Registration Dossier

Administrative data

Description of key information

In conclusion, although no systemic toxicity is seen after treatment up to 1000 mg/kg for 90-days, based on mortality and the local test item-related morphologic alterations in the stomach of males and females, a NOAEL of 300 mg/kg was established.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 January 2016 to 21 June 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
See below
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
See below
Qualifier:
according to
Guideline:
other: Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2011 by MHLW (0331 No.7), METI (H23.03.29 SeiKyoku No. 5) and MOE (No. 110331009).
Deviations:
yes
Remarks:
See below
Principles of method if other than guideline:
Deviations:
1 Deviations from the daily mean relative humidity occurred.
Evaluation: Laboratory historical data do not indicate an effect of the deviations.
2 On several days it was not possible to use the DCS system for verification of the dosing procedure.
Evaluation: DCS is an additional tool to check the dosing procedure and the other checking procedures remained intact, the study integrity is considered not to be affected.
3 All females had a daily lavage from 03 June 2016 (Day 79) up to and including 21 June 2016 (Day 97) instead of to 20 June 2016 (Day 96).
Evaluation: The lavage on the day of necropsy did not adversely affect the study outcome.
4 The brain of a high dose male (an. no. 32) was not weighed at necropsy and lumbar spinal cord of a control female (an. no. 41), thymus of a high dose male (an. no. 40) and urinary bladder of high dose male (an. no. 34) were not available for histopathology.
Evaluation: Sufficient data was available for evaluation of these organs.

In the range finding study (project 510572) no deviations from the study plan were observed:The study integrity was not adversely affected by the deviations.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Specific details on test material used for the study:
Appearance: White crystalline powder
Batch: 2011001922
Test item storage: At room temperature
Stable under storage conditions until 30 April 2017 (retest date)
Purity/composition correction factor: No correction factor required
Test item handling: No specific handling conditions required
Stability at higher temperatures: Yes

Species:
rat
Strain:
Wistar
Remarks:
Crl:WI(Han)
Details on species / strain selection:
Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 6 weeks.
- Weight at study initiation: Males: group mean weights of 150 g to 152 g. Females: group mean weights of 127 g to 130 g.
- Fasting period before study: not specified
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap-water.
- Acclimation period: At least 5 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): a relative humidity of 40 to 70%
- Air changes (per hr): at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): a 12-hour light/12-hour dark cycle. The light/dark cycle was interrupted for study related activities.

IN-LIFE DATES: From: 17 March 2016 To: 21 June 2016
Route of administration:
oral: gavage
Details on route of administration:
Oral gavage, using a plastic feeding tube.
Formulations were placed on a magnetic stirrer during dosing.
A dose control system (DCS) was used to verify the dosing procedure.
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at Charles River Den Bosch and on information from the Sponsor.
- Concentration in vehicle: 20 mg/mL, 60 mg/mL, 200 mg/mL
- Amount of vehicle (if gavage): 5mL/kg
- Lot/batch no.: Not specified
- Purity: Not specified, specific gravity = 1.036.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
An inductively coupled plasma - mass spectrometric (ICP-MS) method for the quantitative analysis of the test item, based on Bismuth (Bi), in vehicle was developed.
Analytical conditions:
Instrument: Agilent 7500CE (Agilent Technologies, Tokyo, Japan)
Cone: Nickel
Nebulizer
Type: MicroMist
Material: quartz
Spray chamber
Material: quartz
Temperature: 15°C
Torch: 2.5 mm i.d.
Detection of Bi:
Reaction gas: none
Integration time: 0.1 seconds per replicate
Replicates per analysis: 5
Quantitation on m/z: 209

Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations, in Weeks 1, 6 and 13). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions and over 8 days in the refrigerator under protection from light was also determined (highest and lowest concentration, in Week 1).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.


Duration of treatment / exposure:
At least 90 days.
Animals were dosed up to the day prior to necropsy.
Frequency of treatment:
Once daily, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
ten animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels are based on results of a 14-day oral range finding study with Bismuth Trinitrate by daily gavage in the rat (project 510572).
- Rationale for animal assignment: By computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
- Rationale for selecting satellite groups: Not applicable
- Post-exposure recovery period in satellite groups: Not applicable
- Section schedule rationale (if not random): Not applicable

Positive control:
None
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes - mortality/viability
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The time of death was recorded as precisely as possible.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals at least immediately after dosing (no peak effect of occurrence of clinical signs was observed in the dose range finding study (project 510572)). Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena (collected under Project 510573 for logistic reasons and reported under Project 510571). The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Time schedule for examinations: food consumption: Weekly

FOOD EFFICIENCY:
Not evaluated.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: \Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Following instillation of tropicamide solution (5 mg/mL, Minims® Tropicamide 0.5% w/v, Bausch&Lomb Pharma, Brussel, Belgium) both eyes were examined by means of an ophthalmoscope (Heine Beta 200S):
at pretest : All animals
at Week 13 : Group 1 and 4 animals
Since no treatment-related ophthalmologic findings were noted in Week 13, the eyes of the rats of Groups 2 and 3 were not examined.


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the study (Day 90). Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL).
- Anaesthetic used for blood collection: Blood samples were collected under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m. at the end of the treatment.
- Animals fasted: Animals were deprived of food overnight (for a maximum of 24 hours), but water was available.
- How many animals: All animals from each test and control group.
- Parameters examined.
The following haematology parameters were determined:
White blood cells (WBC)
Differential leucocyte count:
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
Red blood cells
Reticulocytes
Red blood cell distribution width (RDW)
Haemoglobin
Haematocrit
Mean corpuscular volume (MCV)
Mean corpuscular haemoglobin (MCH)
Mean corpuscular haemoglobin concentration (MCHC)
Platelets
The following clotting parameters were determined:
Prothrombin Time (PT)
Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the study (Day 90). Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.75 mL) was collected into serum tubes for determination of bile acids.
- Anaesthetic used for blood collection: Blood samples were collected under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m. at the end of the treatment.
- Animals fasted: Animals were deprived of food overnight (for a maximum of 24 hours), but water was available.
- How many animals: All animals from each test and control group.
- Parameters examined:
The following clinical biochemistry parameters were determined:
Alanine aminotransferase (ALAT)
Aspartate aminotransferase (ASAT)
Alkaline Phosphatase (ALP)
Total protein
Albumin
Total Bilirubin
Bile acids
Urea
Creatinine
Glucose
Cholesterol
Sodium
Potassium
Chloride
Calcium
Inorganic Phosphate (Inorg. Phos)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During Week 12-13 of treatment.
- Dose groups that were examined: All animals after dosing at no specific time point, but within a similar time period after dosing for the respective animals (based on the absence of a peak effect in occurrence of clinical signs in the dose range finding study (project 510572))
- Battery of functions tested:
• hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
• fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (Series M4-10, Mark-10 Corporation, J.J. Bos, Gouda, The Netherlands).
• locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.

OTHER:
Estrous cycle determination
All females had a daily lavage from 03 June 2016 (Day 79) up to and including 21 June 2016 (Day 97) to determine the stage of estrous (according to SOP DIE/H/147, with exception of Procedure B (microscopic assessment of presence of sperm cells)).

Testosterone Hormone Assessment
At the end of the study (Day 90). Blood samples were drawn from the retro-orbital sinus.An blood sample (0.75 mL) was collected into serum tubes for determination of testosterone.
The following clinical biochemistry parameters were determined in serum, using the IMMULITE® 1000 (Siemens Healthcare Diagnostics B.V., Den Haag, The Netherlands): Testosterone.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy
Animals surviving to the scheduled day of necropsy and all moribund animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination. Animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Rats found dead were subjected to a full post mortem examination as soon as possible after death and always within 24 hours.

Organ Weights
The following organ weights and terminal body weight were recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands
Spleen
Brain
Testes
Epididymides
Thymus
Heart
Uterus (including cervix)
Kidneys
Prostate
Liver
Seminal vesicles including coagulating glands
Ovaries
Thyroid including parathyroid

SPERM ANALYSIS
From all males of Group 1 and 4, additional slides of the testes were prepared to examine staging of spermatogenesis. The testes were processed, sectioned at 3-4 micrometers, and stained with PAS/haematoxylin (Klinipath, Duiven, The Netherlands).

HISTOPATHOLOGY: Yes
Identification marks: not processed
Ovaries
Adrenal glands
Pancreas
Aorta
Peyer's patches [jejunum, ileum] if detectable
Brain [cerebellum, mid-brain, cortex] (7 levels)
Pituitary gland
(Preputial gland)
Caecum
Cervix
Prostate gland
(Clitoral gland)
Rectum
Colon
Salivary glands - mandibular, sublingual
Duodenum
Sciatic nerve
Epididymides
Seminal vesicles including coagulating gland
Eyes with optic nerve [if detectable] and Harderian gland
(Skeletal muscle)
Skin
Female mammary gland area
Spinal cord -cervical, midthoracic, lumbar
(Femur including joint)
Spleen
Heart
Sternum with bone marrow
Ileum
Stomach
Jejunum
Testes
Kidneys
Thymus
Larynx
Thyroid including parathyroid [if detectable]
(Lacrimal gland, exorbital)
(Tongue)
Liver
Trachea
Lung, infused with formalin
Urinary bladder
Lymph nodes - mandibular, mesenteric
Uterus
(Nasopharynx)
Vagina
Oesophagus
All gross lesions

Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.

All organ and tissue samples, as defined under Histopathology (following), were processed, embedded in paraffin wax (Klinipath, Duiven, The Netherlands), cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).

The following slides were examined by a pathologist:
• all tissues collected at the scheduled sacrifice from all Group 1 and 4 animals,
• all tissues from one male of Group2 which died spontaneously and two males and three females of Group 4 which died spontaneously or were terminated in extremis,
• the additional slides of the testes of Group 1 and 4 males to examine staging of spermatogenesis,
• stomach of all males and females, spleen of all males and thymus of all females of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4,
• all gross lesions.
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Histopathology was subjected to a peer review.

Statistics:
The following statistical methods were used to analyze the data:
• If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
• The Fisher Exact-test was applied to frequency data.
• The Kruskal-Wallis nonparametric ANOVA test (Ref. 4) was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test (Ref. 5) was applied to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Surviving animals at 1000 mg/kg showed rales during the entire treatment period. Other symptoms noted towards the end of the treatment period were hunched posture, piloerection and labored or deep respiration.
For clinical signs of the decedents see under Mortality below.
No toxicologically relevant symptoms were noted in clinical observations of the animals at 100 and 300 mg/kg and no findings were noted at all dose levels during the arena observations in this study.
Salivation seen after dosing among males at 300 and 1000 mg/kg and females at 1000 mg/kg was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test item rather than a sign of systemic toxicity.
Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
Four unscheduled deaths at 1000 mg/kg were considered to be related to the test item: 1 female (an. no. 72) was found death after 61 days of treatment (necropsy at day 62) and 2 males and 1 females were sacrificed moribund after 47 or 75 days of treatment (male no.40 and female no.76, necropsy at day 47 and male no.35, necropsy at day 75 respectively). These animals showed test item-related macroscopic changes in the gastro-intestinal tract (black-brown/red foci on the glandular stomach, irregular surface of the glandular stomach, black content in the cecum, GI-tract distended with gas) and in the thymus and/or spleen (reduced size of thymus and/or spleen). Microscopically, these findings correlated with ulcerations/erosions and hemorrhage(s) in the stomach and lymphoid depletion/atrophy in thymus and/or spleen. Additional microscopic findings were lymphoid depletion in the mesenteric lymph nodes and mandibular lymph node in a single female and increased adipocytes in the bone marrow. Especially the ulcerations/erosions in the stomach has been largely responsible for the cause of death/moribundity of the early sacrifices. Most likely secondary to the moribund conditions of these animals the lymphoid tissues were affected.
These animals showed salivation at most of the days during treatment period. The following symptoms were noted in the days or up to two weeks prior to death: hunched posture, labored or deep respiration, rales, gasping, squeaking, scabs in the neck, piloerection, lean or pale appearance, swelling of the abdomen, hypothermia.
In addition two other unscheduled deaths were observed during the treatment phase. At 100 mg/kg, one male (an. no. 13) was found death after 42 days of treatment and necropsied at Day 43, and at 1000 mg/kg, one female (an.no. 79) was sacrificed after 41 days of treatment (necropsy at day 42).
Animal 13 showed fluid in the thoracic cavity at necropsy and moderate granulocytic inflammatory infiltrates in the tissue adjacent to the aorta containing foreign material, which is suggestive for a treatment accident. Labored respiration was noted during clinical observations in this animal on Days 42 and 43.
Animal 79 showed lymphoid depletion/atrophy in several lymphoid organs and slight granulocytic inflammatory infiltrates, minimal erosion of the epithelium and hemorrhages in the esophagus which is suggestive for a treatment accident that may have attributed to the moribundity of the animal. This animal showed salivation during the treatment period and the following were noted a few days prior to death: hunched posture, labored respiration, rales, swelling of the abdomen and lean appearance.
Two control females (an. nos. 43 and 44) died during blood sampling at the day of necropsy, which was considered to be an accidental death.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Body weights and body weight gain of females at 1000 mg/kg were slightly lower compared to control females.
Body weights and body weight gain of males were considered to have been unaffected by treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after correction for body weight remained similar to the control level over the study period.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
No effects were observed.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmology findings were noted that were considered to be related to treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Slightly lower red blood cell count was suggested for males at 1000 mg/kg accompanied with a slightly higher reticulocyte count and a slightly higher mean corpuscular volume. The females at 300 and 1000 mg/kg showed a slightly higher haematocrit level only.
Any other statistically significant changes in haematology parameters were considered to be unrelated to treatment as these were within the normal range for these kind of rats.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Clinical biochemistry investigation revealed higher cholesterol level in males at 1000 mg/kg. In females, higher level of total protein, albumin, total bilirubin and creatinine were noted at 1000 mg/kg.
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Motor activity of males and females at 1000 mg/kg was slightly lower compared to control groups, which was only statistically significant for the total movements of females. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was similar between control and high dose animals.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no test item-related alterations in organ weights.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic examination revealed test item-related black content in the stomach and/or cecum in surviving males and females at 300 and 1000 mg/kg (4/10 males and 2/10 females at 300 mg/kg and 8/8 males and 7/7 females at 1000 mg/kg). A single female at 1000 mg/kg showed a reduced size of thymus and spleen.
The remainder of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Test item-related microscopic findings were noted in the glandular stomach of males and females at 1000 mg/kg.
Findings in the glandular stomach were observed at 1000 mg/kg and consisted of erosion/ulceration (present in all early deaths only), necrosis (present in all early death males only) and increased hemorrhage(s) (minimal hemorrhage is considered to be background; present in all early death males and 1 early death female and 1 male surviving the treatment period).
Microscopic findings that are likely secondary to the moribund condition of the animal are reported in several lymphoid organs (thymus, spleen, mesenteric lymph node, mandibular lymph nodes and bone marrow) and consisted of lymphoid depletion/atrophy or increased adipocytes (bone marrow). The spleen, mesenteric and mandibular lymph nodes and bone marrow were affected in the unscheduled deaths only. Lymphoid depletion in the thymus was noted in all unscheduled sacrifices and in a single female of the scheduled sacrifices.
Microscopically the single female of the scheduled sacrifices with marked lymphoid depletion did not show lymphoid depletion/atrophy in any of the other lymphoid organs and no other characteristics that could explain the marked lymphoid depletion in the thymus were found in the other organs. Therefore, the thymus depletion in this particular female rat was likely an incidental finding rather than a test item-related finding.
Remaining histologic changes were considered to be incidental findings or within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment related changes in estrous cycle length were noted across the dose groups during the period in which estrous cycle length was determined (Day 79 up to and including Day 97).
One control female (no. 42), and one high dose female (no. 72) showed acyclic estrous cycle length and three females at 100 mg/kg (nos. 52, 55 and 59) showed an irregular estrous cycle length. All other females showed a normal (regular) estrous cycle of 4 or 5 days. The incidence of irregular or acyclic estrous cycle length showed no relationship to the dose, and was therefore considered unrelated to treatment.
Details on results:
Analysis of Dose Preparations
Accuracy of preparation:
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%).
A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulations. It was not considered to derive from the formulation since a similar response was obtained in the analytical blanks.
Homogeneity:
The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).
Stability:
Analysis of Group 2 and Group 4 formulations after storage yielded a relative difference of ≤ 10%. Based on this, the formulations were found to be stable during storage at room temperature under normal laboratory light conditions for at least 5 hours and in a refrigerator for 8 days.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
gross pathology
histopathology: non-neoplastic
Key result
Critical effects observed:
no

Figures and tables are attached below under 'Attached Background Material'.

Conclusions:
Wistar rats were treated with Bismuth Trinitrate for at least 90 consecutive days by daily oral gavage at dose levels of 0, 100, 300 and 1000 mg/kg.
Formulation analyses confirmed that formulations of test item in propylene glycol were prepared accurately and homogenously, and were stable over at least 5 hours at room temperature under normal laboratory light conditions and over 8 days in a refrigerator.
Test item-related mortality occurred in this study. Unscheduled deaths were observed at 1000 mg/kg (males 35, 40 and females 72, 76). These animals showed macroscopic changes in the gastro-intestinal tract (black-brown/red foci on the glandular stomach, irregular surface of the glandular stomach, black content in the cecum, GI-tract distended with gas) and thymus and/or spleen (reduced size). This correlated microscopically with ulcerations/erosions, necrosis and hemorrhage(s) in the glandular stomach and lymphoid depletion/atrophy in thymus and/or spleen. Additional microscopic findings were lymphoid depletion in the mesenteric lymph nodes and mandibular lymph node in a single female and increased adipocytes in the bone marrow, which was most likely secondary to the moribund condition of the animals. Particularly the ulcerations/erosions in the stomach are considered to be largely responsible for the cause of death/moribundity of the early sacrifices. The animals showed salivation at most of the days during treatment period. The following symptoms were noted in the days or up to two weeks prior to death: hunched posture, labored or deep respiration, rales, gasping, squeaking, scabs in the neck, piloerection, lean or pale appearance, swelling of the abdomen, hypothermia.
Two unscheduled deaths treated at 100 mg/kg (male 13) and 1000 mg/kg (female 79) showed macroscopic and/or microscopic findings suggestive for a dosing error (male 13: fluid in the thoracic cavity, granulocytic inflammatory infiltrates with foreign material in the tissue adjacent to the aorta; female 79: granulocytic infiltration, epithelial erosion and hemorrhages in the esophagus). Animal no.13 showed labored respiration on Days 42 and 43. And animal no.79 showed salivation during the treatment period and hunched posture, labored respiration, rales, swelling of the abdomen and lean appearance, a few days prior to death.
Surviving animals
The surviving animals showed at 1000 mg/kg rales during the treatment period. Towards the end of the treatment period additional symptoms were noted such as hunched posture, piloerection and labored or deep respiration. Salivation was also noted in these animals (and at 300 mg/kg), however this symptom was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). Functional observation tests revealed no adverse changes. The lower motor activity of females at 1000 mg/kg, and a trend towards lower motor activity for males at 1000 mg/kg were considered not to represent an adverse effect on neurobehaviour. These results were not supported by clinical observations or other functional observation tests, were slight in nature (within the normal range for rats of this age and strain), and had no supportive morphological correlates in examined neuronal tissues.
The females at 1000 mg/kg showed slightly lower body weight gain compared to control animals, which was not supported by an effect in food consumption
Macroscopic and microscopic examination revealed test item-related black content in the stomach and/or cecum in surviving males and females at 300 and 1000 mg/kg. A single female at 1000 mg/kg showed a reduced size of thymus and spleen. No microscopic correlate was found for the black content of stomach and/or cecum, the microscopic correlate for reduced thymus was lymphoid depletion. The single female of the scheduled sacrifices with marked lymphoid depletion did not show lymphoid depletion/atrophy in any of the other lymphoid organs and no other characteristics that could explain the marked lymphoid depletion in the thymus were found in the other organs. Therefore, the thymus depletion in this particular female rat was likely an incidental finding rather than a test item-related finding.
Microscopic examination revealed slight hemorrhage(s) in the glandular stomach in a single male at 1000 mg/kg (minimal hemorrhage was considered to be background), which was considered not to be adverse based on the absence of any other findings in the stomach.
Slight effects in haematology and clinical biochemistry were observed (i.e. changes in red blood cell count, reticulocyte count, mean corpuscular volume, haematocrit level and changes in levels of cholesterol, total protein, albumin, total bilirubin and creatinine). These changes were slight and with no clear related morphological changes and therefore considered to be not toxicologically significant.

There were no indications of possible reproductive toxicity based on the parameters determined in this study. All females showed a normal (regular) estrous cycle of 4 days during the period in which estrous cycle length was determined (Day 79 up to and including Day 97), and histopathological examination of the male and female reproductive organs (including spermatogenesis staging) did not show treatment-related lesions .

In conclusion, although no systemic toxicity is seen after treatment up to 1000 mg/kg for 90-days, based on mortality and the local test item-related morphologic alterations in the stomach of males and females, a NOAEL of 300 mg/kg was established.

Executive summary:

Repeated dose 90-day oral toxicity study with Bismuth Trinitrate by daily gavage in the rat.

Guidelines:

The study was based on the following guidelines:

·        EC No 440/2008, B.26 Repeated Dose (90 days) Toxicity (oral), 2008.

·        OECD 408, Repeated Dose 90-day Oral Toxicity Study in Rodents, 1998.

·        OPPTS 870.3100, EPA 712-C-98-199, 90-Day Oral Toxicity in Rodents, 1998.

·        Japanese Chemical Substances Control Law 1973, Notification of Mar. 31 2011 by MHLW (0331 No.7), METI (H23.03.29 SeiKyoku No. 5) and MOE (No. 110331009).

Rationale for dose levels:

Based on the results of a 14-day range finding study, the dose levels for this 90-day oral gavage study were selected to be 0, 100, 300 and 1000 mg/kg.

Study outline:

The test item, formulated in propylene glycol, was administered daily for at least 90 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females.

Evaluated parameters:

Chemical analyses of formulations were conducted in Weeks 1, 6 and 13 to assess accuracy, homogeneity and stability over 5 hours at room temperature under normal laboratory light conditions and over 8 days in the refrigerator protected from light.

The following parameters were evaluated: clinical signs daily; functional observation tests in Week 12-13; body weight and food consumption weekly; ophthalmoscopy at pretest and in Week 13; estrous cycle determination during the last 2 weeks; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Results:

Formulation analyses confirmed that formulations of test item in propylene glycol were prepared accurately and homogenously, and were stable over at least 5 hours at room temperature under normal laboratory light conditions and over 8 days in a refrigerator.

Test item-related mortality occurred in this study. Unscheduled deaths were observed at 1000 mg/kg (males 35, 40 and females 72, 76). These animals showed macroscopic changes in the gastro-intestinal tract (black-brown/red foci on the glandular stomach, irregular surface of the glandular stomach, black content in the cecum, GI-tract distended with gas) and thymus and/or spleen (reduced size). This correlated microscopically with ulcerations/erosions, necrosis and hemorrhage(s) in the glandular stomach and lymphoid depletion/atrophy in thymus and/or spleen. Additional microscopic findings were lymphoid depletion in the mesenteric lymph nodes and mandibular lymph node in a single female and increased adipocytes in the bone marrow, which was most likely secondary to the moribund condition of the animals. Particularly the ulcerations/erosions in the stomach are considered to be largely responsible for the cause of death/moribundity of the early sacrifices. The animals showed salivation at most of the days during treatment period. The following symptoms were

noted in the days or up to two weeks prior to death: hunched posture, labored or deep respiration, rales, gasping, squeaking, scabs in the neck, piloerection, lean or pale appearance, swelling of the abdomen, hypothermia.

Two unscheduled deaths treated at 100 mg/kg (male 13) and 1000 mg/kg (female 79) showed macroscopic and/or microscopic findings suggestive for a dosing error (male 13: fluid in the thoracic cavity, granulocytic inflammatory infiltrates with foreign material in the tissue adjacent to the aorta; female 79: granulocytic infiltration, epithelial erosion and hemorrhages in the esophagus). Animal no.13 showed labored respiration on Days 42 and 43. And animal no.79 showed salivation during the treatment period and hunched posture, labored respiration, rales, swelling of the abdomen and lean appearance, a few days prior to death.

 

Surviving animals:

The surviving animals showed at 1000 mg/kg rales during the treatment period. Towards the end of the treatment period additional symptoms were noted such as hunched posture, piloerection and labored or deep respiration. Salivation was also noted in these animals (and at 300 mg/kg), however this symptomwas not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing).Functional observation tests revealed no adverse changes. The lower motor activity of females at 1000 mg/kg, and a trend towards lower motor activity for males at 1000 mg/kg were considered not to represent an adverse effect on neurobehaviour. These results were not supported by clinical observations or other functional observation tests, were slight in nature (within the normal range for rats of this age and strain), and had no supportive morphological correlates in examined neuronal tissues.

The females at 1000 mg/kg showed slightly lower body weight gain compared to control animals, which was not supported by an effect in food consumption 

Macroscopic and microscopic examination revealed test item-related black content in the stomach and/or cecum in surviving males and females at 300 and 1000 mg/kg. A single female at 1000 mg/kg showed a reduced size of thymus and spleen. No microscopic correlate was found for the black content of stomach and/or cecum, the microscopic correlate for reduced thymus was lymphoid depletion. The single female of the scheduled sacrifices with marked lymphoid depletion did not show lymphoid depletion/atrophy in any of the other lymphoid organs and no other characteristics that could explain the marked lymphoid depletion in the thymus were found in the other organs. Therefore, the thymus depletion in this particular female rat was likely an incidental finding rather than a test item-related finding.

Microscopic examination revealed slight hemorrhage(s) in the glandular stomach in a single male at 1000 mg/kg (minimal hemorrhage was considered to be background), which was considered not to be adverse based on the absence of any other findings in the stomach.  

Slight effects in haematology and clinical biochemistry were observed (i.e. changes in red blood cell count, reticulocyte count, mean corpuscular volume, haematocrit level and changes in levels of cholesterol, total protein, albumin, total bilirubin and creatinine). These changes were slight and with no clear related morphological changes and therefore considered to be not toxicologically significant.

There were no indications of possible reproductive toxicity based on the parameters determined in this study. All females showed a normal (regular) estrous cycle of 4 days during the period in which estrous cycle length was determined (Day 79 up to and including Day 97), and histopathological examination of the male and female reproductive organs (including spermatogenesis staging) did not show treatment-related lesions.

 

Conclusion:

In conclusion, although no systemic toxicity is seen after treatment up to 1000 mg/kg for 90-days, based on mortality and the local test item-related morphologic alterations in the stomach of males and females, a NOAEL of 300 mg/kg was established.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
300 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 1 study.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
no data available
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Route of exposure is not relevant for risk assessment.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Conduction of a 13-week intratracheal intermittent bismuth dose toxicity study.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: Crj:CD(SD)IGS
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
Route of administration:
other: intratracheal instillation
Vehicle:
other: 0.5% carboxymethylcellulose sodium solution
Remarks on MMAD:
MMAD / GSD: no data
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.
Remarks:
Doses / Concentrations:
20 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
4 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.8 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
The dosing suspensions were intratracheally administered to the 12 animals of each dosing group.
Control animals:
yes, concurrent vehicle
Details on study design:
- 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), heamatocrit 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, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
No abnormal clinical signs attributable to bismuth administration were observed in any treated group during the testing period. 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.
Loss of hair was observed in one and two animals in the 4 and 20 mg/kg groups, respectively.

BODY WEIGHT AND WEIGHT GAIN
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
No abnormal food consumption attributable to bismuth administration were observed in any treated group during the testing period.

FOOD EFFICIENCY
no data

WATER CONSUMPTION
no data

OPHTHALMOSCOPIC EXAMINATION
no data

HAEMATOLOGY
A slight increase in erythrocyte count and MCHC were observed in the 20 mg/kg group, and slight increases in haemoglobin concentration and haematocrit 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 CHEMISTRY
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
There were no significant differences between the control group and any treated group in the urinalysis.

NEUROBEHAVIOUR
no data

ORGAN WEIGHTS
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 PATHOLOGY
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.

HISTOPATHOLOGY: NON-NEOPLASTIC
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 haemosiderin pigment by Berlin blue staining.

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 elimination
from the lungs as 4.47, 3.25, 2.10 days for the doses of 20, 100, 500 mg/kg, respectively.
Dose descriptor:
other:
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)
Critical effects observed:
not specified
Conclusions:
The author concluded that dose-dependent, but not specific adverse effects, were attributable to read-across substance, bismuth, administered by inhalation in the rat.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Quality of whole database:
Considered not reliable (Klimisch 3) as route of exposure (intratracheal) is not relevant for risk assessment..

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
no data available
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Route of exposure is not relevant for risk assessment.
Qualifier:
no guideline followed
Principles of method if other than guideline:
Conduction of a 13-week intratracheal intermittent bismuth dose toxicity study.
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
other: Crj:CD(SD)IGS
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
Route of administration:
other: intratracheal instillation
Vehicle:
other: 0.5% carboxymethylcellulose sodium solution
Remarks on MMAD:
MMAD / GSD: no data
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.
Remarks:
Doses / Concentrations:
20 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
4 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0.8 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
The dosing suspensions were intratracheally administered to the 12 animals of each dosing group.
Control animals:
yes, concurrent vehicle
Details on study design:
- 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), heamatocrit 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, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
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
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
No abnormal clinical signs attributable to bismuth administration were observed in any treated group during the testing period. 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.
Loss of hair was observed in one and two animals in the 4 and 20 mg/kg groups, respectively.

BODY WEIGHT AND WEIGHT GAIN
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
No abnormal food consumption attributable to bismuth administration were observed in any treated group during the testing period.

FOOD EFFICIENCY
no data

WATER CONSUMPTION
no data

OPHTHALMOSCOPIC EXAMINATION
no data

HAEMATOLOGY
A slight increase in erythrocyte count and MCHC were observed in the 20 mg/kg group, and slight increases in haemoglobin concentration and haematocrit 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 CHEMISTRY
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
There were no significant differences between the control group and any treated group in the urinalysis.

NEUROBEHAVIOUR
no data

ORGAN WEIGHTS
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 PATHOLOGY
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.

HISTOPATHOLOGY: NON-NEOPLASTIC
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 haemosiderin pigment by Berlin blue staining.

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 elimination
from the lungs as 4.47, 3.25, 2.10 days for the doses of 20, 100, 500 mg/kg, respectively.
Dose descriptor:
other:
Based on:
test mat.
Sex:
male
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)
Critical effects observed:
not specified
Conclusions:
The author concluded that dose-dependent, but not specific adverse effects, were attributable to read-across substance, bismuth, administered by inhalation in the rat.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Quality of whole database:
Considered not reliable (Klimisch 3) as route of exposure (intratracheal) is not relevant for risk assessment.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Repeated oral toxicity:

No published data or studies for determination the effects of repeated oral doses of bismuth trinitrate are available.

Annex XI of the REACH Regulation specifies rules for adaptation of the standard testing requirements. Point 1.5 of Annex XI permits read-across from substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity.

In an available publication, treatment with bismuth had no significant effects on clinical signs, body weights, food consumption, haematology, clinical chemistry, urinalysis, organ weights, necropsy, or histopathological findings in a 28-day repeated oral dose toxicity study. As a result of the findings, the NOAEL of bismuth was determined to be 1000 mg/kg for males and females.

A read-across concept is applicable from bismuth to bismuth trinitrate considering that they both contain bismuth, the moiety of toxicological concern, and that the substances have similar physicochemical properties.

In addition to fulfil the data requirements for a substance imported or manufactured in quantities 100 to 1000 t/y according to the Annex IX of the regulation (EC) 1907/2006, a 90-day oral toxicity study in rats according to OECD guideline 408 is proposed using read-across substance, bismuth subnitrate.

Repeated dermal toxicity:

A dermal repeated dose toxicity study for bismuth trinitrate is considered to be scientifically unjustified considering that dermal absorption of bismuth is considered to be negligible.

Repeated inhalation toxicity:

No reliable or relevant studies or data are available for bismuth trinitrate. One publication about intratracheal administration of bismuth to rats for 13 weeks is available, however, the exposure route is not reliable for risk assessment. The results showed that dose-dependent, but not specific adverse effects, were attributable to bismuth administration in the rat.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only 1 study is available for read-across substance, bismuth.
A 90-day repeated dose toxicity study using read-across substance, bismuth subnitrate, is also proposed.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
There is a 90-day toxicity study in Sprague-Dawley rats available with intratracheal instillation of bismuth metal. This study was not considered for risk characterization, because although some unspecific effects of local toxicity were observed these could not be attributed to bismuth and are considered as not representative for inhalation exposure.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
There is a 90-day toxicity study in Sprague-Dawley rats available with intratracheal instillation of bismuth metal. This study was not considered for risk characterization, because although some unspecific effects of local toxicity were observed these could not be attributed to bismuth and are considered as not representative for inhalation exposure.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
The oral route is considered the most appropriate route of exposure.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
The oral route is considered the most appropriate route of exposure.

Justification for classification or non-classification

The above oral repeated dose toxicity study has been ranked reliability 2 according to the Klimisch et al system. This ranking was deemed appropriate because sufficient dose ranges and numbers are detailed; hence it is appropriate for use based on reliability and animal welfare grounds. By read across, the above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008).