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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
reproductive toxicity, other
Remarks:
OECD Guideline 408 study with additional reproductive toxicity endpoints
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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 408 study with additional reproductive toxicity endpoints
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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Identification: Bismuth Trinitrate
Chemical name (IUPAC), synonym or trade name: Bismuth(3+) trinitrate, Bismuth trinitrate, Bismuth(III) nitrate pentahydrate
CAS Number: 10361-44-1

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

Test animals

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on 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.
Details on mating procedure:
Not applicable
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:
Frequency of treatment
Once daily, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Details on study schedule:
Estrous cycling determination, testosterone analysis and sperm analysis were conducted in this study.
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day
Dose / conc.:
300 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
No. of animals per sex per dose:
10 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
Positive control:
None

Examinations

Parental animals: Observations and examinations:
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 re corded 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 M 4-10, 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.


Oestrous cyclicity (parental animals):
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)).
Sperm parameters (parental animals):
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).
At necropsy: Weights of testes, epididymides and seminal vesicles were recorded. Histopathological examination of these organs was conducted.
Litter observations:
None
Postmortem examinations (parental animals):
Y: 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.
Postmortem examinations (offspring):
None
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 thecomparison 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.
Reproductive indices:
Estrous cycling determination, testosterone analysis and sperm analysis were conducted in this study.
Offspring viability indices:
None

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

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.
Dermal irritation (if dermal study):
not examined
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:
effects observed, treatment-related
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:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
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.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Histopathological examination of the male and female reproductive organs (including spermatogenesis staging) did not show treatment-related lesions .
Reproductive performance:
not examined

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: NOAEL for general toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: There were no indications of possible reproductive toxicity up to the highest dose tested (1000 mg/kg) based on the parameters determined in this study.
Remarks on result:
other: NOAEL for reproductive toxicity

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

Not applicable

Effect levels (F1)

Key result
Dose descriptor:
other:
Remarks:
Not evaluated
Generation:
F1
Remarks on result:
not measured/tested

Target system / organ toxicity (F1)

Key result
Critical effects observed:
not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:
not examined
Dermal irritation (if dermal study):
not examined
Mortality / viability:
not examined
Body weight and weight changes:
not examined
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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 on 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. There were no indications of possible reproductive toxicity up to the highest dose tested (1000 mg/kg) based on the parameters determined in this study.
Executive summary:

Repeated dose 90-day oral toxicity study with Bismuth Trinitrate by daily gavage in the rat (with additional reproductive toxicity endpoints).

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 gastrointestinal 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.

There were no indications of possible reproductive toxicity up to the highest dose tested (1000 mg/kg) based on the parameters determined in this study.