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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Route of exposure is not relevant for risk assessment.
Cross-reference
Reason / purpose:
read-across: supporting information
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Route of exposure is not relevant for risk assessment.
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in Section 13 and 'Cross-reference'.
Reason / purpose:
read-across source
Qualifier:
no guideline followed
Principles of method if other than guideline:
Bismuth was administered at dose levels of 0, 0.8, 4, 20 mg/kg to male Crj:CD(SD)IGS rats (SPF) by intratracheal intermittent administration once a week for thirteen weeks to investigate its potential toxic effects; especially for specific adverse effects and changes related to pre-neoplastic lesions.
GLP compliance:
no
Limit test:
no
Specific details on test material used for the study:
particle size diameter: 1–2 μm
Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Male Crj:CD(SD)IGS rats (SPF) were used in the study.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Rats were obtained from Charles River Japan, Inc.
- Age at study initiation: 6 weeks old
- Weight at study initiation: 197-229 g
- Diet: ad libitum; the animals were fed a pellet diet (MF, Oriental Yeast Co., Ltd.)
- Water: tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 15
- Photoperiod: 12 hours dark/light cycle
- Ventilation frequency: about 12 changes per hour
Route of administration:
other: intratracheal instillation
Vehicle:
other: 0.5% carboxymethylcellulose sodium solution
Mass median aerodynamic diameter (MMAD):
other: No data
Remarks on MMAD:
MMAD not reported, Particle size diameter: 1-2 µm
Details on inhalation exposure:

Bismuth was weighed in to amounts of 0.032, 0.16 and 0.8 g and mixed with 20 mL of the vehicle for the dosing suspensions. The dose volume was set at 0.5 m//kg and the individual dose volume was calculated on the basis of the body weight measured just before administration on each administration day.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
13 weeks
Frequency of treatment:

Considering the accumulation of bismuth in the lungs by repeated administration and its effects on the physical condition of the animals, it was decided that the dose interval should be once a week to keep a steady-state concentration of bismuth in the lungs, because in the single dose study, the bismuth concentration in the lungs decreased rapidly in each treatment group up to 8 days after administration, but thereafter it did not fluctuate largely.
Dose / conc.:
0 other: mg/kg
Remarks:
Control group
Dose / conc.:
0.8 other: mg/kg
Dose / conc.:
4 other: mg/kg
Dose / conc.:
20 other: mg/kg
No. of animals per sex per dose:
The dosing suspensions were administered to the twelve animals of each dosing group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose selection rationale: The highest dose level was chosen as 20 mg/kg, since the result of a single dose toxicity study (Sano, 2005) showed that 20 mg/kg did not cause death.
Positive control:

No positive control substance was tested.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical signs of the animals were observed twice a day (before and after administration) throughout the dosing period, and once a day in the morning during the other period.
BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of all animals were measured once a week.
FOOD CONSUMPTION:- Time schedule for examinations: The gross weights of the feeders were measured once a week.
FOOD EFFICIENCY:- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data
WATER CONSUMPTION: No data
OPHTHALMOSCOPIC EXAMINATION: No data
HAEMATOLOGY: Yes
- Time schedule for collection of blood: On days of the scheduled necropsy, the animals were anesthetised; thereafter, blood samples were collected via the posterior vena cava.
- Anaesthetic used for blood collection: Yes (identity); intraperitoneal injection of sodium thiopental
- Parameters examined: For haematology, the erythrocyte count (RBC), haemoglobin concentration (Hb), haematocrit value (Ht), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocyte count, platelet count (PLT), prothrombin Time (PT), activated partial thromboplastin time (APTT), leukocyte count (WBC), and differential leukocyte count were measured.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On days of the scheduled necropsy, the animals were anesthetised; thereafter, blood samples were collected via the posterior vena cava.
- Parameters examined: For blood chemistry, the aspartatate aminotransferase (ASAT), alanine arninotransferase (AI-,aT), y-glutamyltrasferase (1GT), alkaline phosphatase (ALP), total bilirubin, blood urea nitrogen (BUN), creatinine, glucose, total cholesterol, triglycerides, total protein, albumin, A/G ratio, calcium, inorganic phosphorus, sodium (Na), potassium (K), and chlorine (Cl) were measured.
URINALYSIS: Yes
- Time schedule for collection of urine: Fresh urine samples from all the surviving animals were collected on the day before scheduled necropsy.
- Metabolism cages used for collection of urine: No data
- Parameters examined: pH, protein, glucose, ketone bodies, bilirubin, occult blood, and urobilinogen were measured.
NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:

GROSS PATHOLOGY: Yes; After the blood sampling, all animals were sacrificed by exsanguinations via the abdominal aorta, and then subjected to necropsy. The following organs of all animals were weighed: brain, liver, kidney, spleen, and testis. Relative organ weights were calculated from body weights on each necropsy day.
HISTOPATHOLOGY: Yes; Histopathological examinations on the lung, liver, kidney, spleen, brain, and testis from the control and 20 mg/kg groups and gross lesions from all animals including the control group. The organs except for testes were fixed by 10% neutral phosphate-buffered formalin solution. The testes were fixed in Bouin's solution. The haematoxylin and eosin stained specimens were prepared according to the standard procedure and then microscopically examined. Furthermore, PAS stained specimens of the kidneys from three animals were prepared to confirm the change of the glomerulus. Berlin blue stained specimens of the spleen from three animals were prepared to confirm that the brown pigments found in the spleen were hemosiderin.
Other examinations:
No further examinations were made.
Statistics:
A multiple comparison test to analyse statistical significances in the numerical data (body weight, food consumption, haematology, blood chemistry,and organ weights) was used. If there was statistical significances in the data between groups, Dunnett's test or Dunnett type rank-sum test was conducted. Statistical significance in graded categorical data (urinalysis, necropsy findings and histopathological findings) was analyzed by a x b chi-square test. If statistically significant data were found, Cochran-Armitage trend test was conducted. Significance levels of 5% and 1% were chosen for all statistical analyses.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No abnormal clinical signs attributable to bismuth administration were observed in any treated group during the testing period. Loss of hair was observed in one and two animals in the 4 and 20 mg/kg groups, respectively.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two animals of the control group, two animals of the 20 mg/kg group, one animal of the 4 mg/kg group, and one animal of the 0.8 mg/kg group died. A possible cause of death in these animals was excessive anaesthesia at administration or suffocation just after the administration associated with the intratracheal dose.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Suppression of body weight gain was observed at Day 29 and thereafter in the 20 mg/kg group, but it was not statistically significant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No abnormal food consumption attributable to bismuth administration were observed in any treated group during the testing period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A slight increase in erythrocyte count and MCHC were observed in the 20 mg/kg group, and slight increases in hemoglobin concentration and hematocrit value were observed in the 4 mg/kg and higher groups.
Higher rates of segmented neutrophil were observed in the 4 mg/kg and higher groups, and lower rates of lymphocytes were observed in the 0.8 mg/kg and higher groups.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
An increase in urea nitrogen was observed in the 20 mg/kg group. A decrease in ASAT in the 4 mg/kg group was observed, however, this was considered to be an incidental change, because it was not observed in the high dose group.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no significant differences between the control group and any treated group in the urinalysis.
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The absolute and relative lung weights increased in the 0.8 mg/kg and higher groups. Increases in the relative brain and kidney weights were observed in the 20 mg/kg group, however, it was considered to be an incidental change related to decreased body weight, since suppression of body weight gain was also observed in the 20 mg/kg group and there was no difference in the absolute organ weight.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Pathological changes attributed to bismuth administration were observed in the lungs and bronchial lymph nodes. In the lungs, a brown patch was observed in 3, 4 and 8 animals of the 0.8, 4 and 20 mg/kg groups, respectively. Black patches originating from the colour of bismuth and collapses were observed in all animals of the 4 and 20 mg/kg groups. Furthermore, enlargement of bronchial lymph nodes was observed in 6, 5 and 10 animals of the 0.8, 4 and 10 mg/kg groups, respectively. A brown patch in the lungs also observed in one animal in the control group, and slight focal inflammatory cell infiltration with small haemorrhage was observed in this lesion microscopically.A white patch in the liver was observed in 1, 2 and 3 animals of the 0.8, 4 and 20 mg/kg groups, respectively. Although, it was not statistically significant, there was a dose dependent trend of increase in number of animals which had a white patch. This necropsy finding was possibly related to bismuth. As an incidental change, abnormal lobulation of the liver and cyst in the kidney were observed in one animal of the 0.8 mg/kg group. In the necropsy of 4 of 6 animals that died during the administration period, the following changes were observed in one animal of the 4 mg/kg group: dilatation and haemorrhage of the urinary bladder, possibly related to death, and other changes such as renal pelvic dilatation, ascites, small sized thymus and spleen, and black patch, dark red change and collapse of the lungs. The cause of death in this animal may have been anuria; it was considered to be an incidental mortality, since it occurred in only one animal.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological changes attributed to bismuth were observed in the lungs, bronchial lymph nodes and kidneys. Focal inflammatory cell infiltration in the lungs was observed in all animals in the 0.8 mg/kg and higher groups. The incidence of lesions increased dose-dependently. In the lesions of inflammatory cell infiltration, hyperfrophy of alveolar epithelial cells was observed in the 0.8 mg/kg and higher groups, and hyperplasia of bronchial epithelial cells was observed in the 4 mg/kg and higher groups. Inflammatory cell infiltration in the 0.8 mg/kg and higher groups, hypertrophy of alveolar epithelium in the 4 mg/kg and higher groups, and hyperplasia of bronchial epithelial cell in the 20 mg/kg group showed statistically significant differences, as compared with the control group. Aggregation of foamy cells was observed in all groups including the control group and increased significantly in the 20 mg/kg group. Microscopic accumulation of bismuth was observed in all animals of the 4 and 20 mg/kg groups, and showed statistically significant differences as compared with the control group. In enlarged bronchial lymph nodes observed macroscopically, hyperplasia of the para-cortical area was observed, and increased pigment-laden phagocytic cells including the test substance was observed in 3 and 10 animals of the 4 and 20 mg/kg groups, respectively. In other changes, hyaline droplet of the proximal tubular epithelium in the kidney was observed in all groups including the control group, and its incidence decreased significantly in the 20 mg/kg group. The kidneys of representative animals of the control and 20 mg/kg groups were stained by PAS method, since a change in the glomerulus was suspected. In the result, there were no changes in the glomerulus and the hyaline droplets were PAS negative. Additional examination of the liver was conducted, since an effect of bismuth on the liver was suspected. Focal fatty degeneration of hepatic cells was observed in 1 and 2 animals of the 4 and 20 mg/kg groups, respectively. Brown pigment of the spleen was confirmed to be a hemosiderin pigment by Berlin blue staining.
Histopathological findings: neoplastic:
not specified
Details on results:
OTHER FINDINGS: The kinetics of bismuth can be described with a one-compartment pharmacokinetic model, since the logarithms of the lung concentrations of bismuth versus time plotted for each dosing group yield straight lines. From the slope of these lines, we calculated the half life of bismuth eliminationfrom the lungs as 4.47, 3.25, 2.10 days for the doses of 20, 100, 500 mg/kg, respectively.
Conclusions:
Dose-dependent foreign body inflammation of the lungs due to intratracheal administration of bismuth and physical change related to the pulmonary lesions were induced by intratracheal administration of bismuth once a week for 13 wk.
Executive summary:

Bismuth was administered at dose levels of 0, 0.8, 4, 20 mg/kg to male Crj:CD(SD)IGS rats (SPF) by intratracheal intermittent administration once a week for thirteen weeks to investigate its potential toxic effects; especially for specific adverse effects and changes related to pre-neoplastic lesions.

The study is well documented and was published in a peer review journal, however is not considered to be reliable (Klimisch 3) or sufficient for a key study, due to the novel route of administration. It could be used in a weight-of-evidence (WoE) approach for repeat dose toxicity.

Data source

Reference
Reference Type:
publication
Title:
A 13-week toxicity study of bismuth in rats by intratracheal intermittent administration
Author:
Sano, Y. et al.
Year:
2005
Bibliographic source:
J Occup Health 2005 ; 47: 242-248

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
Bismuth was administered at dose levels of 0, 0.8, 4, 20 mg/kg to male Crj:CD(SD)IGS rats (SPF) by intratracheal intermittent administration once a week for thirteen weeks to investigate its potential toxic effects; especially for specific adverse effects and changes related to pre-neoplastic lesions.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- CAS: 7440-69-9
- Purity: 99.9%
- Source: Kojundo Chemical Laboratory Co. Ltd.
- Storage: stored at room temperature
Specific details on test material used for the study:
particle size diameter: 1–2 μm

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Details on species / strain selection:
Male Crj:CD(SD)IGS rats (SPF) were used in the study.
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Rats were obtained from Charles River Japan, Inc.
- Age at study initiation: 6 weeks old
- Weight at study initiation: 197-229 g
- Diet: ad libitum; the animals were fed a pellet diet (MF, Oriental Yeast Co., Ltd.)
- Water: tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 55 +/- 15
- Photoperiod: 12 hours dark/light cycle
- Ventilation frequency: about 12 changes per hour

Administration / exposure

Route of administration:
other: intratracheal instillation
Vehicle:
other: 0.5% carboxymethylcellulose sodium solution
Mass median aerodynamic diameter (MMAD):
other: No data
Remarks on MMAD:
MMAD not reported, Particle size diameter: 1-2 µm
Details on inhalation exposure:

Bismuth was weighed in to amounts of 0.032, 0.16 and 0.8 g and mixed with 20 mL of the vehicle for the dosing suspensions. The dose volume was set at 0.5 m//kg and the individual dose volume was calculated on the basis of the body weight measured just before administration on each administration day.
Analytical verification of doses or concentrations:
not specified
Details on analytical verification of doses or concentrations:
No data
Duration of treatment / exposure:
13 weeks
Frequency of treatment:

Considering the accumulation of bismuth in the lungs by repeated administration and its effects on the physical condition of the animals, it was decided that the dose interval should be once a week to keep a steady-state concentration of bismuth in the lungs, because in the single dose study, the bismuth concentration in the lungs decreased rapidly in each treatment group up to 8 days after administration, but thereafter it did not fluctuate largely.
Doses / concentrationsopen allclose all
Dose / conc.:
0 other: mg/kg
Remarks:
Control group
Dose / conc.:
0.8 other: mg/kg
Dose / conc.:
4 other: mg/kg
Dose / conc.:
20 other: mg/kg
No. of animals per sex per dose:
The dosing suspensions were administered to the twelve animals of each dosing group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose selection rationale: The highest dose level was chosen as 20 mg/kg, since the result of a single dose toxicity study (Sano, 2005) showed that 20 mg/kg did not cause death.
Positive control:

No positive control substance was tested.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical signs of the animals were observed twice a day (before and after administration) throughout the dosing period, and once a day in the morning during the other period.
BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of all animals were measured once a week.
FOOD CONSUMPTION:- Time schedule for examinations: The gross weights of the feeders were measured once a week.
FOOD EFFICIENCY:- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data
WATER CONSUMPTION: No data
OPHTHALMOSCOPIC EXAMINATION: No data
HAEMATOLOGY: Yes
- Time schedule for collection of blood: On days of the scheduled necropsy, the animals were anesthetised; thereafter, blood samples were collected via the posterior vena cava.
- Anaesthetic used for blood collection: Yes (identity); intraperitoneal injection of sodium thiopental
- Parameters examined: For haematology, the erythrocyte count (RBC), haemoglobin concentration (Hb), haematocrit value (Ht), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocyte count, platelet count (PLT), prothrombin Time (PT), activated partial thromboplastin time (APTT), leukocyte count (WBC), and differential leukocyte count were measured.
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: On days of the scheduled necropsy, the animals were anesthetised; thereafter, blood samples were collected via the posterior vena cava.
- Parameters examined: For blood chemistry, the aspartatate aminotransferase (ASAT), alanine arninotransferase (AI-,aT), y-glutamyltrasferase (1GT), alkaline phosphatase (ALP), total bilirubin, blood urea nitrogen (BUN), creatinine, glucose, total cholesterol, triglycerides, total protein, albumin, A/G ratio, calcium, inorganic phosphorus, sodium (Na), potassium (K), and chlorine (Cl) were measured.
URINALYSIS: Yes
- Time schedule for collection of urine: Fresh urine samples from all the surviving animals were collected on the day before scheduled necropsy.
- Metabolism cages used for collection of urine: No data
- Parameters examined: pH, protein, glucose, ketone bodies, bilirubin, occult blood, and urobilinogen were measured.
NEUROBEHAVIOURAL EXAMINATION: No data
Sacrifice and pathology:

GROSS PATHOLOGY: Yes; After the blood sampling, all animals were sacrificed by exsanguinations via the abdominal aorta, and then subjected to necropsy. The following organs of all animals were weighed: brain, liver, kidney, spleen, and testis. Relative organ weights were calculated from body weights on each necropsy day.
HISTOPATHOLOGY: Yes; Histopathological examinations on the lung, liver, kidney, spleen, brain, and testis from the control and 20 mg/kg groups and gross lesions from all animals including the control group. The organs except for testes were fixed by 10% neutral phosphate-buffered formalin solution. The testes were fixed in Bouin's solution. The haematoxylin and eosin stained specimens were prepared according to the standard procedure and then microscopically examined. Furthermore, PAS stained specimens of the kidneys from three animals were prepared to confirm the change of the glomerulus. Berlin blue stained specimens of the spleen from three animals were prepared to confirm that the brown pigments found in the spleen were hemosiderin.
Other examinations:
No further examinations were made.
Statistics:
A multiple comparison test to analyse statistical significances in the numerical data (body weight, food consumption, haematology, blood chemistry,and organ weights) was used. If there was statistical significances in the data between groups, Dunnett's test or Dunnett type rank-sum test was conducted. Statistical significance in graded categorical data (urinalysis, necropsy findings and histopathological findings) was analyzed by a x b chi-square test. If statistically significant data were found, Cochran-Armitage trend test was conducted. Significance levels of 5% and 1% were chosen for all statistical analyses.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No abnormal clinical signs attributable to bismuth administration were observed in any treated group during the testing period. Loss of hair was observed in one and two animals in the 4 and 20 mg/kg groups, respectively.
Mortality:
mortality observed, treatment-related
Description (incidence):
Two animals of the control group, two animals of the 20 mg/kg group, one animal of the 4 mg/kg group, and one animal of the 0.8 mg/kg group died. A possible cause of death in these animals was excessive anaesthesia at administration or suffocation just after the administration associated with the intratracheal dose.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Suppression of body weight gain was observed at Day 29 and thereafter in the 20 mg/kg group, but it was not statistically significant.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No abnormal food consumption attributable to bismuth administration were observed in any treated group during the testing period.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
A slight increase in erythrocyte count and MCHC were observed in the 20 mg/kg group, and slight increases in hemoglobin concentration and hematocrit value were observed in the 4 mg/kg and higher groups.
Higher rates of segmented neutrophil were observed in the 4 mg/kg and higher groups, and lower rates of lymphocytes were observed in the 0.8 mg/kg and higher groups.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
An increase in urea nitrogen was observed in the 20 mg/kg group. A decrease in ASAT in the 4 mg/kg group was observed, however, this was considered to be an incidental change, because it was not observed in the high dose group.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no significant differences between the control group and any treated group in the urinalysis.
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The absolute and relative lung weights increased in the 0.8 mg/kg and higher groups. Increases in the relative brain and kidney weights were observed in the 20 mg/kg group, however, it was considered to be an incidental change related to decreased body weight, since suppression of body weight gain was also observed in the 20 mg/kg group and there was no difference in the absolute organ weight.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Pathological changes attributed to bismuth administration were observed in the lungs and bronchial lymph nodes. In the lungs, a brown patch was observed in 3, 4 and 8 animals of the 0.8, 4 and 20 mg/kg groups, respectively. Black patches originating from the colour of bismuth and collapses were observed in all animals of the 4 and 20 mg/kg groups. Furthermore, enlargement of bronchial lymph nodes was observed in 6, 5 and 10 animals of the 0.8, 4 and 10 mg/kg groups, respectively. A brown patch in the lungs also observed in one animal in the control group, and slight focal inflammatory cell infiltration with small haemorrhage was observed in this lesion microscopically.A white patch in the liver was observed in 1, 2 and 3 animals of the 0.8, 4 and 20 mg/kg groups, respectively. Although, it was not statistically significant, there was a dose dependent trend of increase in number of animals which had a white patch. This necropsy finding was possibly related to bismuth. As an incidental change, abnormal lobulation of the liver and cyst in the kidney were observed in one animal of the 0.8 mg/kg group. In the necropsy of 4 of 6 animals that died during the administration period, the following changes were observed in one animal of the 4 mg/kg group: dilatation and haemorrhage of the urinary bladder, possibly related to death, and other changes such as renal pelvic dilatation, ascites, small sized thymus and spleen, and black patch, dark red change and collapse of the lungs. The cause of death in this animal may have been anuria; it was considered to be an incidental mortality, since it occurred in only one animal.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Histopathological changes attributed to bismuth were observed in the lungs, bronchial lymph nodes and kidneys. Focal inflammatory cell infiltration in the lungs was observed in all animals in the 0.8 mg/kg and higher groups. The incidence of lesions increased dose-dependently. In the lesions of inflammatory cell infiltration, hyperfrophy of alveolar epithelial cells was observed in the 0.8 mg/kg and higher groups, and hyperplasia of bronchial epithelial cells was observed in the 4 mg/kg and higher groups. Inflammatory cell infiltration in the 0.8 mg/kg and higher groups, hypertrophy of alveolar epithelium in the 4 mg/kg and higher groups, and hyperplasia of bronchial epithelial cell in the 20 mg/kg group showed statistically significant differences, as compared with the control group. Aggregation of foamy cells was observed in all groups including the control group and increased significantly in the 20 mg/kg group. Microscopic accumulation of bismuth was observed in all animals of the 4 and 20 mg/kg groups, and showed statistically significant differences as compared with the control group. In enlarged bronchial lymph nodes observed macroscopically, hyperplasia of the para-cortical area was observed, and increased pigment-laden phagocytic cells including the test substance was observed in 3 and 10 animals of the 4 and 20 mg/kg groups, respectively. In other changes, hyaline droplet of the proximal tubular epithelium in the kidney was observed in all groups including the control group, and its incidence decreased significantly in the 20 mg/kg group. The kidneys of representative animals of the control and 20 mg/kg groups were stained by PAS method, since a change in the glomerulus was suspected. In the result, there were no changes in the glomerulus and the hyaline droplets were PAS negative. Additional examination of the liver was conducted, since an effect of bismuth on the liver was suspected. Focal fatty degeneration of hepatic cells was observed in 1 and 2 animals of the 4 and 20 mg/kg groups, respectively. Brown pigment of the spleen was confirmed to be a hemosiderin pigment by Berlin blue staining.
Histopathological findings: neoplastic:
not specified
Details on results:
OTHER FINDINGS: The kinetics of bismuth can be described with a one-compartment pharmacokinetic model, since the logarithms of the lung concentrations of bismuth versus time plotted for each dosing group yield straight lines. From the slope of these lines, we calculated the half life of bismuth eliminationfrom the lungs as 4.47, 3.25, 2.10 days for the doses of 20, 100, 500 mg/kg, respectively.

Applicant's summary and conclusion

Conclusions:
Dose-dependent foreign body inflammation of the lungs due to intratracheal administration of bismuth and physical change related to the pulmonary lesions were induced by intratracheal administration of bismuth once a week for 13 wk.
Executive summary:

Bismuth was administered at dose levels of 0, 0.8, 4, 20 mg/kg to male Crj:CD(SD)IGS rats (SPF) by intratracheal intermittent administration once a week for thirteen weeks to investigate its potential toxic effects; especially for specific adverse effects and changes related to pre-neoplastic lesions.

The study is well documented and was published in a peer review journal, however is not considered to be reliable (Klimisch 3) or sufficient for a key study, due to the novel route of administration. It could be used in a weight-of-evidence (WoE) approach for repeat dose toxicity.