did not produce thyroid gland neoplasia at doses up to 400 mg/kgay in rats or 2000 mg/kg/day in mice treated for 2 years. The various sulfonamides that have been tested had no apparent genotoxic effects, and the observed thyroid gland neoplasia using goitrogenic dosages were considered to be secondary to hormone imbalance.
The goitrogenic effect of the sulfonamides is known to be highly species-specific. Some species (including rats, mice, hamsters, dogs and swine) are sensitive, whereas no goitrogenic effect is observed in other species (including chickens, guinea pigs or monkeys). In the primate, no effect on thyroid function or morphology was observed in rhesus monkeys treated with sulfamethoxazole for 1 year at doses up to 300 mg/kg/day nor in cynomolgus monkeys treated for 4 weeks with sulfamonomethoxine at doses up to 300 mg/kg/day. In humans, no clinically significant effects on thyroid function have been observed with sulfonamides at therapeutic dose levels. Although a small decrease in patients treated with sulfamethoxazole in contrimoxazole, no increase in TSH was observed. In addition, no abnormalities in thyroid function were observed in a group of young patients receiving chronic treatment with cortimoxazole.
In another study, species differences between monkey and rodents using propylthiouracil (PTU) and sulfomonomethoxine (SMM) were investigated. Treated rats exhibited decreased T3 and T4 values and markedly elevated TSH values accompanied by follicular cell hyperplasia and increased thyroid gland weight at 30 or 300 mg/kg/day of either compound. In contrast, no effect on thyroid function or morphology was observed in monkeys receiving 300 mg/kg/day of the substances. In vitro there was a marked difference in the inhibition of microsomal thyroid peroxidise (TPO) depending upon the source of the enzyme. The concentration of SMM required to inhibit monkey TPO was approximately 450 times greater than that required to inhibit TPO from rats which explains why the sulfonamides are goitrogenis in rats at relatively low dosages but do not produce effects in monkeys at very high dosages or in humans at therapeutic dosages. The biochemical basis for the observed species differences in the goitrogenic effects of sulfonamides id thus due to marked species difference in the inhibition of TPO.
Sulfamethazine:
As mentioned above, sulfamethazine is goitrogenic in rodents and produces thyroid gland neoplasia in rats at doses of 600 ppm or more and in mice at a dosage of 4800 ppm. Since sulfamethazine is used in food producing animals in the, it is subject to the provisions of the Delaney amendments prohibiting the use of food or colour additives and animal drugs found to induce cancer in animals or humans. The residue level defined for sulfamethazine by the linear extrapolation procedure was well below the currently permitted level of 100 ppb and would preclude the use of sulfonamides in the animal health market. Sulfamethazine was shown to be an inhibitor of thyroid gland microsomal peroxidise, an important enzyme in the biosynthesis of thyroid hormone. Studies of thyroid gland function demonstrated that thyroid hormone imbalance occurs under the conditions of the rodent bioassay commensurate with a minimal to moderate tumour response (decreased thyroid hormone, increased TSH, increased thyroid weights and follicular cell hypertrophy and hyperplasia). All morphological effects on the thyroid gland were completely reversed after the withdrawal of sulfamethazine treatment. These morphological changes in the thyroid gland of sulfamethazine treated rats were similar in appearance to that observed in rats fed a low iodine diet. The supplemental dietary administration of thyroid hormone completely inhibited the fuctional and morphological changes observed with sulfamethazine treatment at doses that normalized but did not suppress TSH. Further, no detectable thyroid gland effects were observed in hypophysectomised rats treated with sulfamethazine. In vitro, sulfamethazine did not increase cell proliferation in FRTL_% cells in the absence of TSH. In accord with the other studies with sulfonamides in monkeys, no effect on thyroid gland function in cynomolgus monkeys was observed at dosages of up to 300 mg/kg/day for 13 weeks suppoting the conclusion that the effect of sulfamethazine on the thyroid gland is species-specific.
Dose-response characteristics:
A 4-week study was conducted in rats to investigate the dose-response characteristics for the effects of sulfamethazine ranging from 15 to 12000 ppm. A characteristic log dose-response relationship was observed in all the parameters of thyroid function measured. There were no significant effects at lower dosages, followed by a sharp linear rise at higher dosages. The results of this experiment clearly demonstrate that the effects of sulfamethazine on all aspects of thyroid function assessed, as well as the morphological changes in the thyroid gland exhibit a non-linear response from high to low doses. The dose-response characteristics observed in this experiment are consistent with a process that exhibits an apparent threshold.Description of key information
In a 12 week study in humans a significant increase in serum thyroxine and triiodothyronine was observed between the start and end of the study for the 9 mg(Br-)/kg bw/day dose group females but remained within normal limits at all concentrations. No changes were observed in measurements of hormones produces by the adrenals, the gonads and the pituitary gland.
Given the hormone changes observed, despite that fact that they are considered to remain within normal limits, a precautionary LOAEL of 9 mg(Br-)/kg bw/day has been determined. The NOAEL from this study is therefore 4 mg(Br-)/kg bw/day.
Additional information
The lowest LOAEL was obtained in a 12 week study in humans (Van Gelderen et al., 1993):
No findings of clinical effects relevant to the investigation were found, except for some incidences of nausea associated with bromide-capsule ingestion.Haematological, clinical chemistry and urine analysis parameters remained within normal limits for all dose groups.
A significant increase in serum thyroxine and triiodothyronine between the start and end in the study for the 9 mg(Br-)/kg bw/day dose group females but all concentrations remained within normal limits. No changes were observed in measurements of hormones produces by the adrenals, the gonads and the pituitary gland.
All neurophysiological findings were within normal limits.
Given the hormone changes observed, despite that fact that they are considered to remain within normal limits, a precautionary LOAEL of 9 mg(Br-)/kg bw/day has been determined. The NOAEL from this study is therefore 4 mg(Br-)/kg bw/day.
A lower NOAEL of 1 mg(Br-)/kg bw/day was found in a shorter (8 week) human volunteer study, however, only one dose was assessed in this study and no LOAEL was determined.
An additional 12 week in humans study concluded that the NOAEL is 9 mg(Br-)/kg bw/day. Given that a LOAEL has been determined at this dose the NOAEL was not used.
In summary:
LOAEL: 9 mg(Br-)/kg bw/day - hormone level alterations in females.
NOAEL: 4 mg(Br-)/kg bw/day.
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