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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

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

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Identification : Bismuth Subnitrate
CAS Number : 1304-85-4
EC Number : 215-136-8
Physical State/Appearance : White powder
Chemical Name : Bismuth hydroxide nitrate oxide
Purity : 99.8%
Batch Number : 2015000647
Date Received : 27 February 2015

Test animals

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.

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

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

Maternal developmental toxicity

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

Effect levels (maternal animals)

open allclose all
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).

Results (fetuses)

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.

Effect levels (fetuses)

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).

Fetal abnormalities

Key result
Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.