Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information
No further studies are available.
Link to relevant study records
Reference
Endpoint:
toxicity to reproduction
Remarks:
other: 90 day repeated dose study with additional reproductive toxicity endpoints
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:
guideline study
Qualifier:
according to
Guideline:
other: OECD 408 study with additional reproductive toxicity endpoints
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
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).
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on 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.
Details on mating procedure:
Not applicable
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
Details on study schedule:
Estrous cycling, testosterone analysis and sperm analysis were conducted in this study.
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
200 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:
10 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
Parental animals: Observations and examinations:
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:

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.


Oestrous cyclicity (parental animals):
Estrous Cycle Assessment
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 estrus was recorded for each day.

Sperm parameters (parental animals):
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.
Litter observations:
None
Postmortem examinations (parental animals):
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.

HISTOPATHOLOGY/ORGAN WEIGHTS: Yes
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

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.
Postmortem examinations (offspring):
None
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.
Reproductive indices:
Estrous cycling, testosterone analysis and sperm analysis were conducted in this study.
Offspring viability indices:
None
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
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 and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
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.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Visual inspection of water bottles did not reveal any intergroup differences.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Opthalmoscopic examination of animals of both sexes from the control and 1000 mg/kg bw/day dose groups did not indicate any treatment-related difference
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):

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 biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
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
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
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.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
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 pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
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.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
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.
Histopathological findings: neoplastic:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
There was no effect of treatment with Bismuth Subnitrate at any dose level on plasma concentrations of testosterone in males.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
There was no effect of treatment with Bismuth Subnitrate at any dose level on estrous cycling activity in females as assessed over the last three weeks of dosing.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
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.
Reproductive performance:
not examined
Key result
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)
Key result
Critical effects observed:
no
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: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Not applicable
Key result
Dose descriptor:
other:
Remarks:
Not evaluated
Generation:
F1
Remarks on result:
not measured/tested
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
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
Key result
Reproductive effects observed:
no

Tables 1 to 17 are attached below under 'Attached background information'.

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).

Additional reproductive toxicity endpoints were evaluated in this study including estrous cycling, sperm analysis and testosterone analysis.

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, 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).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP study conducted in accordance with OECD and EU Guidelines. Rated as Klimisch 2 because it is a read across study.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Quality of whole database:
Oral route is considered the most appropriate route.
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a 90 day repeated dose oral toxicity study conducted in accordance with OECD Guideline 408, bismuth subnitrate 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). 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. Additional reproductive toxicity endpoints were evaluated in this study: estrous cycling, sperm analysis and testosterone analysis. 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).


Short description of key information:
In a 90 day repeated dose oral toxicity study with additional reproductive toxicity endpoints conducted in accordance with OECD Guideline 408, bismuth subnitrate had no toxicological effects on sperm or on testosterone levels in male rats or on the estrous cycle in female rats. The NOAEL in this study was 1000 mg/kg bw/day. Therefore, bismuth subnitrate is not predicted to have any toxic effects on fertility.

Justification for selection of Effect on fertility via oral route:
Longest duration - 90 days.

Justification for selection of Effect on fertility via inhalation route:
Oral route is considered the most appropriate route.

Justification for selection of Effect on fertility via dermal route:
Oral route is considered the most appropriate route.

Effects on developmental toxicity

Description of key information
The oral administration of bismuth subnitrate to pregnant rats by gavage during gestation at dose levels of 100, 300 or 1000 mg/kg bw/day was well tolerated. The ‘No Observed Effect Level’ (NOEL) for maternal toxicity was considered to be 1000 mg/kg bw/day.
No treatment-related changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity, was therefore, considered to be 1000 mg/kg bw/day.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 September 2015 to 04 November 2015.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
See below for details.
Principles of method if other than guideline:
Deviations:
Environment
According the Study Plan, the target ranges for relative humidity were to be between 50 ± 20%. An episode of higher relative humidity was noted during this study on 12 September 2015 during which the actual values ranged between 73.52 to 77.48% RH. Although this incident was less than ideal, it was of short duration and occurred only once during the study. Actual relative humidity values were only slightly above the target range whilst clinical condition of the animals was considered to have remained unaffected by this increase in relative humidity. This deviation from the Study Plan was therefore considered not to have any impact on the integrity of the study or results obtained.
Uterine Examination
According the Study Plan, fetal sex and placental weight were to be recorded for each individual fetus at necropsy on Day 20 of gestation. External fetal sex for two foetuses (fetal ID: V9 and V10) from Litter 63 was not recorded, in error. The sexes for these two foetuses were, however, determined during the processes performed for visceral or skeletal examination. As internal sexing is normally considered to be more definitive, this omission during necropsy examination was considered not to have any effect on the integrity of the study. Placental weights for two foetuses (fetal ID: V7 and V9 from Litters 28 and 33, respectively) were not recorded, in error. Sufficient relevant data were, however, available from the remaining litters for an adequate scientific interpretation of the results. There was no effect of treatment with the test item at any dose level on placental weights and this deviation from the study plan was considered not to have any effect on the integrity of the study or results obtained.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Sprague-Dawley Crl:CD®(SD) IGS BR
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited, Margate, Kent. Ninety-six time-mated female were delivered in two batches containing females prior to Day 3 of gestation. The day that positive evidence of mating was observed was designated Day 0 of gestation.
- Age at study initiation: No data
- Weight at study initiation: 171 to 269g.
- Fasting period before study: No fasting
- Housing: Individually housed in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
- Diet: Ad libitum. A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK) was used.
- Water: Tap water ad libitum. Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Acclimation period: None

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Target temperature: 22 ± 3 °C
- Humidity (%): Target humidity: 30 to 70%. An episode of higher relative humidity was noted during this study on 12 September 2015 during which the actual values ranged between 73.52 to 77.48% RH. Although this incident was less than ideal, it was of short duration and occurred only once during the study. Actual relative humidity values were only slightly above the target range whilst clinical condition of the animals was considered to have remained unaffected by this increase in relative humidity.
- Air changes (per hr): At least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): Twelve hours of continuous artificial light in each twenty-four hour period
Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).

IN-LIFE DATES: From 06 September 2015 (first day of treatment) to 23 September 2015 (final day of necropsy).

The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on 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 100 mg/kg bw/day, concentration was 25 mg/ml. At dose of 300 mg/kg bw/day, the concentration was 75 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
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 were taken of test item formulations on two occasions and were analyzed for concentration of Bismuth Subnitrate

The formulations investigated during the study were found to comprise test item in the range of 94% to 102% 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.
Details on mating procedure:
- Impregnation procedure: Purchased timed pregnant. Time-mated females were delivered in two batches containing females prior to Day 3 of gestation.
Duration of treatment / exposure:
From Day 5 (post-implantation) to Day 19 of gestation (the day prior to necropsy).
Frequency of treatment:
Once daily
Duration of test:
20 days after mating
Dose / conc.:
40 mg/kg bw/day (actual dose received)
Dose / conc.:
200 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:
24 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen in consultation with the Study Monitor based on the results from previous toxicity work including a 28 day toxicity study in rat with recovery groups (Sano et al., 2005) and a 90 day toxicity study in rat (Study Number: 41500380). In the 28 day study, administration of bismuth to male and female Crj:CD (SD) IGS rats (SPF) at dose levels of 40, 200 or 1000 mg/kg/day was well tolerated. There was no effect of treatment with the test item on body weight development and food consumption and no adverse findings were identified at any dose level in hematology, clinical chemistry and histopathology evaluations. In the 90 day study, 10 weeks of bismuth subnitrate dosing to male and female Wistar Han™:RccHan™:WIST strain rats at dose levels of 40, 200 or 1000 mg/kg/day had not resulted in any clinical signs related to the toxicity of the test item or any adverse effects on body weight performance or associated dietary intake. A dose level of 1000 mg/kg bw/day was therefore considered to be suitable for investigation in this OECD 414 study together with 100 and 300 mg/kg bw/day as the low and intermediate dose levels, respectively.
- Rationale for animal assignment: The animals were randomly allocated to treatment groups using a randomization procedure based on stratified body weight to ensure similarity between the treatment groups. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.
Maternal examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Following arrival, all animals were examined for overt signs of toxicity, ill-health or behavioral changes once daily during the gestation period. Additionally, during the dosing period, observations were recorded immediately before and soon after dosing and one hour post dosing. All observations were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded on Day 3 (before the start of treatment) and on Days 5, 6, 7, 8, 11, 14 and 17 of gestation. Body weights were also recorded for animals at terminal kill (Day 20).

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Food consumption was recorded for each individual animal at Day 3, 5, 8, 11, 14, 17 and 20 of gestation.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water intake was observed daily by visual inspection of the water bottles for any overt changes.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20: All animals were killed by carbon dioxide asphyxiation followed by cervical dislocation on Day 20 of gestation. All animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded.

Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes.
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

The uteri of any apparently non-pregnant females were immersed in 0.5% ammonium polysulphide solution to reveal evidence of implantation.
Implantation types were divided into:
Early Death: No visible distinction between placental/decidual tissue and embryonic tissue
Late Death: Separate embryonic/fetal and placental tissue visible
Dead Fetus: A fetus that had died shortly before necropsy. These were included as late deaths for reporting purposes
All implantations and viable fetuses were numbered according to their intrauterine position.
Fetal examinations:
- External examinations: Yes
- Soft tissue examinations: Yes
- Skeletal examinations: Yes

The fetuses were killed by subcutaneous injection of a suitable barbiturate agent. Fetuses from each litter were divided into two groups and examined for skeletal alterations and soft tissue alterations. Alternate fetuses were identified using an indelible marker and placed in Bouin’s fixative. Fetuses were subsequently transferred to distilled water and examined for visceral anomalies under a low power binocular microscope and then stored in 10% Buffered Formalin. The remaining fetuses were identified using cardboard tags marked with chinagraph pencil and placed 70% IMS in distilled water. The fetuses were subsequently eviscerated, processed and the skeletons stained with alizarin red S before being transferred to 50% glycerol for examination of skeletal development and anomalies and storage.
Statistics:
The following parameters were analyzed statistically, where appropriate, using the test methods outlined below:
Female body weight change, food consumption and gravid uterus weight: Shapiro Wilk normality test and Bartlett’s test for homogeneity of variance and one way analysis of variance, followed by Dunnett’s multiple comparison test or, if unequal variances were observed, on alternative multiple comparison test.
All caesarean necropsy parameters and fetal parameters: Kruskal-Wallis non-parametric analysis of variance; and a subsequent pairwise analysis of control values against treated values using the Mann-Whitney ‘U’ test, where significance was seen.
Fetal evaluation parameters, including skeletal or visceral findings: Kruskal-Wallis nonparametric analysis of variance and Mann-Whitney ‘U’ test.
Probability values (p) are presented as follows:
p<0.05 *
p≥0.05 (not significant)
Indices:
Reproductive Indices:
Pre-implantation loss = ((number of corpora lutea - number of implantations)/number of corpora lutea) x 100
Post-implantation loss = ((number of implantations - number of live fetuses)/number of implantations) x 100
% Male fetuses (sex ratio) = (number of male fetuses/total number of fetuses) x 100
Clinical signs:
no effects observed
Description (incidence and severity):
Throughout the study, there were no clinical signs for any of the animals.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths on the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Throughout the dosing period, there was no effect of treatment with the test item at any dose level on body weight development. Group mean gravid uterus weights and adjusted body weight gains across all dose groups including controls were comparable.
Group mean cumulative body weight gain for the 1000 mg/kg bw/day females over Days 5 to 7 of gestation was statistically significantly higher than controls (p<0.05). This was due to slightly higher weight gains for these females over Days 5 to 6 and 6 to 7 of gestation in relation to controls; these differences did not achieve statistical significance and were deemed likely due to biological variation.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Throughout the treatment period, there was no effect of treatment with the test item at any dose level on food consumption.
At 300 mg/kg bw/day, group mean dietary intake over Days 11 to 14 of gestation was statistically significantly higher than controls (p<0.05). The difference was only marginal and in the absence of any dose-relationship, this finding was considered likely to be incidental.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Daily visual inspection of water bottles did not reveal any overt intergroup differences.
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:
no effects observed
Description (incidence and severity):
There were no macroscopic findings for any of the females.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not specified
Number of abortions:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on in utero offspring survival.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on pre- and post-implantation losses.
Implantation loss (%) (group mean litter):
Control: pre-: 0.6%, post-:3.0%
100 mg/kg: pre-: 2.4%, post-: 1.9%
300 mg/kg: pre-: 1.4%, post-: 3.8%
1000 mg/kg: pre-: 1.4%, post-: 1.9%
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on in utero offspring survival, as assessed by the mean numbers of early or late resorptions.
Early or late resorptions:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on in utero offspring survival, as assessed by the mean numbers of early or late resorptions.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on in utero offspring survival.
Number of embryonic/fetal deaths (group mean litter):
Control: 0.4
100 mg/kg: 0.3
300 mg/kg: 0.5
1000 mg/kg: 0.3
Changes in pregnancy duration:
not examined
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not examined
Changes in number of pregnant:
effects observed, non-treatment-related
Description (incidence and severity):
In total, 24, 23, 24 and 24 females from the control, 100, 300 and 1000 mg/kg bw/day dose groups, respectively, were found to be pregnant at scheduled necropsy on Day 20 of gestation. Female 47 from the 100 mg/kg bw/day was found to be non-pregnant; however, this was an isolated incidence considered to be within the background ranges for this strain of animals and thus unrelated to treatment with the test item.
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No treatment-related adverse effects detected at highest dose tested (1000 mg/kg bw/day)
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: No treatment-related adverse effects detected at highest dose tested (1000 mg/kg bw/day).
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Fetal weights were unaffected by treatment with the test item at any dose level.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): Fetal weights were unaffected by treatment with the test item at any dose level.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on in utero offspring survival, as assessed by the mean numbers of early or late resorptions, live litter size and post-implantation losses.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
Fetal sex ratios across all treated groups were similar to controls.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on live litter size.
Fetal weights were also unaffected by treatment with the test item at any dose level.
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Description (incidence and severity):
For all dose groups, there were no treatment-related trends in the proportion of fetuses (or litters) with evidence of external abnormalities. Any statistically significant differences for external observations were considered to be incidental as the affected fetuses were limited to control litters.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
An analysis of the skeletal evaluation data did not identify any treatment-related differences. Whilst the total percentage of fetuses with skeletal observations in the 100 or 300 mg/kg bw/day litter were marginally but statistically significantly higher than controls (p<0.05), there was no dose-relationship and, in absence of any treatment-related differences for any of the skeletal parameters, this finding was deemed likely to be incidental.
Visceral malformations:
no effects observed
Description (incidence and severity):
For all dose groups, there were no treatment-related trends in the proportion of fetuses (or litters) with evidence of external or visceral abnormalities. Any statistically significant differences for external observations were considered to be incidental as the affected fetuses were limited to control litters.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Litter Data and Litter Placental and Fetal Weights
In total, 24, 23, 24 and 24 females from the control, 100, 300 and 1000 mg/kg bw/day dose groups, respectively, were found to be pregnant at scheduled necropsy on Day 20 of gestation. Female 47 from the 100 mg/kg bw/day was found to be non-pregnant; however, this was an isolated incidence considered to within the background ranges for this strain of animals and thus unrelated to treatment with the test item. The following assessment is based on fetuses from maternal termination on Day 20 of gestation.
There were no treatment-related effects on in utero offspring survival, as assessed by the mean numbers of early or late resorptions, live litter size and post-implantation losses. Preimplantation losses and fetal sex ratios across all treated groups were similar to controls. Fetal and placental weights were also unaffected by treatment with the test item at any dose level.

Fetal Examination
For all dose groups, there were no treatment-related trends in the proportion of fetuses (or litters) with evidence of external or visceral abnormalities. Any statistically significant differences for external observations were considered to be incidental as the affected fetuses were limited to control litters. An analysis of the skeletal evaluation data did not identify any treatment-related differences. Whilst the total percentage of fetuses with skeletal observations in the 100 or 300 mg/kg bw/day litter were marginally but statistically significantly higher than controls (p<0.05), there was no dose-relationship and, in absence of any treatment-related differences for any of the skeletal parameters, this finding was deemed likely to be incidental.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
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).
Key result
Abnormalities:
not specified
Key result
Developmental effects observed:
no

Tables 1 to 11 are attached below under 'Attached background information'.

Conclusions:
The oral administration of Bismuth Subnitrate, to pregnant rats by gavage during gestation at dose levels of 100, 300 or 1000 mg/kg bw/day was well tolerated. The ‘No Observed Effect Level’ (NOEL) for maternal toxicity was considered to be 1000 mg/kg bw/day.
No treatment-related changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity, was therefore, considered to be 1000 mg/kg bw/day.
Executive summary:

Introduction

The study was performed according to the study plan and was designed to investigate the effects of the test item on embryonic and fetal development following repeated administration by gavage to the pregnant female during gestation including the period of organogenesis.

The study was designed to comply with the following guidelines: OECD Guidelines for Testing of Chemicals, No 414, ‘Prenatal Developmental Toxicity Study’ (adopted 22 January 2001).

Methods

The test item was administered by gavage to three groups each of twenty-four time mated Sprague-Dawley Crl:CD® (SD) IGS BR strain rats, between Days 5 and 19 of gestation inclusive at dose levels 100, 300, and 1000 mg/kg bw/day. A further group of twenty-four time mated females was exposed to the vehicle only (Arachis oil BP) to serve as a control.

Clinical signs, body weight change, food and water consumptions were monitored during the study.

All females were terminated on Day 20 of gestation and subjected to gross necropsy including examination of the uterine contents. The number of corpora lutea, number, position and type of implantation, placental weights, fetal weight, sex and external and internal macroscopic appearance were recorded. Half of each litter were examined for detailed skeletal development and the remaining half were subjected to detailed visceral examination.

Results

Mortality

There were no unscheduled deaths during the study.

Clinical Observations

Throughout the study, there were no clinical observations for any of the animals.

Body Weight

There was no effect of treatment with the test item at any dose level on body weight development.

Food Consumption

There was no effect of treatment with the test item at any dose level on food consumption.

Water Consumption

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

Post Mortem Studies

There were no macroscopic findings for any of the females on the study.

Litter Data and Litter Placental and Fetal Weights

No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in fetal growth and development.

Fetal Examination

No treatment-related effects were detected on external development or in the type and incidence of skeletal or visceral findings.

Conclusion

The oral administration of Bismuth Subnitrate, to pregnant rats by gavage during gestation at dose levels of 100, 300 or 1000 mg/kg bw/day was well tolerated. The ‘No Observed Effect Level’ (NOEL) for maternal toxicity was considered to be 1000 mg/kg bw/day.

No treatment-related changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity, was therefore, considered to be 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch 1 study.
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In a study conducted in accordance with OECD Guideline 414, the oral administration of bismuth subnitrate to pregnant rats by gavage during gestation at dose levels of 100, 300 or 1000 mg/kg bw/day was well tolerated. There were no unscheduled deaths during the study. Throughout the study, there were no clinical observations for any of the animals. There was no effect of treatment with the test item at any dose level on body weight development. There was no effect of treatment with the test item at any dose level on food consumption. Visual inspection of water bottles did not reveal any intergroup differences. There were no macroscopic findings for any of the females on the study. No treatment-related effects were detected in the uterine parameters examined, in fetal viability or in fetal growth and development. No treatment-related effects were detected on external development or in the type and incidence of skeletal or visceral findings. The ‘No Observed Effect Level’ (NOEL) for maternal toxicity was considered to be 1000 mg/kg bw/day. No treatment-related changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity, was therefore, considered to be 1000 mg/kg bw/day.

In a supporting study published as an abstract only, it was concluded that no adverse effects of read-across substance, bismuth citrate,upon pre- or post-implantation loss, numbers of viable foetuses or foetal development were apparent in either AHA rats or Dutch rabbits. Rat post-natal development was also unaffected. It is not possible to assess the reliability of this study as it is only available as an abstract.


Justification for selection of Effect on developmental toxicity: via oral route:
GLP study conducted in accordance with OECD Guideline.

Justification for selection of Effect on developmental toxicity: via inhalation route:
Oral route is considered the most appropriate route.

Justification for selection of Effect on developmental toxicity: via dermal route:
Oral route is considered the most appropriate route.

Justification for classification or non-classification

A 90 day repeated dose oral toxicity study was conducted in accordance with OECD Guideline 408 with bismuth subnitrate. Additional reproductive toxicity endpoints were evaluated in this study: estrous cycling, sperm analysis and testosterone analysis. 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).

In a study conducted in accordance with OECD Guideline 414, the oral administration of bismuth subnitrate to pregnant rats by gavage during gestation at dose levels of 100, 300 or 1000 mg/kg bw/day was well tolerated. The ‘No Observed Effect Level’ (NOEL) for maternal toxicity was considered to be 1000 mg/kg bw/day. No treatment-related changes were detected in the offspring parameters measured. The ‘No Observed Effect Level’ (NOEL) for developmental toxicity,was therefore, considered to be 1000 mg/kg bw/day.

Therefore, based on the results of these two studies, classification under the CLP Regulation for reproductive toxicity/ developmental toxicity is not required.