Registration Dossier

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Diss Factsheets

Administrative data

Description of key information

Oral (WoE, 90 d, rat): NOAEL 5000 mg/kg bw/d (female/male)

Oral (WoE, 90 d, rat): NOAEL 1000 mg/kg bw/d (female/male)

Read-across from source substance bismuth hydroxide nitrate oxide (CAS 1304-85-4)

Dermal (WoE, 28 d, rabbit): NOAEL = 500 mg/kg bw/d (female/male)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
8 May 2015 - 7 Mar 2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
/ Purity of test item not specified
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study 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. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Wistar Han™:RccHan™:WIST
Sex:
male/female
Details on test animals or test system 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 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
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:
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
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.
Clinical signs:
no effects observed
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.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
no effects observed
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):
Compound intake was not exmained
Food efficiency:
not examined
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.
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.
Compound intake was not exmained
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examination of animals of both sexes from the control and 1000 mg/kg bw/day dose groups did not indicate any treatment-related differences.
Haematological findings:
no effects observed
Description (incidence and severity):
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 biochemistry findings:
no effects observed
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):
no effects observed
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:
no effects observed
Description (incidence and severity):
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 pathological findings:
no effects observed
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:
no effects observed
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):
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.
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

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

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).

Discussion

The oral (gavage) administration of Bismuth Subnitrate to rats, at dose levels of 40, 200 or 1000 mg/kg bw/day was well tolerated. Clinical signs were confined to a few instances of increased post-dose salivation in animals of either sex treated with 1000 mg/kg bw/day, which may be due to a slightly unpalatable or irritant test item formulation. Whilst overall group mean body weight gains in males and females receiving 1000 mg/kg bw/day were marginally lower than controls, this was not associated with an effect on food intake and this finding was considered not to be of an adverse nature.

Hematological and blood chemistry investigations did not reveal any adverse effect of treatment in animals of either sex at any dose level. Some statistically significant intergroup differences were evident in males and/or females, however, individual values in most instances were within the historical control data ranges and in the absence of any histopathology correlates, these findings were deemed to be of no toxicological significance.

At the end of the treatment period, a decrease in liver weights was apparent in males treated with 200 or 1000 mg/kg bw/day. The corresponding values in females were similar to controls and as most individual values from the test item-treated males were within the control data ranges and there were no histopathology correlates, these observations were deemed unlikely to be of any toxicological relevance.

At necropsy, green colored contents were observed in the caecum for most animals of either sex treated with 1000 mg/kg bw/day as well as one female from the 200 mg/kg bw/day dose group. One female receiving 1000 mg/kg bw/day also showed green colored contents in the stomach. Microscopic examination of these tissues did not reveal any treatment-related findings and these observations were considered unlikely to be of any toxicological significance.

When compared with controls, there was no effect of treatment with the test item at any dose level on estrous cycling activity in females over the last three weeks of dosing or sperm analysis parameters examined in males. Additionally, testosterone assessment in males did not identify any treatment-related differences.

Overall, it is considered that a dose level of 1000 mg/kg bw/day could be assigned a No Observed Adverse Effect Level (NOAEL).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
(basic information given on housing conditions and administration of dosages; no clinical biochemistry performed; not all organs recommended in the OECD TG 408 and the number of animals recommended in the OECD TG 408 were investigated in gross necropsy and histopathological examination)
Qualifier:
no guideline followed
Principles of method if other than guideline:
Male and femalre rats were exposed daily for 5 days/ week to the test substance via gavage for a exposure period of 90 days. The animals were observed for evidence of clinical signs. The body weights and food consumption were recorded. Haematological analysis was performed initially and monthly thereafter on 5 animals from each group. At the end of the experimental period the animals were sacrificed and a gross examination was performed.
GLP compliance:
no
Species:
rat
Strain:
other: Holtzman
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Weight at study initiation: Ranges: 59 - 72 g (females) and 62 - 83 g (males)
- Housing: The animals were individually housed in raised wire mesh cages.
- Diet: Ground Fox Blox, ad libitum
- Water: ad libitum
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was suspended in distilled water.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
90 d
Frequency of treatment:
once daily, 5 days/week
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
5 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes

BODY WEIGHT: Yes
- Time schedule for examinations: Body weight was recorded weekly.

FOOD CONSUMPTION: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Analysis was performed initially and monthly thereafter on 5 animals from each group.
- Parameters checked: Haematocrit, red blood count, white blood count, differential count (neutrophils, lymphocytes, monocytes, eosinophils, basophils).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, at the end of the experimental period the animals were sacrificed and a gross examination was performed.

HISTOPATHOLOGY: Yes, kidneys, adrenal gland, liver, spleen and small intestine were removed from the animals used for blood analysis and fixed in 10% formalin. After fixation the tissue was dehydrated and embedded in paraffin. After removal of paraffin the tissue was stained in an aqueous solution of Harris hametoxylin and counterstained in eosin.
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
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
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
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
5 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects were observed in the highest tested dose level.
Key result
Critical effects observed:
no
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study from a reference substance with similar intrinsic properties and consistent studies from the test substance itself. Read-across is justified based on common characteristic chemical properties. The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
In a scientifically acceptable and well documented repeated dermal toxicity test, the test substance (50% formulation in water) was administered to the abdominal abraded and intact skin of rabbits.
The dose levels of 125, 250 and 500 mg/kg bw were dermally administrated to two male and two female albino rabbits each (abraded and intact skin). The control group with the same amount of animals received 0.5 mL/kg bw of distilled water in a similar regimen.
GLP compliance:
no
Specific details on test material used for the study:
Appearance: A viscous, silver liquid
Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Dutchland Laboratory Animals, Inc., Denver, Pennsylvania, USA
- Age at study initiation: Adult
- Weight at study initiation: 2.5 to 3.3 kg
- Diet: Purina Rabbit Chow ad libitum
- Water: ad libitum
Type of coverage:
occlusive
Vehicle:
water
Details on exposure:
TEST SITE
- Area of exposure: Abdominal skin (intact and abraded)
- Type of wrap: Nonabsorbent binder (rubber damming or butcher - paper), gauze, and elastic adhesive tape
- Time intervals for clippings: As necessary, usually once a week

REMOVAL OF TEST SUBSTANCE
- Washing: No

TEST MATERIAL
- Concentration: 50% formulation of the test substance
- Constant volume or concentration used: Yes
- For solids, paste formed: Yes (viscous)

VEHICLE
- Amount applied: 0.5 mL/kg bw
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
24 hours
Frequency of treatment:
Once daily, five days per week for a period of three weeks (Total of 15 applications)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
2 (abraded and intact skin)
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Observations were recorded once daily for three weeks

DERMAL IRRITATION: Yes
- Time schedule for examinations: Observations were recorded 24 hours after each application and on Sundays (48 hours after the Friday application).

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded initially, weekly, and terminally.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Initially and at termination on all control and test animals
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Initially and terminally on all animals
- Animals fasted: No data
- How many animals: All
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Initially and terminally on all animals
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, on all animals at the time of sacrifice by intravenous air embolism on the third day following the last application.
Tissue preservation (see table 4)

HISTOPATHOLOGY: Yes (see table 5)
Statistics:
Statistical analysis of the following parameters was performed by the t-test at the 5.0% probability level; group mean body weight data, hematology data, blood biochemistry data, and urine analysis data.
(Reference: Wilfred J. Dixon and Frank J. Massey, Jr., Introduction to Statistical Analysis, 123-124, McGraw Hill, 1957.)
Clinical signs:
no effects observed
Dermal irritation:
effects observed, non-treatment-related
Description (incidence and severity):
Grossly, all test groups demonstrated persistent erythema, atonia, and desquamation which the control group failed to demonstrate.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Dosages of 0.125, 0.25, and 0.5 g/kg to the intact and abraded skin surface of rabbits resulted in a slight increase in acanthosis and hyperkeratosis in the high dose (0.5 g/kg) rabbits. A variable degree of acanthosis and - hyperkeratosis was noted in rabbits at the lower dose levels and these alterations were generally of comparable severity to those noted in the control group.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at any dose level.
Key result
Critical effects observed:
no

Result tables are provided in the attachment.

Summary

In a scientifically acceptable and well documented repeated dermal toxicity test, the test substance (50% formulation in water) was administered to the abdominal abraded and intact skin of rabbits.

The dose levels of 125, 250 and 500 mg/kg bw were dermally administrated to two male and two female albino rabbits each (abraded and intact skin). The control group with the same amount of animals received 0.5 mL/kg bw of distilled water in a similar regimen.

Generally, all rabbits demonstrated body weight losses over the test period, however, mean body weight of the test groups were comparable to those of the controls at all intervals. All animals essentially appeared normal throughout the study. Signs of dermal irritation included slight to moderate erythema, slight atonia, and slight desquamation with dermal irritation more persistent and varied in the test group as compared to controls.

Results of the clinical studies and gross pathology findings were essentially unremarkable in the test groups as compared to controls.

Microscopically, a slight increase in the incidence of acanthosis and hyperkeratosis of the exposure site was evident in group No. 4 animals (500 mg/kg bw) as compared to controls. All other observations of the test groups were comparable to those of the controls.

Therefore, the NOAEL for dermal repeated toxicity was determined to be 500 mg/kg bw.

Endpoint:
short-term repeated dose toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
In a scientifically acceptable and well documented repeated dermal toxicity test, the test substance (70% formulation in water) was administered to the abdominal abraded and intact skin of rabbits.
The dose levels of 125, 250 and 500 mg/kg bw were dermally administrated to two male and two female albino rabbits each (abraded and intact skin). The control group with the same amount of animals received 1 mL/kg bw of castor oil in the first 13 applications, after which they received 0.5 g/kg/application.
GLP compliance:
no
Specific details on test material used for the study:
Appearance:
BCL 1579D (a silver paste)

Species:
rabbit
Strain:
New Zealand White
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Dutchland Laboratory Animals, Inc., Denver, Pennsylvania, USA
- Age at study initiation: Adult
- Weight at study initiation: 2.7 to 3.3 kg
- Diet: Purina Rabbit Chow ad libitum
- Water: ad libitum


Type of coverage:
occlusive
Vehicle:
castor oil
Remarks:
KAYDOL MINERAL OIL
Details on exposure:
TEST SITE
- Area of exposure: Abdominal skin (intact and abraded)
- Type of wrap: Nonabsorbent binder (rubber damming or butcher - paper), gauze, and elastic adhesive tape
- Time intervals for clippings: Weekly

REMOVAL OF TEST SUBSTANCE
- Washing: No

TEST MATERIAL
- Concentration: 70% formulation of the test substance
- Constant volume or concentration used: Yes
- For solids, paste formed: Yes

VEHICLE
- Justification for use and choice of vehicle: Commercial use
- Amount applied: 1.0 mL/kg/application 13th application, after which received 0.5 g/kg/application
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
24 hours
Frequency of treatment:
Once daily five days per week for a period of three weeks (Total of 15 applications)
Dose / conc.:
125 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
2 (abraded and intact)
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Observations were recorded once daily throughout the study.

DERMAL IRRITATION: Yes
- Time schedule for examinations: Observations were recorded 24 hours after each application and 48 hours after the last application of each week.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded initially, at one and two weeks, and terminally.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Initially and terminally on all animals
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: All
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Initially and terminally on all animals
- Animals fasted: No data
- How many animals: All
- Parameters checked in table 2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Initially and terminally on all animals
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters checked in table 3 were examined.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Gross necropsies were performed on all animals sacrificed (intravenous air embolism).
Tissue preservation (see table 4)

HISTOPATHOLOGY: Yes (see table 5)
Clinical signs:
no effects observed
Dermal irritation:
effects observed, non-treatment-related
Description (incidence and severity):
Dermal signs of irritation such as slight to moderate erythema, slight edema, slight to moderate atonia, and/or desquamation were noted at a comparable incidence in the control and test groups.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed at any dose level.
Key result
Critical effects observed:
no

Result tables can be found in the attachment.

Summary

In a scientifically acceptable and well documented repeated dermal toxicity test, the test substance (70% formulation in castor oil) was administered to abraded and intact abdominal skin of three groups of two male and two female rabbits each. The dosage levels were 125, 250 and 500 mg/kg bw per application for five applications per week for three weeks. One group of two male and two female rabbits served as controls and received applications of the test material vehicle (1 mL/kg through the 13th application, 0.5 g/kg for the last two applications).

Mean body weight changes in the test groups as compared to controls were not remarkable. Incidental signs of systemic toxicity included anorexia, slight wheezing, and soft faeces in animals surviving to termination; however, there were no consistent compound-related trends in these signs. Dermal signs of irritation such as slight to moderate erythema, slight oedema, slight to moderate atonia, and/or desquamation were noted at a comparable incidence in the control and test groups.

No compound-related differences between the test groups and the controls were noted in the haematological or biochemical studies, urine analyses, gross necropsy findings, or microscopic findings.

Therefore, the NOAEL for dermal repeated toxicity was determined to be 500 mg/kg bw.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
The available information comprises adequate, reliable (Klimisch score 2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for read-across

There are data available regarding repeated dose toxicity following dermal and oral route for bismuth chloride oxide (CAS 7787-59-9).In addition read-across from an appropriate substance is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5. in order to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VIII, 8.6. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

For repeated dose toxicity following oral route information will also be taken into account from the analogue substancebismuth hydroxide nitrate oxide (CAS 1304-85-4) to fulfil the standard data requirements defined in Regulation (EC) No 1907/2006, Annex VIII, 8.6.

Oral route

CAS 1304-85-4

There is an oral 90-day repeated dose toxicity study in rats with the analogue substancebismuth hydroxide nitrate oxide (CAS 1304-85-4) available. Male and female Wistar rats were exposed to 40, 200 and 1000 mg/kg bw/day, administered by gavage for 90 days. The study was conducted according to OECD TG 408 and in compliance with GLP criteria (reference 7.5.1-1). No mortality and clinical signs were observed during treatment. Occasional effects on body weight or body weight gains were not considered to be treatment-related. No adverse effects in food consumption and dietary intake were seen. Gross necropsy and ophthalmoscopic examination revealed no abnormalities. No adverse or treatment-related changes in organ weights, hematology or clinical chemistry parameters in male or female rats and no treatment-related macroscopic and histopathological changes were observed in the present study. Under the conditions of this study and based on the toxicological endpoints evaluated, the NOAEL was considered to be 1000 mg/kg bw/day for male and female rats.

The available data of the appropriate read-across substancebismuth hydroxide nitrate oxide (CAS 1304-85-4) do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.

 

CAS 7787-59-9

There is an oral 90-day repeated dose toxicity study in rats with bismuth chloride oxide (CAS 7787-59-9) available (reference 7.5.1-2). Ten male and ten female Holtzman rats were exposed to 250, 1000 and 5000 mg/kg bw/day. The test substance was suspended in distilled water and administered intragastrically once daily on five days a week for a 90-day period. No mortality and clinical signs were observed during treatment. No adverse effects on body weight were seen in the study and no treatment-related changes occurred after gross and histopathological examination. Under the conditions of this study and based on the toxicological endpoints evaluated, the NOAEL was considered to be 5000 mg/kg bw/day for male and female rats.

 

Dermal route

CAS 7787-59-9

There is a dermal 28-day repeated dose toxicity study in rabbits with the target substance bismuth chloride oxide (CAS 7787-59-9) available (reference 7.5.3-1). In this study water was used as vehicle. Male and female NZW rabbits were exposed to 125, 250 and 500 mg/kg bw for 24 h once daily on five days per week for a period of three weeks on the back skin (abraded and intact) under occlusive conditions. All test groups demonstrated persistent erythema, atonia, and desquamation during the treatment period which the control group failed to demonstrate. No mortality and clinical signs, and effects on body weight or body weight gains were observed during treatment. Gross necropsy revealed no abnormalities. No adverse or treatment-related changes in urine analysis, hematology or clinical chemistry parameters in male or female rabbits and no treatment-related macroscopic and histopathological changes were present in the study. Under the conditions of this study and based on the toxicological endpoints evaluated, the NOAEL was considered to be 500 mg/kg bw/day for male and female rabbits.

In a second dermal 28-day repeated dose toxicity study in rabbits with the target substance bismuth chloride oxide (CAS 7787-59-9) castor oil was used as vehicle (reference 7.5.3-2). Male and female NZW rabbits were exposed to 125, 250 and 500 mg/kg bw for 24 h once daily on five days per week for a period of three weeks on the back skin (abraded and intact) under occlusive conditions. Dermal signs of irritation such as slight to moderate erythema, slight edema, slight to moderate atonia, and/or desquamation were noted at a comparable incidence in the control and test groups. No mortality and clinical signs, and effects on body weight or body weight gains were observed during treatment. Gross necropsy revealed no abnormalities. No adverse or treatment-related changes in urine analysis, hematology or clinical chemistry parameters in male or female rabbits and no treatment-related macroscopic and histopathological changes were present in the study. Under the conditions of this study and based on the toxicological endpoints evaluated, the NOAEL was considered to be 500 mg/kg bw/day for male and female rabbits.

As supporting evidence, there is a dermal 90-day repeated dose toxicity study in rabbits with the target substance bismuth chloride oxide (CAS 7787-59-9) available (reference 7.5.3-3). Five rabbits were exposed to 0.2 mL and 2 mL/kg bw of a 25% suspension in distilled water, respectively. The applied doses were equivalent to 50 and 500 mg/kg bw/d, respectively. The test substance was applied once daily for 90 days to the clipped back skin of the rabbits. Neither mortality, nor clinical signs, nor effects on body weight nor body weight gain were observed during treatment. Macroscopic and microscopic examination revealed no abnormalities. Under the conditions of this study and based on the toxicological endpoints evaluated, the NOAEL was considered to be 2 mL/kg bw/day corresponding to 500 mg/kg bw/d for male and female rabbits.

Justification for classification or non-classification

Based on the analogue approach, the available data with the source substance bismuth subnitrate (CAS 1304-85-4) and with the test substance bismuth chloride oxide (CAS 7787-59-9) do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and are therefore conclusive but not sufficient for classification.