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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
26 February 2010 to 02 June 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP guideline study As this study is being used for read-across, the reliability has been amended to reflect this. Read-across from calcium carbonate to sugar factory lime (SFL) is justified on the following basis. SFL is primarily composed of inorganic substances. The major constituent is calcium carbonate, along with sand and a small amount of other inorganic salts (including calcium salts) and the remainder is composed of organic plant material. SFL is not classified for human health. Of its components, only calcium oxalate is classified as acutely toxic via the oral and dermal routes (category 4); however, this does not affect the overall classification of SFL. As a result, it is considered that the properties of SFL are governed by those of calcium carbonate. It is therefore considered appropriate for this data to be used for read-across purposes and any further testing would be scientifically unjustified.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Reference substance name:
Calcium carbonate (nano)
IUPAC Name:
Calcium carbonate (nano)
Details on test material:
- Name of test material (as cited in study report): Calcium carbonate (nano)
- Description: White powder
- Analytical purity: 98.5% w/w
- Batch No.: GICM014427
- Expiration date of the batch: 31 July 2014
- Storage condition of test material: Room temperature in the dark

The nano form of calcium carbonate was tested because this form was anticipated to represent the worst case as it is likely to be more soluble than the bulk form due to the smaller particle size and hence greater surface area for systemic absorption.

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Blackthorn, Bicester, Oxon, UK
- Age at study initiation: approximately 12 weeks old
- Weight at study initiation: Males: 299 - 376 g; Females: 191 - 227 g
- Housing: Initially, all animals were housed in groups of five in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). During the mating phase, animals were transferred to polypropylene grid floor cages suspended over trays lined with absorbent paper on a one male: one female basis within each dose group. Following evidence of successful mating, the males were returned to their original cages. Mated females were housed individually during gestation and lactation, in solid floor polypropylene cages with stainless steel mesh lids and softwood flakes. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK) except for mated females during gestation and lactation.
- Diet: A pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used and was available ad libitum.
- Water: Mains drinking water was supplied from polycarbonate bottles attached to the cage and was available ad libitum.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 55± 15%
- Air changes: at least fifteen air changes per hour
- Photoperiod: low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: From: 02 March 2010 (first day of treatment) To: 18 April 2010 (final necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: For the purpose of this study the test material was prepared at the appropriate concentrations as a suspension in Distilled water. The stability and homogeneity of the test material formulations were previously determined by Harlan Laboratories Ltd. (Harlan Laboratories Ltd. Project Number: 2974-0011). Results from the previous study showed the formulations to be stable for at least fourteen days. Formulations were therefore prepared weekly and stored at 4 ºC in the dark.
The treatment volume for each animal was 5 mL/kg.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each test material formulation were taken and analysed for concentration of Calcium carbonate (nano).

Due to the complex nature of the test material and its limited solubility in organic and aqueous media, a substance specific quantitative method of analysis could not be developed. The concentration of Calcium Carbonate (nano) in the test material formulations was determined using a gravimetric technique.

The results indicate that the prepared formulations were within ± 6% of the nominal concentration.
Duration of treatment / exposure:
Up to 48 consecutive days (including a two week maturation phase, pairing, gestation and early lactation for females).
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 animals/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of previous toxicity work (Harlan Project Number: 2974-0011).
- Rationale for animal assignment: The animals were allocated to dose groups using a randomisation procedure based on stratified bodyweights and the group mean bodyweights were then determined to ensure similarity between the dose groups.

Examinations

Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill-health and behavioural change immediately before dosing, up to thirty minutes after dosing, and one and five hours after dosing, during the working week. Animals were observed immediately before dosing, soon after dosing, and one hour after dosing at weekends and public holidays (except for females during parturition where applicable). All observations were recorded.
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed: gait, hyper/hypothermia, tremors, skin colour, twitches, respiration, convulsions, palpebral closure, bizarre/abnormal/stereotypic behaviour, urination, salivation, defecation, pilo-erection, transfer arousal, exophthalmia, tail elevation, lachrymation

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 (prior to dosing) and then weekly for males until termination and weekly for females until mating was evident. Bodyweights were then recorded for females on Days 0, 7, 14 and 20 post coitum, and on Days 1 and 4 post partum.

FOOD CONSUMPTION:
- During the maturation period, weekly food consumption was recorded for each cage of non-recovery adults. This was continued for males after the mating phase. For females showing evidence of mating, food consumption was recorded for the periods covering post coitum Days 0-7, 7-14 and 14-20. For females with live litters, food consumption was recorded on Days 1 and 4 post partum.

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: Yes - Food efficiency was calculated retrospectively for males throughout the study period and for females during the premating phase. Due to offspring growth and milk production, food efficiency could not be accurately calculated during gestation and lactation.

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 42 for males and Day 4 post partum for females
- How many animals: five males and five females selected from each test and control group
- Parameters examined:
* Haemoglobin (Hb)
* Erythrocyte count (RBC)
* Haematocrit (Hct)
* Erythrocyte indices - mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
* Total leucocyte count (WBC)
* Differential leucocyte 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: Day 42 for males and Day 4 post partum for females
- How many animals: five males and five females selected from each test and control group
- Parameters examined:
* 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 (Bile)
* Calcium (Ca++)

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/behavioural toxicity. Functional performance tests were also performed on five selected males and females from each dose level, prior to termination, together with an assessment of sensory reactivity to various stimuli.
- Dose groups that were examined: All animals in all dose groups and five selected males and females from each dose level, prior to termination.
- Battery of functions tested: sensory reactivity (grasp response, touch escape, vocalisation, pupil reflex, toe pinch, blink reflex, tail pinch, startle reflex, finger approach) grip strength, motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes: Adult males were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 43. Adult females were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination on Day 5 post partum. All adult animals and offspring, including those dying during the study, were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
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
* Pituitary (post fixation)
* Brain
* Seminal vesicles
* Epididymides
* Spleen
* Heart
* Testes
* Kidneys
* Thymus
* Liver
* Thyroid (weighed post-fixation with Parathyroid)
* Ovaries
* Uterus (weighed with Cervix)
* Prostate

HISTOPATHOLOGY: Yes: Samples of the following tissues were removed from all animals and preserved:
* Adrenals
* Muscle (skeletal)
* Aorta (thoracic)
* Oesophagus
* Bone & bone marrow (femur including stifle joint)
* OVARIES
* Bone & bone marrow (sternum)
* Pancreas
* Brain (including cerebrum, cerebellum, medulla oblongata and pons)
* PITUITARY
* PROSTATE
* Caecum
* Rectum
* CERVIX
* Salivary glands (submaxillary)
* COAGULATION GLAND
* Sciatic nerve
* Colon
* SEMINAL VESICLES
* Duodenum
* Skin (hind limb)
* EPIDIDYMIDES
* Spinal cord (cervical, mid-thoracic and lumbar)
* Eyes
* Gross lesions
* Spleen
* Heart
* Stomach
* Ileum
* TESTES
* Jejunum
* Thymus
* Kidneys
* Thyroid/parathyroid
* Liver
* Trachea
* Lungs (with bronchi)
* Urinary bladder
* Lymph nodes (cervical and mesenteric)
* UTERUS
* MAMMARY TISSUE
* VAGINA

The tissues from five selected control and 1000 mg/kg bodyweight/day dose group animals, any animals dying during the study were prepared as paraffin blocks, sectioned at nominal thickness of 5 μm and stained with haematoxylin and eosin for subsequent microscopic examination. The tissues shown in capital letters from the remaining control and 1000 mg/kg bodyweight/day were also processed. In addition, sections of testes and epididymides from all control and 1500 mg/kg bodyweight/day males were also stained with Periodic Acid-Schiff (PAS) stain and examined.
Statistics:
Data for males and females prior to pairing, and functional performance test data, where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA and ANCOVA and Barletts’s test. The transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (non-parametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY
There were no unscheduled deaths that were considered to be related to test material toxicity.
One male treated with 1000 mg/kg bodyweight/day was killed in extremis on Day 39. Histopathological examinations of this animal revealed the cause of death to be due to a misplaced gavage with perforation leading to necrotizing inflammation around the trachea, oesophagus, lungs and thymus. This was therefore considered to be unrelated to test material toxicity.

CLINICAL OBSERVATIONS
There were no toxicologically significant changes detected.
Episodes of generalised fur loss were evident in three females treated with 1000 mg/kg bodyweight/day and two females treated with 100 mg/kg bodyweight/day. One female treated with 300 mg/kg bodyweight/day had a missing upper front tooth between Days 31 and 35. These incidences in isolation were considered not to be of toxicological significance. Two control females also had fur loss between Day 32 and Day 45. One male treated with 300 mg/kg bodyweight/day had an open wound from Day 27 onwards, followed by scab formation and fur loss from Day 28. Observations of this nature are commonly observed in group housed animals and are not considered to be related to treatment.
The male that was killed in extremis on Day 39 had noisy respiration on Days 36 and 39 and pilo erection, a decreased respiration rate, lethargy and hunched posture prior to termination.

BODY WEIGHT AND WEIGHT GAIN
There were no treatment related effects detected in bodyweight development.
Statistical analysis of the data did not reveal any significant intergroup differences.

FOOD CONSUMPTION
No adverse effect on food consumption was detected for males during the treatment period, or for females during the pre-mating, gestation or lactation phases of the study.

FOOD EFFICIENCY
Food efficiency (the ratio of bodyweight gain to dietary intake) was not affected for males throughout the treatment period, or for females during the pre-mating phase.

WATER CONSUMPTION
No treatment-related intergroup differences in water intake were detected for treated animals when compared to controls.

HAEMATOLOGY
No toxicologically significant effects were detected.
Males treated with 1000 mg/kg bodyweight/day showed a statistically significant reduction in mean corpuscular haemoglobin and mean corpuscular volume. All individual values were within the normal ranges for rats of the strain and age used and in isolation were considered not to be of toxicological importance.

CLINICAL CHEMISTRY
No toxicologically significant effects were detected.
Males treated with 1000 mg/kg bodyweight/day showed a statistically significant reduction in total protein and a statistically significant increase in chloride concentration. Males from all treatment groups also showed statistically significant reductions in phosphorus. All individual values were within the normal ranges for rats of the strain and age used and in isolation were considered not to be of toxicological importance.

NEUROBEHAVIOUR
- Behavioural Assessments: Weekly open field arena observations did not reveal any treatment-related effects for treated animals when compared to controls.
- Functional Performance Tests: There were no treatment related changes in functional performance.
- Sensory Reactivity Assessments: There were no treatment-related changes in sensory reactivity.

ORGAN WEIGHTS
No toxicologically significant effects were detected in the organ weights measured.
Males treated with 100 mg/kg bodyweight/day showed a statistically significant reduction in spleen weight both absolute and relative to terminal bodyweight. Females treated with 300 mg/kg bodyweight/day showed a statistically significant increase in relative brain weight. In the absence of a true dose related response or any associated histology correlates the intergroup differences were considered not to be of toxicological significance.

GROSS PATHOLOGY
Adults: There were no toxicologically significant macroscopic abnormalities detected in terminal kill animals.
Three males treated with 300 mg/kg bodyweight/day had red lungs at necropsy. A further male from this treatment group had pale lungs and dark cervical lymph nodes. One male treated with 100 mg/kg bodyweight/day also had dark cervical lymph nodes and hydronephrosis in the right kidney. In the absence of a true dose related response or any associated histology correlates the intergroup differences were considered not to be of toxicological importance. One female treated with 1000 mg/kg bodyweight/day, two females treated with 100 mg/kg bodyweight/day and two control females showed fur loss at necropsy. Observations of this nature are commonly observed following lactation and in conjunction with the observation also being present in control females the intergroup differences were considered unrelated to treatment.
The male that was killed in extremis on Day 39 showed thickening in the stomach, white fluid in the thoracic cavity, dark kidneys, red lungs and flaccid testes.

HISTOPATHOLOGY: NON-NEOPLASTIC
There were no treatment related microscopic abnormalities detected in terminal kill animals.
All findings noted in this study were considered to be incidental findings commonly noted in rats of this strain and age or findings associated with the status post partum.
The cause of death in the male that was killed in extremis was considered to be due to a misplaced gavage with perforation leading to necrotizing inflammation around the trachea, oesophagus, lungs and thymus. This was therefore considered to be unrelated to test material toxicity.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration of Calcium Carbonate (nano) to rats by gavage, at dose levels of 100, 300 and 1000 mg/kg bodyweight/day, resulted in treatment-related effects at all dose levels. These effects were considered not to represent an adverse effect of treatment, hence the 'No Observed Adverse Effect Level' (NOAEL) for systemic toxicity was considered to be 1000 mg/kg bodyweight/day.