Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 May 2015 to 7 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in accordance with OECD and EU Guidelines.

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
See below for details.
Principles of method if other than guideline:
Deviations:
The target ranges for relative humidity and temperature were to be between 50 ± 20% and 22 ± 3°C, respectively. Instances of higher relative humidity were noted during this study on twenty three occasions between 17 June 2015 and 22 August 2015. During these episodes, the relative humidity ranged between 70.53 to 78.94% RH. One instance of lower temperature, where values ranged between 17.55 to 18.76 °C, was noted on 10 June 2015 whilst a single instance of
higher temperature (25.15 °C) was also noted on 01 July 2015. Although these episodes of higher relative humidity or lower/higher temperature were less than ideal, they were of short duration with the majority of relative humidity incidents and the single high temperature incident lasting for a maximum of up to two hours. The high temperature incident was considered to be due to a technical fault with the air conditioning system and specific measures were put into place immediately to rectify the situation on 01 July 2015 when the technical fault had occurred. The low temperature incident also occurred on one occasion only and lasted for a maximum of up to six hours. Clinical condition of the animals was considered to have remained unaffected by these episodes and this deviation from the Study Plan was therefore considered not to have any impact on the integrity of the study or results obtained.
According to the Study Plan, samples of the homogenate (from testis) were to be examined microscopically to determine the number of homogenisation resistant spermatids present. This was a typographical error in the Study Plan as an automated semen analyser is utilized at the Test Facility for this purpose. This deviation from the Study Plan therefore did not have any impact on the integrity of the study or results obtained.
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Identification : Bismuth Subnitrate
Physical State/Appearance : White powder
Chemical Name : Bismuth hydroxide nitrate oxide
CAS Number : 1304-85-4
EC Number : 215-136-8

Test animals

Species:
rat
Strain:
other: Wistar Han™:RccHan™:WIST
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK.
- Age at study initiation: approximately six to eight weeks old.
- Weight at study initiation: the males weighed 198 to 238g, the females weighed 131 to 167g,
- Fasting period before study: Not specified
- Housing: The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
- Diet: The animals were allowed free access to food. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used.
- Water: The animals were allowed free access to water. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: The animals were acclimatized for at least nine days (before the start of treatment) during which time their health status was assessed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): target range: 22 ± 3 °C. One instance of lower temperature, where values ranged between 17.55 to 18.76 °C, was noted on 10 June 2015 whilst a single instance of higher temperature (25.15 °C) was also noted on 01 July 2015. Clinical condition of the animals was considered to have remained unaffected by these episodes.
- Humidity (%): target range: 50 ± 20%
- Air changes (per hr): The rate of air exchange was at least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): The low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness.
Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records.

IN-LIFE DATES: From: To: 2 June 2015 (first day of treatment) and 11 September 2015 (final day of necropsy).

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Most of the lab's background data was with Arachis oil hence this was there preferred vehicle.
- Concentration in vehicle: At dose level of 40 mg/kg bw/day, concentration was 10 mg/ml. At dose of 200 mg/kg bw/day, the concentration was 50 mg/ml. At dose level of 1000 mg/kg bw/day, the concentration was 250 mg/ml.
- Amount of vehicle (if gavage): Treatment volume: 4 ml/kg
- Lot/batch no. (if required): Not provided
- Purity: Not provided

The test item was administered daily, for ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP. The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Since the method used for formulation analysis was non-stability indicating, test item formulation stability was not determined, and therefore, fresh formulations were prepared each day and dosed within two hours of preparation. It is assumed that the formulation was stable for this duration. As stability was not determined, this is an exception with regards to GLP and has been reflected in the GLP compliance statement. Homogeneity of the test item formulations was demonstrated by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services.

Due to the complex nature of the test item and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis could not been developed. The concentration of test item in the formulations was determined using a gravimetric technique. The test item formulations were weighed into tared glass sintered crucibles and then rinsed with acetone to leave a test item residue. The samples were then dried in an oven at approximately 105 degrees C before allowing to cool over silica gel in a dessicator and re-weighed.

Samples of Arachis oil BP were accurately fortified with known amounts of test item equivalent to the lowest and highest anticipated dose concentrations.

The fortified samples of Arachis oil BP were found to have a recovery value of +/- 10% of the fortification.
The formulations investigated during the study were found to comprise test item in the range of 93% to 103% and thus the required content limit of +/- 10% with reference to the nominal content was met.

The results indicate the accurate use of the test item and Arachis oil BP as vehicle during the study. the formulations were found to be homogeneously prepared.

The analytical procedure had acceptable recoveries of test item in the vehicle. The method of analysis was validated and proven to be suitable for use.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
40 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
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: Based on doses used in 28 day study with Bismuth (Sano et al., 2005)
- Rationale for animal assignment: The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
- 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

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was recorded for each cage group at weekly intervals throughout the study.

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

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-treatment and before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye. Following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.
- Dose groups that were examined: Th.e eyes of all control and high dose animals were examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91.
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: All animals from each test and control group.
- Parameters examined.
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH), - mean corpuscular volume (MCV), - mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
- Methylene blue stained slides were prepared but reticulocytes were not assessed
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91.
- Animals fasted: No
- How many animals: All animals from each test and control group.
- Parameters examined:
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.)
Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation)
Creatinine (Creat)
Sodium (Na+)
Total cholesterol (Chol)
Potassium (K+)
Total bilirubin (Bili)
Chloride (Cl-)
Bile acids
Calcium (Ca++)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: All animals.
- Battery of functions tested:
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait
Hyper/Hypothermia
Tremors
Skin color
Twitches
Respiration
Convulsions
Palpebral closure
Bizarre/Abnormal/Stereotypic behavior
Urination
Salivation
Defecation
Pilo-erection
Transfer arousal
Exophthalmia
Tail elevation
Lachrymation

This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979).
Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.
The following parameters were observed:
Grasp response
Touch escape
Vocalization
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach

OTHER:
Oestrus cycling
Vaginal smears were taken daily for 21 days, on all test and control group females, during the final three weeks of the study. The stage of oestrus was recorded for each day.

Testosterone Hormone Assessment
On Day 90 of dosing, whole blood samples (ca. 0.35ml to yield approximately 0.15 ml of plasma) was taken from the lateral tail vein from all males into labelled lithium heparin coated blood tubes. All samples were mixed gently, by inverting several times, and placed on a roller before being centrifuged (approximately 2570 g, 10 minutes, room temperature). The plasma was separated off, collected into Eppendorf tubes and immediately placed on dry ice (within approximately 30 minutes of obtaining the blood sample). As soon as practical thereafter, plasma samples were stored in the freezer (approximately -70°C) before shipment, packed in dry ice, to the Test Site for analysis.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Necropsy
On completion of the dosing period all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals
Ovaries
Brain
Spleen
Right Epididymis
Right Testis
Heart
Thymus
Kidneys
Uterus
Liver

SPERM ANALYSIS
At necropsy, the left testis and epididymis were removed from all males, dissected from connective tissue and weighed separately.
For the epididymis, the distal region was incised and a sample of the luminal fluid was collected and transferred to a buffer solution for analysis of sperm motility. The semen sample was assessed using an automated semen analyser to determine the numbers of motile, progressively motile and non-motile sperm.
For the testis, the tunica albuginea was removed and the testicular tissue was stored frozen at approximately -20°C. The tissue was later thawed and homogenized in a suitable saline/detergent mixture. Samples of the homogenate were examined to determine the number of homogenization resistant spermatids present; see deviations from Study Plan.
The cauda epididymis was separated from the body of the epididymis and weighed. The cauda epididymis was frozen at approximately -20°C. The tissue was later thawed and homogenized in an appropriate saline/detergent to determine the numbers of homogenization resistant spermatids.
Morphological assessment was performed on a sample of a minimum of 200 sperm to determine the number with apparent structural anomalies.
Assessment of homogenization resistant spermatids and morphological evaluation were only performed for control and 1000 mg/kg bw/day males. As there were no treatment-related findings, these evaluations were not extended to males from other dose groups.

HISTOPATHOLOGY: Yes
Samples of the following tissues were removed from all animals and preserved in buffered 10%
formalin, except where stated:
Adrenals, Ovaries, Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint), Pituitary, Bone & bone marrow (sternum), Prostate, Brain (including cerebrum, cerebellum and pons), Rectum, Caecum, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Right Epididymis, Skin, Esophagus, Spinal cord (cervical, mid-thoracic and lumbar), eyes, Gross lesions, Spleen, Heart, Stomach, Ileum (including Peyer’s patches), Right Testis, Jejunum Thymus, Kidneys Thyroid/Parathyroid, Liver, Tongue, Lungs (with bronchi), Trachea, Lymph nodes (mandibular and mesenteric), Urinary bladder, Mammary glands, Uterus (with cervix), Muscle (skeletal), Vagina.

All tissues were dispatched to the Test Site (Envigo CRS Limited) for processing (Principal Investigator: D Roberts). All tissues from control and 1000 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 μm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.
Microscopic examination was conducted by the Study Pathologist (W Henderson). A peer review of the findings was conducted by Peter Millar (Peter Millar Associates Ltd. Edinburgh) at the histopathology peer review test site.
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters: Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights, Sperm Analysis Parameters,
Testosterone Concentrations.

Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows: Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for nonparametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-
Whitney U test (non-parametric). Sperm analysis parameters and testosterone concentrations were statistically analyzed using the R Environment for Statistical Computing. Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pairwise comparisons using Dunnett’s test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Compound intake was not exmained
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Compound intake was not exmained
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during the study.
Throughout the treatment period, there were no clinical signs at any dose level considered to be related to the toxicity of the test item.
Clinical observations were confined to a few instances of increased post-dose salivation for individual males treated with 1000 mg/kg bw/day during Weeks 5, 7 and 10 of dosing. A single incident of increased post-dose salivation was also observed for one female from this dose group during Week 10. Such observations are often observed following the oral gavage administration of an unpalatable or slightly irritant test item formulation and are considered to be of no toxicological importance.

BODY WEIGHT AND WEIGHT GAIN
When compared with controls, males treated with 1000 mg/kg bw/day showed statistically significantly lower group mean body weight gains during Weeks 6 and 13 of dosing (p<0.05). Females receiving 200 or 1000 mg/kg bw/day also showed statistically significantly reduced body weight gains during Weeks 10 and 13 (p<0.05 or p<0.01). Minor group mean body weight losses were observed for both sexes receiving 1000 mg/kg bw/day during Week 13. This resulted in marginally reduced overall group mean body weight gains for animals of either sex receiving 1000 mg/kg bw/day in relation to their respective controls (approximately 7% each).
The majority of individual body weight gain values for the test item-treated animals were, however, similar to controls and taking into consideration the small magnitude of these differences, this finding was deemed not to be of an adverse nature.

FOOD CONSUMPTION
Throughout the treatment period, weekly food consumption values for the test item-treated males and females were generally comparable with their respective controls. Any differences in food conversion efficiency were deemed to be reflective of fluctuations in body weight gains and/or
dietary intake.

WATER CONSUMPTION
Visual inspection of water bottles did not reveal any intergroup differences.

OPHTHALMOSCOPIC EXAMINATION
Opthalmoscopic examination of animals of both sexes from the control and 1000 mg/kg bw/day dose groups did not indicate any treatment-related differences.

HAEMATOLOGY
Males receiving the test item at all dose levels and females treated with 200 or 1000 mg/kg showed statistically significant decreases in mean corpuscular hemoglobin concentrations relative to controls (p<0.01 for females receiving 1000 mg/kg bw/day and p<0.05 in all other instances). There was no dose-relationship in males and whilst the majority of individual values from the test item-treated animals of either sex were within the historical control data ranges, 3/10 control males and 2/10 control females showed atypically high values which may explain these differences. Males treated with 200 or 1000 mg/kg bw/day also showed statistically significantly higher mean corpuscular volume in comparison with controls (p<0.05) albeit without any dose-dependence and with all individual values remaining within the background data ranges. In the absence of any alteration in related hematology parameters, these findings were considered to be of no toxicological significance. When compared with controls, group mean prothrombin times in females treated with 200 or 1000 mg/kg bw/day were statistically significantly higher than controls (p<0.05) in a dose related manner. Most individual values were within the background data ranges whilst the corresponding group mean values in males were similar to controls. In the absence of any related histopathology findings, this observation was considered to be of no toxicological relevance.

CLINICAL CHEMISTRY
At 1000 mg/kg bw/day, animals of either sex, in particular the females, showed statistically significantly higher plasma levels of urea when compared with controls (P<0.05). Group mean plasma concentration of creatinine in these females was also statistically significantly higher than controls (P<0.05), however males from this dose group showed comparable creatinine values to their respective controls. Females treated with 200 mg/kg bw/day also showed slightly higher plasma concentrations of urea and creatinine in relation to controls but without achieving statistical significance. Whilst these differences in females were dose-related and with most individual values for the 1000 mg/kg bw/day females outside the historical data ranges,
microscopic examination of relevant tissues did not identify any treatment-related findings and as such these observations were considered not to be of any toxicological importance. When compared with controls, males and females treated with 1000 mg/kg bw/day showed slightly higher plasma levels of glucose albeit without any dose-dependence and with statistical significance only achieved in females (p<0.01). Although most individual values for the 1000 mg/kg bw/day females were outside the historical control data ranges, in the absence of any histopathological correlates, this finding was considered to be of no toxicological significance. At all dose levels, females showed statistically significantly lower plasma levels of bilirubin with respect to controls (p<0.01). Whilst a dose-relationship was apparent, all individual values were within the control data ranges and group mean values in the corresponding males were similar to controls. Other statistically significant intergroup differences in relation to controls were confined to the 1000 mg/kg bw/day females and included a reduction in group mean plasma alkaline phosphatase level (p<0.05) and an increase in plasma chloride concentration (p<0.01). All individual values from the test item-treated females were within the background data ranges whilst the corresponding parameters in males from this dose group were similar to controls. As there were no treatment-related microscopic observations in any relevant tissues, these findings were deemed to be of no toxicological importance.

NEUROBEHAVIOUR
Behavioral Assessments
There were no changes in the behavioral parameters considered to be related to treatment with Bismuth Subnitrate at any dose level.
Functional Performance Tests
There were no intergroup differences considered to be related to treatment with the test item. When compared with controls, males treated with 1000 mg/kg bw/day showed a statistically significant decrease in forelimb strength in 1/3 tests during Week 12 of the treatment period (p<0.05). Although a dose-relationship was apparent, similar intergroup differences were not evident in the remaining limb strength tests for these males or for any of the female dose groups and, in the absence of any signs of neurotoxicity on this study, this finding was considered likely to be incidental.
Sensory Reactivity Assessments
Sensory reactivity scores across all test item-treated dose groups were similar to controls.

ORGAN WEIGHTS
Males treated with 200 or 1000 mg/kg bw/day showed statistically significantly lower absolute and body weight-related liver weights in relation to controls (p<0.05 and p<0.01, respectively).
A dose-relationship was evident, but most individual values from the test item-treated animals were within the historical control data ranges. In the absence of any histopathology correlates, this finding was considered unlikely to be of any toxicological significance.
Males treated with 40 mg/kg bw/day also showed statistically significantly higher thymus weights with respect to controls (p<0.05). The corresponding values from the remaining test item-treated male dose groups were similar to controls and as such this finding was considered to
be incidental.

GROSS PATHOLOGY
At necropsy, most animals of either sex given 1000 mg/kg bw/day and 1/10 females treated with 200 mg/kg bw/day showed green colored contents in the caecum with one of the 1000 mg/kg bw/day females also showing green colored contents in the stomach. Microscopic evaluation of these tissues did not identify any treatment-related findings and as such these observations were considered unlikely to be of any toxicological significance.
A small number of males and females from all dose groups including controls showed red discoloration of lungs. One male from the 200 mg/kg bw/day dose group was observed with small/flaccid testes and small epididymides. These findings were deemed unlikely to be related to treatment with the test item.

HISTOPATHOLOGY
No consistent changes were noted which could be related to treatment with the test item. No histopathological changes were found to account for the clinical chemistry alterations nor were any associated with the caecal changes.

OTHER FINDINGS
Oestrus Cycling
There was no effect of treatment with Bismuth Subnitrate at any dose level on oestrus cycling activity in females as assessed over the last three weeks of dosing.
Testosterone Analysis
There was no effect of treatment with Bismuth Subnitrate at any dose level on plasma concentrations of testosterone in males.
Sperm Analysis
At necropsy, sperm analysis did not indicate any appreciable differences in group mean sperm concentration and motility at any dose level. An evaluation of homogenization resistant spermatids and morphology in males from the control and 1000 mg/kg bw/day dose groups also did not reveal any treatment-related differences.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related adverse effects detected at highest dose tested (1000 mg/kg bw/day)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Tables 1 to 17 are attached below under 'Attached Background Material'.

Applicant's summary and conclusion

Conclusions:
The oral (gavage) administration of Bismuth Subnitrate to male and female Wistar Han™:RccHan™:WIST strain rats at dose levels up to 1000 mg/kg bw/day was well tolerated.
There was no adverse effect of treatment on body weight development and dietary intake in animals of either sex. Hematology, blood chemistry, testosterone hormone assessment, estrous cycle assessment in females, sperm analysis in males and microscopic examination of the selected tissues did not identify any findings of toxicological relevance. A dose level of 1000 mg/kg bw/day is therefore considered to be the ‘No Observed Adverse Effect Level’ (NOAEL).
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity of the test item and is compatible with the following regulatory guidelines:

i) The OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dose levels of 40, 200 and 1000 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study. Plasma concentrations of testosterone were evaluated for all males on Day 90 of dosing. Ophthalmoscopic examination was also performed on control group and high dose animals. In addition, sperm concentrations and motility were analyzed for males at necropsy followed by an evaluation of morphology and homogenization resistant spermatid counts in control and high dose males. Estrous cycling was also evaluated for females toward the end of the treatment period.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed.

Results

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

Throughout the treatment period, there were no clinical signs deemed to be indicative of test item toxicity.

Behavioral Assessment

Behavioral assessment scores across the test-item treated animals of either sex remained similar to the respective controls.

Functional Performance Tests

There were no treatment-related changes in functional performance at any dose level.

Sensory Reactivity Assessments

Sensory reactivity scores were comparable across all dose groups including controls.

Body Weight

There was no adverse effect of treatment with Bismuth Subnitrate at any dose level on body weight development in animal of either sex.

Food Consumption

There was no adverse effect of treatment with Bismuth Subnitrate at any dose level on food consumption or food conversion efficiency in animal of both sexes.

Water Consumption

Visual inspection of water bottles did not reveal any intergroup differences.

Ophthalmoscopy

Ophthalmoscopic examination of males and females from control and 1000 mg/kg bw/day dose group during Week 12 of the study did not reveal any treatment-related differences.

Estrous Cycling

There was no effect of treatment with the test item on estrous cycling activity assessed over the last three weeks of dosing in females.

Hematology

Hematology evaluations did not reveal any toxicologically significant effects in animals of either sex resulting from treatment with the test item.

Blood Chemistry

Blood chemistry evaluations did not indicate any effects of toxicological relevance in animals of both sexes resulting from test item administration.

Testosterone Hormone Assessment

There was no effect of treatment with the test item at any dose level on plasma levels of testosterone.

Necropsy

Changes noted in the colour of the caecal contents in a number of animals of either sex given 200 (one female) or 1000 mg/kg bw/day were not associated with any microscopic observations and as such this findings was considered to be no toxicological relevance. Any other macroscopic findings observed at necropsy were considered unlikely to be related to treatment with the test item.

Organ Weights

There were no intergroup differences considered to be of toxicological relevance.

Sperm Analysis

Analyses of sperm concentration, motility, morphology and homogenization resistant spermatids did not identify any treatment-related differences.

Histopathology

No findings were observed at histopathology which could be related to treatment with Bismuth Subnitrate within the confines of this study.

Conclusion

The oral (gavage) administration of Bismuth Subnitrate, to male and female Wistar Han™:RccHan™:WIST strain rats at dose levels up to 1000 mg/kg bw/day was well tolerated.

There was no adverse effect of treatment on body weight development and dietary intake in animals of either sex. Hematology, blood chemistry, testosterone hormone assessment, oestrus cycle assessment in females, sperm analysis in males and microscopic examination of the selected tissues did not identify any findings of toxicological relevance. A dose level of 1000 mg/kg bw/day is therefore considered to be the ‘No Observed Adverse Effect Level’ (NOAEL).