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Endpoint summary

Description of key information

Daily oral administration of micro calcium carbonate and nano calcium carbonate (Read-across substances) for 28 days to mice did not produce any obvious symptoms of toxicity or mortality even at the highest dose of 1300 mg/kg bw/day administered. A careful and extensive necropsy revealed no gross organ changes. The NOAEL for both micro calcium carbonate and nano calcium carbonate were reported to be 1300 mg/kg bw/day.

Furthermore, in a repeated dose oral study equivalent to OECD 408, all rats dosed with 250, 500 or 1000 mg/kg bw/day for 90 days survived, and no abnormal symptoms related to nano calcium carbonate administration were observed through general clinical observation and eye examination. Moreover, there were no significant differences in body weight, food consumption, urine, hematological measures, blood biochemistry, and organ weight, and no lesions following gross anatomical examination and histopathological analysis. Thus, the NOAEL for nano calcium carbonate is >1000 mg/kg bw/day.

Inhalation exposure to 0.399 mg/L calcium carbonate (nano) resulted in treatment-related changes in the lower airways, characterized by an increased lung weight accompanied by slight increases in BAL derived inflammation and cytotoxicity biomarkers. These changes were largely, but not fully, reversible within a 4 week recovery period after the last exposure. Based on these observations, the NOAEC for local effects of sub-chronic inhalation exposure to calcium carbonate (nano) was placed at 0.212 mg/L. Since exposure to the test material did not induce any systemic toxicity, the NOEC for systemic effects was 0.399 mg/L.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26 February 2010 to 02 June 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

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)

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

Dose / conc.:

100 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

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.

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).

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

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Other effects:

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.

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 toxicologically significant effects observed at any dose level.

Key result

Critical effects observed:

no

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.

Reference 2

Endpoint:

short-term repeated dose 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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

not specified

Principles of method if other than guideline:

The study examined whether the bioavailability of calcium carbonate and calcium citrate could be improved by reducing the particle size. Because nanoscale supplements are novel formulas in health foods, the acute toxicity (see separate IUCLID entry), sub-chronic toxicity and bioavailability (see separate IUCLID entry) needs to be determined in both sexes of mice in advance.
Micro calcium carbonate and nano calcium carbonate were administered by gavage to groups of mice at doses of 1.3, 0.13 or 0.013 g/kg bw. The animals were then observed daily for a period of 28 days.

GLP compliance:

no

Remarks:

No data provided.

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: National Taiwan University Hospital, Taipei, Taiwan
- Age at study initiation: 8-10 weeks
- Fasting period before study: Animals were fasted overnight
- Diet: Pelleted mouse feed available ad libitum
- Water: Reverse osmosis water available ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25 °C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 h/12 h day/night cycle

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

The test materials were administered by gavage.
The control group received the vehicle only.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Dose / conc.:

13 mg/kg bw/day (nominal)

Remarks:

plus Vitamin D3 (261 U/kg bw)
nominal in water

Dose / conc.:

130 mg/kg bw/day (nominal)

Remarks:

plus Vitamin D3 (261 U/kg bw)
nominal in water

Dose / conc.:

1 300 mg/kg bw/day (nominal)

Remarks:

plus Vitamin D3 (261 U/kg bw)
nominal in water

No. of animals per sex per dose:

8 animals/sex/dose

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

All experimental animals were observed every day for general signs and symptoms of toxicity.
The body weights were recorded prior to the commencement of the study and all of the animals were weighed on Day 28.

Sacrifice and pathology:

All animals were sacrificed on Day 28 after an overnight fast.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Gross pathological findings:

no effects observed

Details on results:

Daily oral administration of micro calcium carbonate and nano calcium carbonate did not produce any obvious symptoms of toxicity or mortality even at the highest dose administered. Overall body weight increased slightly but insignificantly throughout the study in all groups as the mice matured. A careful and extensive necropsy revealed no gross organ changes.

Key result

Dose descriptor:

NOAEL

Effect level:

1 300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

Key result

Critical effects observed:

not specified

Table 1: Body weight and mortality during the 28-day toxicity test

Dose	Sex (n)	Initial body weight (g)	Final body weight (g)	Mortality %
Control	Male (8) Female (8)	31.2 ± 3.3 32.5 ± 3.6	35.3 ± 3.9 36.1 ± 3.7	0 0
Micro calcium carbonate (1.3 g/kg bw)	Male (8) Female (8)	33.4 ± 3.2 32.1 ± 3.9	36.8 ± 3.5 36.2 ± 3.6	0 0
Micro calcium carbonate (0.13 g/kg bw)	Male (8) Female (8)	32.4 ± 3.2 31.9 ± 3.1	36.3 ± 3.5 35.2 ± 3.3	0 0
Micro calcium carbonate (0.013 g/kg bw)	Male (8) Female (8)	33.4 ± 3.2 33.1 ± 3.1	37.3 ± 3.7 37.2 ± 3.3	0 0
Nano calcium carbonate (1.3 g/kg bw)	Male (8) Female (8)	32.4 ± 3.6 33.1 ± 2.9	36.4 ± 4.1 36.9 ± 3.7	0 0
Nano calcium carbonate (0.13 g/kg bw)	Male (8) Female (8)	32.4 ± 3.3 33.8 ± 3.6	36.4 ± 3.9 37.2 ± 3.4	0 0
Nano calcium carbonate (0.013 g/kg bw)	Male (8) Female (8)	32.9 ± 3.3 32.5 ± 3.2	36.4 ± 3.4 36.2 ± 3.3	0 0

Data are mean ± SE values

 

Conclusions:

Daily oral administration of micro calcium carbonate and nano calcium carbonate did not produce any obvious symptoms of toxicity or mortality even at the highest dose administered. A careful and extensive necropsy revealed no gross organ changes. The NOAEL for both micro calcium carbonate and nano calcium carbonate were reported to be 1.3 g/kg bw.

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

16 December 2009 to 03 February 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: The study was designed to provide information for Oral (Gavage) Combined Repeat Dose Toxicity Study with Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 422 1996)

Deviations:

no

Principles of method if other than guideline:

The study was a 14-day repeated dose oral (gavage) range-finding toxicity study in the rat for OECD 422.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

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., Oxon, UK
- Age at study initiation: Approximately 13 weeks
- Weight at study initiation: Males: 331 - 360 g; Females: 208 - 229 g
- Housing: The animals were housed in groups of three by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding.
- Diet: Pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was available ad libitum.
- Water: Mains drinking water was supplied from polycarbonate bottles attached to the cage and was available ad libitum.
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 55 ± 15%
- Air changes (per hr): At least fifteen air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 darkness

Route of administration:

oral: gavage

Vehicle:

not specified

Details on oral exposure:

The treatment volume was 5 mL/kg administered by gavage using a stainless steel cannula attached to a disposable plastic syringe.

Control animals were treated in an identical manner with 5 mL/kg bodyweight/day of distilled water.

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 3% of the nominal concentration.

Duration of treatment / exposure:

The test material was administered daily, for 14 consecutive days.

Frequency of treatment:

Once daily for 14 days

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

3 animals/sex/dose

Control animals:

yes, sham-exposed

Details on study design:

- Dose selection rationale: The dose levels were chosen based on previous toxicity work provided by the sponsor.
- 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.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were examined for overt signs of toxicity, ill health or behavioural change immediately before dosing, up to thirty minutes after dosing and one hour after dosing. Additional observations were also made five hours following dosing whenever possible (not at weekends or on public holidays).

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Days 1, 4, 8, 11 and 15.

FOOD CONSUMPTION:
Food consumption was recorded for each cage group for Days 1 to 4, 4 to 8, 8 to 11 and 11 to 15.

WATER CONSUMPTION : Yes
- Time schedule for examinations: Water intake was measured and recorded daily for each cage group.

Sacrifice and pathology:

On completion of the dosing period, all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination and subjected to an internal and external macroscopic examination. Any tissues showing macroscopic abnormalities were preserved in buffered 10% formalin.

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:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no unscheduled deaths during the study. No clinically observable signs of toxicity were detected.

BODY WEIGHT AND WEIGHT GAIN
No adverse effect on bodyweight development was detected.
A slight reduction in overall bodyweight gain was evident in males treated with 1000 mg/kg bodyweight/day. In the absence of any associated clinical observations to suggest a decline in animal health the intergroup difference was not considered to represent an adverse health effect.
No such effects were evident in females treated with 1000 mg/kg bodyweight/day or in animals of either sex treated with 500 or 250 mg/kg bodyweight/day.

FOOD CONSUMPTION AND FOOD EFFICIENCY
No adverse effect on dietary intake or food efficiency (the ratio of bodyweight gain to dietary intake) was detected in treated animals when compared to controls throughout the treatment period.

WATER CONSUMPTION
Daily quantitative measurement of water consumption did not reveal any intergroup differences.

GROSS PATHOLOGY
No toxicologically significant macroscopic abnormalities were detected.
One male treated with 1000 mg/kg bodyweight/day had two small yellow areas on the median lobe of the liver.
One male treated with 250 mg/kg bodyweight/day had a diaphragmatic hernia. In the absence of treatment-related effects this finding is considered not to be of toxicological significance.


No clinically observable signs of toxicity were detected. A slight reduction in overall bodyweight gain was evident in males treated with 1000 mg/kg bodyweight/day. No adverse effects were detected in food consumption and in the absence of any associated clinical signs of toxicity this finding was considered not to be of toxicological importance. One male treated with 1000 mg/kg bodyweight/day had two small yellow areas on the median lobe of the liver. There were no treatment-related macroscopic abnormalities detected in the remaining 1000 mg/kg bodyweight/day animals, or in 500 and 250 mg/kg bodyweight/day animals at terminal kill.

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: Oral administration of the test material, Calcium carbonate (nano) to rats for a period of fourteen consecutive days at dose levels of 1000, 500 and 250 mg/kg bodyweight/day produced no toxicologically significant changes in the parameters measured.

Key result

Critical effects observed:

not specified

Conclusions:

Oral administration of the test material, Calcium carbonate (nano) to rats for a period of fourteen consecutive days at dose levels of 1000, 500 and 250 mg/kg bodyweight/day produced no toxicologically significant changes in the parameters measured. The ‘No Observed Adverse Effect Level’ (NOAEL) and a suitable high dose level for use on future toxicity studies was therefore, considered to be 1000 mg/kg bodyweight/day.

Reference 4

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

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Orientbio
- Age at study initiation: 5 weeks
- Housing: wire mesh cages (250 mm width 350 mm length 180 mm height)
- Diet:ad libitum, radiation-sterilized, solid laboratory animal feed (Teklad Certified Irradiated Global 18% Protein Rodent Diet, Harlan Laboratories Inc., Indianapolis, IN)
- Water: ad libitum, filtered water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 0.9
- Humidity (%): 59.2 +/- 4.1
- Photoperiod (hrs dark / hrs light): 12-h light/dark cycle

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 25, 50, 100 mg/mL
- Amount of vehicle : 10 mL/kg

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Dose / conc.:

250 mg/kg bw/day (nominal)

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 spores/kg bw/day (nominal)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on results of 14-day range-finder

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily after adminstration

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily after adminstration

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: At the time of grouping and during last week of administration
- Dose groups that were examined: All animals at grouping, control and high dose only in final week

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Before necropsy
- Anaesthetic used for blood collection: Yes (CO2)
- Animals fasted: Yes - overnight
- How many animals: All

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Before necropsy
- Animals fasted: Yes - overnight
- How many animals: All

URINALYSIS: Yes
- Time schedule for collection of urine: Final week of adminstration
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see attachment)
HISTOPATHOLOGY: Yes (see attachment)

Statistics:

Differences between vehicle control and dosing groups were examined through parametric or non-parametric multiple comparison procedures. The occurrence rate was converted into percentage. SPSS for Windows (Ver. 12; Chicago, IL) was used for analysis. Continuous data included body weight, food consumption, hematology, blood biochemistry, and organ weight. Group differences were examined using standard oneway analysis of variance; if this test showed statistical significance, a parametric multiple comparison procedure was used to compare the vehicle control group with the other experimental groups. If equal variance was assumed, Duncan’s test was applied; if equal variance was not assumed, Dunnett’s T3 test was applied. Non-continuous data (urinalysis) was analyzed using Kruskal–Wallis’ H-test, a non-parametric multiple comparison procedure. If the H-test indicated statistical significance, a Mann-Whitney U-test was used to compare the vehicle control group with the other experimental groups.

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:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: A mass on the right ear was observed in a male animal in the 1000 mg/kg nanocalcium carbonate group. No other animals showed abnormal clinical signs.

BODY WEIGHT AND WEIGHT GAIN: There were no significant differences between groups in terms of changes in body weight or food consumption.

OPHTHALMOSCOPIC EXAMINATION: No abnormal lesions were observed during ophthalmoscopy for all examined animals (see Supplementary Data Table S1).

HAEMATOLOGY: WBC, LY, and MO counts for female rats in the 500 mg/kg nanocalcium carbonate group were lower than those of the control group ( p 5 0.05; Table S7). However, these differences are not considered to be toxicologically significant, as they are within the normal range, and there was no dose-dependency. No other significant hematological differences were found.

CLINICAL CHEMISTRY: The Ca2+ measured in the blood of female rats treated with 1000 mg/kg nanocalcium carbonate was slightly higher than that
of the control group ( p 5 0.01), however, the level remained within the normal range (9.2–11.9 mg/dl, Table S9) and no similar increase was observed in males. Therefore, the level was not considered toxicologically significant. No other significant differences in blood biochemistry were found.

URINALYSIS: Urine pH of males in the 250, 500 and 1000 mg/kg nanocalcium carbonate group was higher than that of the control group (250 mg/kg, p 5 0.05; 500 and 1000 mg/kg, p 5 0.01; see Supplementary Data Table S2). However, this is not considered to be related to nanocalcium carbonate, as there was no correlation between males and females, and differences were not observed in the kidney, including BUN, CRE, and histopathology. There were no differences in urine color (see Supplementary Data Table S3) or sediments (see Supplementary Data Table S4) between groups.

ORGAN WEIGHTS: Thymus weight for males in the 500 mg/kg nanocalcium carbonate group was higher than that of the control group ( p 5 0.05; Table S15), but within the normal range and not considered to be toxicologically significant. There were no other significant differences in organ weight (Table S15, S16).

GROSS PATHOLOGY: In two males in the 250 mg/kg nanocalcium carbonate group, bilateral small testes and epididymis, and right small testis and epididymis, respectively, were observed (see Supplementary Data Table S12). A male in the 500 mg/kg nanocalcium carbonate group had multifocal cysts in the kidneys (see Supplementary Data Table S10). In 3 females and 4 males in the control group, some remarkable lesions were observed in the liver. However, such lesions appearing in the livers of control group rats are simply local monocytic cell infiltration and historical background lesions. These findings and their incident rates are spontaneous or happen by chance. Compared to those of the control group, 20% of the thyroids from the high dose group had some lesions. However, ultimobranchial cysts (that appear in about 20% of thyroids) are congenital lesions derived during a developmental
stage. Therefore, such lesions did not appear to be associated with nanocalcium carbonate treatment. In two males in the 1000 mg/kg nanocalcium carbonate group, a light brown-colored caudate lobe of the liver with torsion, and a mass with blood exudate in the right ear, respectively, were observed. The heart of a female in the 1000 mg/kg nanocalcium carbonate group was encapsulated in a white substance. No other animals exhibited gross abnormalities.

HISTOPATHOLOGY: In regards to the gross abnormalities observed in the males in the 250 mg/kg nanocalcium carbonate group, degeneration of the
seminiferous tubule, and degeneration of spermatids in the lumen of the epididymis, were observed in the case of small bilateral testes/epididymis. The case of the small right testis and epididymis is considered to be atrophy. The multifocal cysts in the kidneys of the male in the 500 mg/kg nanocalcium carbonate group were found to have inflammatory cell infiltration, tubule dilatation, and hyaline droplets. For the one male in the 1000 mg/kg nanocalcium
carbonate group, diffuse degeneration of hepatocytes was observed in the torsioned region of the caudate lobe. The mass in the right ear of the other male in this group was considered to be auricular chondropathy. The female with the encapsulated heart in the 1000 mg/kg group is considered to be a case of adhesion. In histopathological evaluation of organs (liver, renal cortex, lung, cerebral cortex) of male and female rats from the control group and high-dose group (1000 mg/kg) of nanocalcium carbonate, there were no abnormal histopathological findings in either group (Figure 8).

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: Absence of clinical signs; mortality; body weight; effects on food consumption; ophthalmoscopic examination; haematology; clinical chemistry; urinalysis; gross pathology; organ weights; histopathology.

Key result

Critical effects observed:

not specified

Conclusions:

All animals survived, and no abnormal symptoms related to nanocalcium carbonate adminstration were observed through general clinical observation and eye examination. Moreover, there were no significant differences in body weight, food consumption, urine, hematological measures, blood biochemistry, and organ weight, and no lesions following gross anatomical examination and histopathological analysis. Thus, the NOAEL for nanocalcium carbonate is >1000 mg/kg bw/day.

Executive summary:

In a repeated dose oral study equivalent to OECD 408, all animals dosed with 250, 500 or 1000 mg/kg bw/day for 90 days survived, and no abnormal symptoms related to nanocalcium carbonate adminstration were observed through general clinical observation and eye examination. Moreover, there were no significant differences in body weight, food consumption, urine, hematological measures, blood biochemistry, and organ weight, and no lesions following gross anatomical examination and histopathological analysis. Thus, the NOAEL for nanocalcium carbonate is >1000 mg/kg bw/day.

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:

similar to OECD 408 (read-across)

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

4th June - 18th July, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was designed to provide information for dosing in a 90-day repeated dose toxicity study in rats (OECD TG 413, Subchronic Inhalation Toxicity: 90-Day Study, adopted 7 September 2009).

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The study was conducted with albino rats. The rat was used because this species is normally used in toxicity studies of this type and is accepted by the relevant authorities. The Wistar rat strain was used because it is routinely used at the test facility for this type of studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: No data
- Age at study initiation: Approximately 8 weeks
- Weight at study initiation: Mean body weight at the start of treatment on day 0 was 285 grams for males and 180 grams for females.
- Fasting period before study: None
- Housing: The animals were housed in Makrolon® cages (type IV) with a bedding of wood shavings (Lignocel, Rettenmaier & Söhne GmbH & Co, Rosenberg, Germany) and strips of paper (Enviro-dri, Shepherd Specialty Papers, Michigan, USA) and a wooden block (ABEDD, Vienna, Austria) as environmental enrichment. After allocation, the animals were housed in groups of five of the same sex. During exposure, the animals were kept individually in the exposure unit. Immediately after each exposure, the animals were returned to their home cages.
- Diet: ad libitum except during exposure; Rat & Mouse No. 3 Breeding Diet, RM3 (SDS Special Diets Services, Whitham, England).
- Water: ad libitum except during exposure; domestic mains tap water
- Acclimation period: Approximately 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45-65%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12h/12h

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 1.9 - <= 2.6 µm

Remarks on MMAD:

Aerodynamic particle size was determined weekly for each exposure condition. Overall, the mass median aerodynamic diameter (MMAD) was in the range of 1.9 – 2.6 μm with a tendency towards increasing MMAD with increasing target concentration, and the distribution of particle sizes had geometric standard deviations (gsd) in the range of 1.9 – 2.4. (Tables 1.4.1.1 – 1.4.4.2):
For group 2, MMAD was 1.93 μm (gsd of 2.33) and 2.13 μm (gsd of 2.26).
For group 3, MMAD was 1.92 μm (gsd of 2.14) and 2.50 μm (gsd of 1.94).
For group 4, MMAD was 2.30 μm (gsd of 2.09) and 2.32 μm (gsd of 2.10).
For group 5, MMAD was 2.32 μm (gsd of 2.39) and 2.56 μm (gsd of 2.01).
Thus, particle size was within the range of 1 – 3 μm MMAD with a gsd in the range of 1.5 – 3.0, as recommended by OECD guideline 413.
During preliminary experiments, the content of nanoparticles (< 100 nm) was determined for each exposure condition. As expected, the relative contribution of nanoparticles was very low due to aggregation of the particles. When expressed in particle number (by comparing the number of particles < 100 nm as measured by SMPS, to the total number of particles measured by CPC), the percentage of nanoparticles was 7.3%, 1.8%, 1.4% and 1.1% for the the low-, mid-, high- and top concentration test atmospheres, respectively. In terms of particle mass (by comparing the mass of the particles < 100 nm as measured by SMPS, to the total mass concentration determined gravimetrically) the percentage of nanoparticles was 0.0053%, 0.0048%, 0.0048% and 0.0044% for the low-, mid-, high- and top concentration test atmospheres, respectively.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Groenendijk Kunststoffen BV cylindrical polypropylene or steel column
- Method of holding animals in test chamber: Rodent tube
- Source and rate of air: compressed air
- Method of conditioning air: Humidified and filtered
- System of generating particulates/aerosols: Turntable dust feeder and eductor
- Temperature, humidity, pressure in air chamber: 22+/-3C, 30-70%, slight positive pressure
- Air flow rate: 1 litre/min per animal
- Method of particle size determination: Particle size distribution measurements were carried out using a 10-stage cascade impactor (2110k, Sierra instruments, Carmel Valley, California, USA) at least once weekly during exposure and at least once during preliminary generation of the test atmosphere for each exposure condition. The Mass Median Aerodynamic Diameter (MMAD) and the geometric standard deviation (gsd) were calculated

TEST ATMOSPHERE
- Brief description of analytical method used: Filters weighed before and after loading with test atmosphere. Samples were taken at least three times per day for each exposure condition.
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The actual concentration of the test material in the test atmosphere was determined by means of gravimetric analysis. Representative test atmosphere samples were obtained from the animals’ breathing zone by passing mass flow controlled (Bronkhorst Hi Tec) amounts of test atmosphere at 4.6 Ln/min through fiber glass filters (Sartorius, 13400-47). Samples of 200.1 (group 2), 66.7 (group 3), 20.7 (group 4) or 6.9 (group 5) Ln2 test atmosphere were obtained, respectively. Filters were weighed before sampling, loaded with a sample of test atmosphere, and weighed again. The actual concentration was calculated by dividing the amount of test material present on the filter, by the volume of the sample taken. Samples were taken at least three times per day for each exposure condition.

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

5 days/week over a 14-day period with a total number of 10 exposure days.

Dose / conc.:

0.025 mg/L air (nominal)

Dose / conc.:

0.075 mg/L air (nominal)

Dose / conc.:

0.25 mg/L air (nominal)

Dose / conc.:

0.75 mg/L air (nominal)

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

two recovery groups, also consisting of 5 male and 5 female animals each, were simultaneously exposed with the main study animals of the control and top concentration groups,

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: On exposure days, each animal was observed daily in the morning, prior to exposure. All animals checked again after exposure. During exposure, a group-wise observation was made about half-way through the 6-h exposure period. On weekends, 1 check per day was made.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: See above

BODY WEIGHT: Yes
- Time schedule for examinations: Once before the start of exposure, once prior to exposure on the first day of exposure (day 0) and twice weekly thereafter, for the last time on the day of scheduled sacrifice.

FOOD CONSUMPTION:
- Food consumption measured per cage and expressed in g/animal/day. Food consumption was measured over 2 seven-day periods, starting on day 0.

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

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: Bronchoalveolar lavage and measurements: At necropsy, the lungs of animals of the main groups were lavaged according to a standardized method. In short: the right half of the lungs (after binding of the left lung lobe, which was used for histopathology) of these animals was rinsed three times with a single volume of 26.7 ml saline per kg body weight (one value for each group based on mean body weight). The final amount of lung lining fluid and cells collected was weighed and retained on ice. The bronchoalveolar lavage cells were recovered by centrifugation (250xG) for 5 minutes. The temperature control of the centrifuge was set at 4°C. Each cell pellet thus obtained per animal was resuspended in 0.5 ml saline and used for total white blood cell numbers, viability and cell differentials. The supernatant was used for biochemical determinations.
Biochemical determinations: The volume of the supernatant was determined. Total protein, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), N-acetylglucosaminidase (NAG), and gammaglutamyltransferase (GGT) were determined.
Cellular determinations: Total white blood cell numbers were counted using a Coulter Counter (Beckman Coulter Nederland B.V., Woerden, Netherlands). The number of viable cells was determined using an acridine orange / ethidium bromide staining method in combination with fluorescent microscopic evaluation. The cytospins were made using a Cyto-Tek (Sakura, Netherlands) and stained by May-Grunwald Giemsa. The differential cells were evaluated by light microscopy (absolute numbers were calculated from total white blood cell number and percentage distribution of the different cell types).
Since exposure-related changes were observed in animals of the main study groups, investigation of bronchoalveolar lavage parameters (biochemical and cellular determinations) was extended to animals of the recovery groups (control and top concentration).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Statistics:

Body weight data collected after initiation of treatment: ‘AnCova & Dunnett’s Test’ with ‘Automatic’ data transformation method. Day 0 body weight data were used as covariate in the analysis of the post-treatment data unless removed during data preprocessing.
Pre-treatment body weight, organ weight, and bronchoalveolar lavage data: ‘Generalised Anova/Ancova Test’ with ‘Automatic’ data transformation method.
Incidences of histopathological changes: Fisher’s exact probability test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical abnormalities were observed. One female animal of the high concentration group had a kink in the tail throughout the study, which was unrelated to the exposure to the test material.

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:

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:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period, an exposure-related increase in lung weight was observed in animals of the top concentration group, which reached the level of statistical significance in females for absolute as well as relative organ weight, and in males for relative organ weight only. No exposure-related changes in weight of the lungs, or any of the other organs investiged, were observed at the end of the recovery period.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no macroscopic findings attributable to the exposure to the test material. The few gross changes observed represented background pathology in rats of this strain and age and occurred only incidentally or at random incidence in
the different groups.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic evaluation revealed treatment-related histopathological changes in the lungs, characterized by minimal (multi)focal alveolitis in all males and females of the top concentration main group. No treatment-related changes were found in any of the organs and tissues examined of animals of the high concentration main group.
At the end of the recovery period, minimal focal alveolitis was observed in one female animal exposed to the top concentration. Since this microscopic change is occasionally observed as part of common background pathology in this strain of rats, it was considered to be a chance finding which was not related to exposure to the test material.
The histopathological changes observed in the other organs and tissues were considered unremarkable, because they represented background findings and occurred in only one or a few animals or at about the same incidence as in the
control group.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The following statistically significant differences in bronchoalveolar lavage parameters were observed between animals of the main groups exposed to the test material and unexposed controls:
- A concentration-dependent increase in several biochemical parameters in animals of the high and top concentration groups: an increased level of ALP in both sexes of the high and top concentration groups, increased GGT in females of the high and top concentration and in males of the top concentration group; increased LDH and total protein in females of the high and top concentration, and increased NAG in males and females of the top concentration group.
- An increased number of macrophages and – as a result – total cells in females of the high and top concentration, and in males of the high concentration group.
- An increased number of neutrophils in females of the high and top concentration group. A similar tendency was observed in males of the top concentration group, which did not reach the level of statistical significance because of an outlier showing a relatively high neutrophil count in the control group.
- An increased number of eosinophils in males of the high concentration group.
In response to the changes observed in animals of the main groups, bronchoalveolar lavage parameters were also examined in animals of the recovery
groups (control and top concentration). No exposure-related changes were observed in any of the parameters investigated at the end of the 14-day recovery group.

Key result

Dose descriptor:

NOAEC

Effect level:

0.26 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

organ weights and organ / body weight ratios

other: slight changes in bronchoalveolar lavage parameters

Critical effects observed:

not specified

Conclusions:

Under the conditions of the current study, inhalation exposure to 0.73 mg/L Calcium carbonate (nano) resulted in adverse pulmonary changes, characterized by minimal (multi)focal alveolitis, increased lung weight, and slight changes in bronchoalveolar lavage parameters indicating tissue damage and an early inflammatory response. All changes were reversible within a 14-day recovery period after exposure. Based on these observations, the NOAEC of Calcium carbonate (nano) in rats exposed for 6 hours/day, 5 days/week for a period of 14 days was considered to be 0.26 mg/L.

Executive summary:

The aim of the present 14-day range finding study was to provide data on the toxicity of inhaled Calcium carbonate (nano) in rats, to be able to select a suitable concentration range for a following sub-chronic inhalation toxicity study. Five main groups of 5 male and 5 female rats each were exposed by nose-only inhalation exposure to 0 (control), 0.025 (± 0.002), 0.076 (± 0.004), 0.26 (± 0.01) or 0.73 (± 0.04) mg/L Calcium carbonate (nano) for 6 hours/day, 5 days/week over a 14-day period (10 exposure days). Animals of the main groups were sacrificed on the day after the last exposure. To assess recovery or delayed occurrence of toxicity, two groups of 5 male and 5 female animals each were exposed together with the animals of the control and top concentration groups, and were sacrificed after a 14-day recovery period following the exposure period.

The exposure conditions were close to their respective targets. The aerodynamic particle size (MMAD) of the test atmospheres was in the range of 1.9 – 2.6 μm, with a geometric standard deviation (gsd) between 1.9 – 2.4. The relative contribution of nanoparticles The relative contribution of nanoparticles (<100 nm) in the various test atmospheres was determined to be very low. These particles size measurements were confirmed by SEM analysis of aerosol samples. Although trace amounts of primary particles (60-70 nm) were detected, the test material was primarily present in agglomerates with an estimated average size of about 0.5 – 1.5 μm (i.e. slightly smaller than the aerodynamic particle size, which is expected for particles with a density >1 g/cm3) .

The exposure to the test material was well tolerated by the animals. No treatment-related clinical abnormalities, or changes in growth and food consumption were observed.

Exposure to 0.73 mg/L Calcium carbonate (nano) resulted in exposure-related changes in the lungs, characterized by minimal (multi)focal alveolitis, increased lung weight, and changes in bronchoalveolar lavage parameters indicating tissue damage (elevated levels of all biochemical markers) and an early inflammatory response (as indicated by slightly increased numbers of neutrophils, and possibly a slight influx of eosinophils in males). Although all pulmonary changes were fully reversible within the 14-day recovery period, they were considered to be an adverse effect of the exposure to 0.73 mg/L Calcium carbonate (nano).

Exposure-related findings at the high concentration of 0.26 mg/L were limited to slightly increased levels of a few bronchoalveolar lavage (BAL) parameters. Female animals showed slightly increased levels of most biochemical markers and of the number of macrophages, neutrophils and total cells; changes in males were limited to slightly increased levels of ALP, macrophages and total cells. The increase in macrophages and – as a result4 – in total cells, which was fully reversible within the recovery period even at the top concentration, was considered to represent a physiological adaptation to exposure to the particulate test material indicating enhanced alveolar clearance activity. The severity of the remaining changes was very limited, all changes were transient and fully reversible, and they could not be related to any histopathological observations. Therefore, the exposure-related findings at the high concentration were considered not to be toxicologically relevant and were judged as non-adverse.

Conclusion:

Under the conditions of the current study, inhalation exposure to 0.73 mg/L Calcium carbonate (nano) resulted in adverse pulmonary changes, characterized by minimal (multi)focal alveolitis, increased lung weight, and slight changes in bronchoalveolar lavage parameters indicating tissue damage and an early inflammatory response. All changes were reversible within a 14-day recovery period after exposure. Based on these observations, the No-Observed-Adverse-Effect-Concentration (NOAEC) of Calcium carbonate (nano) in rats exposed for 6 hours/day, 5 days/week for a period of 14 days was considered to be 0.26 mg/L.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 October 2014 to 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Labopratories, The Netherlands
- Age at study initiation: 7 weeks
- Weight at study initiation: 288g for males, 206g for females
- Housing: Makrolon Type IV cages
- Diet (e.g. ad libitum): SDS R&M No. 3 ad libitum
- Water (e.g. ad libitum): mains water ad libitum
- Acclimation period: ~2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2C
- Humidity (%): 45-65%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: 03 November 2014 To: 13 March 2015

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 1.29 - <= 1.35 µm

Remarks on MMAD:

MMAD / GSD: The average (± standard deviation) particle size as determined by Aerodynamic Particle Sizer, was 1.33 (+/- 0.03), 1.35 (+/- 0.02), 1.32 (+/- 0.03) and 1.29 (+/- 0.03) µm MMAD (Mass Median Aerodynamic Diameter) for the low, mid, high and top concentration groups, with corresponding average geometric standard deviations (+/- standard deviation) of 1.53 (+/- 0.10), 1.52 (+/- 0.01), 1.54 (+/- 0.01) and 1.54 (+/- 0.00), respectively. The relative contribution of nanoparticles (< 100 nm) in the various test atmospheres was determined to be very low. Scanning electron microscopy of aerosol samples confirmed that the particles were primarily present in agglomerates, which varied in size (ranging 60 nm – 30 µm, with trace amounts of primary particles) and shape, with little to no difference between the groups.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Groenendijk Kunststoffen BV cylindrical polypropylene or steel column
- Method of holding animals in test chamber: Rodent tube
- Source and rate of air:
- Method of conditioning air: Humidified and filtered
- System of generating particulates/aerosols: Turntable dust feeder and eductor
- Temperature, humidity, pressure in air chamber: 22+/-3C, 30-70%, slight positive pressure
- Air flow rate: 1 litre/min per animal
- Method of particle size determination: Gravimetric

TEST ATMOSPHERE
- Brief description of analytical method used: Filters weighed before and after loading with test atmosphere
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Actual concentration
The actual concentration of the test material in the test atmosphere was determined by means of gravimetric analysis. Representative test atmosphere samples were obtained from the animals’ breathing zone by passing mass flow controlled (Bronkhorst Hi Tec) amounts of test atmosphere at 4.6 Ln/min 2 through fiber glass filters (Sartorius, 13400-47). Samples of 202.4 (group 2), 41 .4 (group 3), 23.0 (group 4) or 13.8 (group 5) Ln test atmosphere were obtained, respectively. Filters were weighed before sampling, loaded with a sample of test atmosphere, and weighed again. The actual concentration was calculated by dividing the amount of test material present on the filter, by the volume of the sample taken. Samples were taken at least three times per day for each exposure condition.

Duration of treatment / exposure:

6 hours per day

Frequency of treatment:

daily, 5 days per week

Dose / conc.:

0.025 mg/L air (nominal)

Remarks:

0.026 (± 0.002) mg/L, actual dose

Dose / conc.:

0.075 mg/L air (nominal)

Remarks:

0.123 (± 0.006) mg/L actual dose

Dose / conc.:

0.25 mg/L air (nominal)

Remarks:

0.212 (± 0.013) mg/L actual dose

Dose / conc.:

0.75 mg/L air (nominal)

Remarks:

0.399 (± 0.019) mg/L actual dose

No. of animals per sex per dose:

10 male and 10 females, in addition 10 males and 10 females for the control and top dose groups acted as recovery group animals.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a 14-day range finding study, taking the cut-off concentrations for classification into account
- Rationale for animal assignment (if not random): Random
- Post-exposure recovery period in satellite groups: Control and top dose groups

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Before exposure, half-way through exposure, after exposure

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before and after exposure
BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

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 / No / No data

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy
- Anaesthetic used for blood collection: Yes (phenobarbital)
- Animals fasted: Yes
- How many animals: All
- Standard parameters were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy
- Animals fasted: Yes
- How many animals: All
- Standard parameters were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Bronchoalveolar lavage and measurements. At necropsy, the lungs of animals of the main groups were lavaged according to a standardized method. In short: the right half of the lungs (after binding of the left lung lobe, which was used for histopathology) of these animals was rinsed three times with a single volume of 26.7 ml saline per kg body weight (one value for each group based on mean body weight). The final amount of lung lining fluid and cells collected was weighed and retained on ice. The bronchoalveolar lavage cells were recovered by centrifugation (250xG) for 5 minutes. The temperature control of the centrifuge was set at 4°C. Each cell pellet thus obtained per animal was resuspended in 0.5 ml saline and used for total white blood cell numbers, viability and cell differentials. The supernatant was used for biochemical determinations.
Biochemical determinations
The volume of the supernatant was determined. Total protein, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), N-acetylglucosaminidase (NAG), and gammaglutamyltransferase (GGT).
Cellular determinations
Total white blood cell numbers were counted using a Coulter Counter (Beckman Coulter Nederland B.V., Woerden, Netherlands). The number of viable cells was determined using an acridine orange / ethidium bromide staining method in combination with fluorescent microscopic evaluation. The cytospins were made using a Cyto-Tek (Sakura, Netherlands) and stained by May-Grunwald Giemsa. The differential cells were evaluated by light microscopy (absolute numbers were calculated from total white blood cell number and percentage distribution of the different cell types).
Since exposure-related changes were observed in animals of the main groups, investigation of bronchoalveolar lavage parameters (biochemical and cellular determinations) was extended to animals of the recovery groups.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

See: Observations and examinations performed and frequency.

Statistics:

Body weight data collected after initiation of treatment: ‘AnCova & Dunnett’s Test’ (abbreviation ANCDUN) with ‘Automatic’ data transformation method (abbreviation AUTO).
Pre-treatment body weight, organ weight, haematology, clinical chemistry and bronchoalveolar lavage data: ‘Generalised Anova/Ancova Test’ (abbreviation GEN AN) with ‘Automatic’
Food consumption: Dunnett’s multiple comparison test.
Incidences of histopathological changes: Fisher’s exact probability test.
Tests were performed as two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Female animals of the top concentration group showed a slightly reduced growth during the first month of exposure, which was no longer observed during the remainder of the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A statistically significantly reduced food consumption was seen during the first week of the study in males of the top concentration group only.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative weights of the lungs were statistically significantly increased in males and females of the top concentration main groups, sacrificed at the end of the exposure period. Lung weights were also increased at the mid-dose in females.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

BODY WEIGHT AND WEIGHT GAIN: Body weight gain was slightly, but statistically significantly reduced in females of the top concentration group when compared to unexposed controls during the first month of the exposure period (on days 10, 14, 17 and 24). After the first month, differences in growth were no longer observed.

FOOD CONSUMPTION: During the first week of the study, food consumption was slightly reduced in male animals of the top concentration group when compared to unexposed controls.

ORGAN WEIGHTS: Absolute and relative weights of the lungs were statistically significantly increased in males and females of the top concentration main groups, sacrificed at the end of the exposure period. Lung weights were also increased at the mid (but not at the high) concentration level in females only.

OTHER FINDINGS:
The following statistically significant differences in bronchoalveolar lavage (BAL) parameters were observed between animals of the main groups exposed to the test material and unexposed controls:
- A concentration-dependent increase in several biochemical parameters in animals of the mid, high and top concentration groups: increased levels of
GGT and total protein in both sexes and increased ALP and LDH in males of the mid, high and top concentration group; in females, LDH was increased at the top concentration and ALP was – not dose-dependently – increased at the mid concentration only. In addition, the level of NAG was increased in male animals of the top concentration group.
- A slightly increased number of neutrophils in males and females of the high and top concentration groups, and – as a result (since BAL fluid usually contains near 100% macrophages in healthy animals) – a decrease in the relative content of macrophages in males at the top concentration and in females at the mid, high and top concentration level.
- A slightly increased number of lymphocytes in females of the top concentration group.
- A decreased volume of BAL fluid in males of the low and high concentration group, which – in the absence of a concentration-response relationship – was not considered to be related to the exposure.
Since exposure-related changes were observed in animals of the main groups, bronchoalveolar lavage parameters were also examined in animals of the recovery groups (control and top concentration). Females of the top concentration recovery group showed a very slight, but statistically significant increase in the level of GGT and NAG, and a slightly decreased percentage of viable cells; total and differential white blood cell numbers were not affected. Male animals did not show any exposure-related changes in BAL parameters at the end of the 1 -month recovery period following the last exposure.

Key result

Dose descriptor:

NOAEC

Remarks:

Local

Effect level:

>= 0.212 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

other: bronchoalveolar lavage (BAL) parameters

Key result

Dose descriptor:

NOEC

Remarks:

systemic

Effect level:

0.399 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No systemic effects noted at highest exposure level

Critical effects observed:

not specified

Conclusions:

Under the conditions of the current study, inhalation exposure to 0.399 mg/L Calcium carbonate (nano) resulted in treatment-related changes in the lower airways, characterized by an increased lung weight accompanied by slight increases in BAL derived inflammation and cytotoxicity biomarkers. These changes were largely, but not fully, reversible within a 4 week recovery period after the last exposure. Based on these observations, the No-Observed-Adverse-Effect-Concentration (NOAEC) for local effects of sub-chronic inhalation exposure to Calcium carbonate (nano) was placed at 0.212 mg/L. Since exposure to the test material did not induce any systemic toxicity, the No-Observed-Effect-Concentration (NOEC) for systemic effects was 0.399 mg/L.

Executive summary:

The aim of the present study was to provide data on the sub-chronic (13‑week) toxicity of inhaled calcium carbonate (nano) in rats. Five main groups of 10 male and 10 female rats each were exposed by nose-only inhalation exposure to 0 (control), 0.026 (± 0.002), 0.123 (± 0.006), 0.212 (± 0.013) or 0.399 (± 0.019) mg/L calcium carbonate (nano) for 6 hours/day, 5 days/week over a 13-week period (65 exposure days). Animals of the main groups were sacrificed on the day after the last exposure. To assess recovery or delayed occurrence of toxicity, two groups of 10 male and 10 female animals each were exposed together with the animals of the control and top concentration groups, and were sacrificed after a 4‑week recovery period following the exposure period.

The exposure conditions were close to their respective targets. The aerodynamic particle size distribution of the test atmospheres was highly comparable across the groups with an average mass median aerodynamic diameter (MMAD) in the range of 1.29 – 1.35 µm and a geometric standard deviation (gsd) of 1.52 – 1.54. The relative contribution of nanoparticles (< 100 nm) in the various test atmospheres was determined to be very low. Scanning electron microscopy of aerosol samples confirmed that the particles were primarily present in agglomerates, which varied in size (ranging 60 nm – 30 µm, with trace amounts of primary particles) and shape, with little to no difference between the groups.

The exposure to the test material was well tolerated by the animals. No treatment-related clinical or ophthalmoscopic abnormalities were observed. A transient decrease in growth (females) or food consumption (males), observed in the top concentration group shortly after the initiation of exposure, was no longer observed after a few weeks. Haematology results, clinical chemistry analysis and necropsy findings did not show any treatment-related changes. No indications for systemic toxicity of inhaled calcium carbonate (nano)were observed in this study. 

Exposure to the test material resulted in local changes in the lower airways. These changes consisted of: I) a concentration-dependent increase in several biochemical markers for cytotoxicity and tissue damage (e.g. ALP, GGT, LDH, total protein) in bronchoalveolar lavage (BAL) fluid of animals of the mid, high and top concentration main groups; II) slight changes in differential white blood cell numbers in BAL fluid of animals of the high and top concentration main groups, characterized by an increase in the number of neutrophils and – for females of the top concentration group only – a slight increase in the number of lymphocytes; and III) an increased lung weight in males and females of the top concentration main group. These findings were not accompanied by any microscopic changes in the lungs; histopathology did not reveal any treatment-related changes in the respiratory tract (or in any other tissues)[1]. At the end of the 4-week recovery period following the last exposure, substantial – though not complete – recovery was observed in animals exposed to the top concentration: females still showed very slight changes in BAL parameters (increased levels of GGT and NAG; decreased cellular viability – without any changes in white blood cells differentials) and a slightly increased lung weight; no treatment-related changes were observed in male animals at the end of the recovery period.

Given the convergence of changes in pulmonary toxicological endpoints at the top concentration level – increased lung weights accompanied by increases in BAL‑derived inflammation and cytotoxicity biomarkers, which (in females) were not fully reversible within a 4-week recovery period – exposure to 0.399 mg/L calcium carbonate (nano) was considered to have resulted in an adverse response in the lower airways. Exposure to 0.212mg/L calcium carbonate (nano) resulted in very limited alterations in BAL parameters only. These findings were not substantiated by any concomitant changes in lung weight or treatment-related histopathology. Therefore, the findings at the high concentration level were considered to be of no toxicological relevance and were judged as non-adverse.

Conclusion

Under the conditions of the current study, inhalation exposure to 0.399 mg/L calcium carbonate (nano) resulted in treatment-related changes in the lower airways, characterized by an increased lung weight accompanied by slight increases in BAL‑derived inflammation and cytotoxicity biomarkers. These changes were largely, but not fully, reversible within a 4‑week recovery period after the last exposure. Based on these observations, the No-Observed-Adverse-Effect-Concentration (NOAEC) for local effects of sub-chronic inhalation exposure to calcium carbonate (nano) was placed at 0.212 mg/L. Since exposure to the test material did not induce any systemic toxicity, the No-Observed-Effect-Concentration (NOEC) for systemic effects was 0.399 mg/L.

[1] BAL measurements are usually a rather sensitive toxicological read-out parameter, and it is not uncommon to observe treatment-related changes at concentrations below a level at which histopathology is induced.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

0.4 mg/L

Study duration:

subchronic

Species:

rat

Quality of whole database:

OECD 413 (read-across)

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

14 October 2014 to 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Labopratories, The Netherlands
- Age at study initiation: 7 weeks
- Weight at study initiation: 288g for males, 206g for females
- Housing: Makrolon Type IV cages
- Diet (e.g. ad libitum): SDS R&M No. 3 ad libitum
- Water (e.g. ad libitum): mains water ad libitum
- Acclimation period: ~2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2C
- Humidity (%): 45-65%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12:12

IN-LIFE DATES: From: 03 November 2014 To: 13 March 2015

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

>= 1.29 - <= 1.35 µm

Remarks on MMAD:

MMAD / GSD: The average (± standard deviation) particle size as determined by Aerodynamic Particle Sizer, was 1.33 (+/- 0.03), 1.35 (+/- 0.02), 1.32 (+/- 0.03) and 1.29 (+/- 0.03) µm MMAD (Mass Median Aerodynamic Diameter) for the low, mid, high and top concentration groups, with corresponding average geometric standard deviations (+/- standard deviation) of 1.53 (+/- 0.10), 1.52 (+/- 0.01), 1.54 (+/- 0.01) and 1.54 (+/- 0.00), respectively. The relative contribution of nanoparticles (< 100 nm) in the various test atmospheres was determined to be very low. Scanning electron microscopy of aerosol samples confirmed that the particles were primarily present in agglomerates, which varied in size (ranging 60 nm – 30 µm, with trace amounts of primary particles) and shape, with little to no difference between the groups.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Groenendijk Kunststoffen BV cylindrical polypropylene or steel column
- Method of holding animals in test chamber: Rodent tube
- Source and rate of air:
- Method of conditioning air: Humidified and filtered
- System of generating particulates/aerosols: Turntable dust feeder and eductor
- Temperature, humidity, pressure in air chamber: 22+/-3C, 30-70%, slight positive pressure
- Air flow rate: 1 litre/min per animal
- Method of particle size determination: Gravimetric

TEST ATMOSPHERE
- Brief description of analytical method used: Filters weighed before and after loading with test atmosphere
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Actual concentration
The actual concentration of the test material in the test atmosphere was determined by means of gravimetric analysis. Representative test atmosphere samples were obtained from the animals’ breathing zone by passing mass flow controlled (Bronkhorst Hi Tec) amounts of test atmosphere at 4.6 Ln/min 2 through fiber glass filters (Sartorius, 13400-47). Samples of 202.4 (group 2), 41 .4 (group 3), 23.0 (group 4) or 13.8 (group 5) Ln test atmosphere were obtained, respectively. Filters were weighed before sampling, loaded with a sample of test atmosphere, and weighed again. The actual concentration was calculated by dividing the amount of test material present on the filter, by the volume of the sample taken. Samples were taken at least three times per day for each exposure condition.

Duration of treatment / exposure:

6 hours per day

Frequency of treatment:

daily, 5 days per week

Dose / conc.:

0.025 mg/L air (nominal)

Remarks:

0.026 (± 0.002) mg/L, actual dose

Dose / conc.:

0.075 mg/L air (nominal)

Remarks:

0.123 (± 0.006) mg/L actual dose

Dose / conc.:

0.25 mg/L air (nominal)

Remarks:

0.212 (± 0.013) mg/L actual dose

Dose / conc.:

0.75 mg/L air (nominal)

Remarks:

0.399 (± 0.019) mg/L actual dose

No. of animals per sex per dose:

10 male and 10 females, in addition 10 males and 10 females for the control and top dose groups acted as recovery group animals.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a 14-day range finding study, taking the cut-off concentrations for classification into account
- Rationale for animal assignment (if not random): Random
- Post-exposure recovery period in satellite groups: Control and top dose groups

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Before exposure, half-way through exposure, after exposure

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before and after exposure
BODY WEIGHT: Yes
- Time schedule for examinations: Twice weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

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 / No / No data

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At necropsy
- Anaesthetic used for blood collection: Yes (phenobarbital)
- Animals fasted: Yes
- How many animals: All
- Standard parameters were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy
- Animals fasted: Yes
- How many animals: All
- Standard parameters were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Bronchoalveolar lavage and measurements. At necropsy, the lungs of animals of the main groups were lavaged according to a standardized method. In short: the right half of the lungs (after binding of the left lung lobe, which was used for histopathology) of these animals was rinsed three times with a single volume of 26.7 ml saline per kg body weight (one value for each group based on mean body weight). The final amount of lung lining fluid and cells collected was weighed and retained on ice. The bronchoalveolar lavage cells were recovered by centrifugation (250xG) for 5 minutes. The temperature control of the centrifuge was set at 4°C. Each cell pellet thus obtained per animal was resuspended in 0.5 ml saline and used for total white blood cell numbers, viability and cell differentials. The supernatant was used for biochemical determinations.
Biochemical determinations
The volume of the supernatant was determined. Total protein, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), N-acetylglucosaminidase (NAG), and gammaglutamyltransferase (GGT).
Cellular determinations
Total white blood cell numbers were counted using a Coulter Counter (Beckman Coulter Nederland B.V., Woerden, Netherlands). The number of viable cells was determined using an acridine orange / ethidium bromide staining method in combination with fluorescent microscopic evaluation. The cytospins were made using a Cyto-Tek (Sakura, Netherlands) and stained by May-Grunwald Giemsa. The differential cells were evaluated by light microscopy (absolute numbers were calculated from total white blood cell number and percentage distribution of the different cell types).
Since exposure-related changes were observed in animals of the main groups, investigation of bronchoalveolar lavage parameters (biochemical and cellular determinations) was extended to animals of the recovery groups.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Other examinations:

See: Observations and examinations performed and frequency.

Statistics:

Body weight data collected after initiation of treatment: ‘AnCova & Dunnett’s Test’ (abbreviation ANCDUN) with ‘Automatic’ data transformation method (abbreviation AUTO).
Pre-treatment body weight, organ weight, haematology, clinical chemistry and bronchoalveolar lavage data: ‘Generalised Anova/Ancova Test’ (abbreviation GEN AN) with ‘Automatic’
Food consumption: Dunnett’s multiple comparison test.
Incidences of histopathological changes: Fisher’s exact probability test.
Tests were performed as two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Female animals of the top concentration group showed a slightly reduced growth during the first month of exposure, which was no longer observed during the remainder of the study.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A statistically significantly reduced food consumption was seen during the first week of the study in males of the top concentration group only.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Absolute and relative weights of the lungs were statistically significantly increased in males and females of the top concentration main groups, sacrificed at the end of the exposure period. Lung weights were also increased at the mid-dose in females.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

BODY WEIGHT AND WEIGHT GAIN: Body weight gain was slightly, but statistically significantly reduced in females of the top concentration group when compared to unexposed controls during the first month of the exposure period (on days 10, 14, 17 and 24). After the first month, differences in growth were no longer observed.

FOOD CONSUMPTION: During the first week of the study, food consumption was slightly reduced in male animals of the top concentration group when compared to unexposed controls.

ORGAN WEIGHTS: Absolute and relative weights of the lungs were statistically significantly increased in males and females of the top concentration main groups, sacrificed at the end of the exposure period. Lung weights were also increased at the mid (but not at the high) concentration level in females only.

OTHER FINDINGS:
The following statistically significant differences in bronchoalveolar lavage (BAL) parameters were observed between animals of the main groups exposed to the test material and unexposed controls:
- A concentration-dependent increase in several biochemical parameters in animals of the mid, high and top concentration groups: increased levels of
GGT and total protein in both sexes and increased ALP and LDH in males of the mid, high and top concentration group; in females, LDH was increased at the top concentration and ALP was – not dose-dependently – increased at the mid concentration only. In addition, the level of NAG was increased in male animals of the top concentration group.
- A slightly increased number of neutrophils in males and females of the high and top concentration groups, and – as a result (since BAL fluid usually contains near 100% macrophages in healthy animals) – a decrease in the relative content of macrophages in males at the top concentration and in females at the mid, high and top concentration level.
- A slightly increased number of lymphocytes in females of the top concentration group.
- A decreased volume of BAL fluid in males of the low and high concentration group, which – in the absence of a concentration-response relationship – was not considered to be related to the exposure.
Since exposure-related changes were observed in animals of the main groups, bronchoalveolar lavage parameters were also examined in animals of the recovery groups (control and top concentration). Females of the top concentration recovery group showed a very slight, but statistically significant increase in the level of GGT and NAG, and a slightly decreased percentage of viable cells; total and differential white blood cell numbers were not affected. Male animals did not show any exposure-related changes in BAL parameters at the end of the 1 -month recovery period following the last exposure.

Key result

Dose descriptor:

NOAEC

Remarks:

Local

Effect level:

>= 0.212 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

other: bronchoalveolar lavage (BAL) parameters

Key result

Dose descriptor:

NOEC

Remarks:

systemic

Effect level:

0.399 mg/L air

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No systemic effects noted at highest exposure level

Critical effects observed:

not specified

Conclusions:

Under the conditions of the current study, inhalation exposure to 0.399 mg/L Calcium carbonate (nano) resulted in treatment-related changes in the lower airways, characterized by an increased lung weight accompanied by slight increases in BAL derived inflammation and cytotoxicity biomarkers. These changes were largely, but not fully, reversible within a 4 week recovery period after the last exposure. Based on these observations, the No-Observed-Adverse-Effect-Concentration (NOAEC) for local effects of sub-chronic inhalation exposure to Calcium carbonate (nano) was placed at 0.212 mg/L. Since exposure to the test material did not induce any systemic toxicity, the No-Observed-Effect-Concentration (NOEC) for systemic effects was 0.399 mg/L.

Executive summary:

The aim of the present study was to provide data on the sub-chronic (13‑week) toxicity of inhaled calcium carbonate (nano) in rats. Five main groups of 10 male and 10 female rats each were exposed by nose-only inhalation exposure to 0 (control), 0.026 (± 0.002), 0.123 (± 0.006), 0.212 (± 0.013) or 0.399 (± 0.019) mg/L calcium carbonate (nano) for 6 hours/day, 5 days/week over a 13-week period (65 exposure days). Animals of the main groups were sacrificed on the day after the last exposure. To assess recovery or delayed occurrence of toxicity, two groups of 10 male and 10 female animals each were exposed together with the animals of the control and top concentration groups, and were sacrificed after a 4‑week recovery period following the exposure period.

The exposure conditions were close to their respective targets. The aerodynamic particle size distribution of the test atmospheres was highly comparable across the groups with an average mass median aerodynamic diameter (MMAD) in the range of 1.29 – 1.35 µm and a geometric standard deviation (gsd) of 1.52 – 1.54. The relative contribution of nanoparticles (< 100 nm) in the various test atmospheres was determined to be very low. Scanning electron microscopy of aerosol samples confirmed that the particles were primarily present in agglomerates, which varied in size (ranging 60 nm – 30 µm, with trace amounts of primary particles) and shape, with little to no difference between the groups.

The exposure to the test material was well tolerated by the animals. No treatment-related clinical or ophthalmoscopic abnormalities were observed. A transient decrease in growth (females) or food consumption (males), observed in the top concentration group shortly after the initiation of exposure, was no longer observed after a few weeks. Haematology results, clinical chemistry analysis and necropsy findings did not show any treatment-related changes. No indications for systemic toxicity of inhaled calcium carbonate (nano)were observed in this study. 

Exposure to the test material resulted in local changes in the lower airways. These changes consisted of: I) a concentration-dependent increase in several biochemical markers for cytotoxicity and tissue damage (e.g. ALP, GGT, LDH, total protein) in bronchoalveolar lavage (BAL) fluid of animals of the mid, high and top concentration main groups; II) slight changes in differential white blood cell numbers in BAL fluid of animals of the high and top concentration main groups, characterized by an increase in the number of neutrophils and – for females of the top concentration group only – a slight increase in the number of lymphocytes; and III) an increased lung weight in males and females of the top concentration main group. These findings were not accompanied by any microscopic changes in the lungs; histopathology did not reveal any treatment-related changes in the respiratory tract (or in any other tissues)[1]. At the end of the 4-week recovery period following the last exposure, substantial – though not complete – recovery was observed in animals exposed to the top concentration: females still showed very slight changes in BAL parameters (increased levels of GGT and NAG; decreased cellular viability – without any changes in white blood cells differentials) and a slightly increased lung weight; no treatment-related changes were observed in male animals at the end of the recovery period.

Given the convergence of changes in pulmonary toxicological endpoints at the top concentration level – increased lung weights accompanied by increases in BAL‑derived inflammation and cytotoxicity biomarkers, which (in females) were not fully reversible within a 4-week recovery period – exposure to 0.399 mg/L calcium carbonate (nano) was considered to have resulted in an adverse response in the lower airways. Exposure to 0.212mg/L calcium carbonate (nano) resulted in very limited alterations in BAL parameters only. These findings were not substantiated by any concomitant changes in lung weight or treatment-related histopathology. Therefore, the findings at the high concentration level were considered to be of no toxicological relevance and were judged as non-adverse.

Conclusion

Under the conditions of the current study, inhalation exposure to 0.399 mg/L calcium carbonate (nano) resulted in treatment-related changes in the lower airways, characterized by an increased lung weight accompanied by slight increases in BAL‑derived inflammation and cytotoxicity biomarkers. These changes were largely, but not fully, reversible within a 4‑week recovery period after the last exposure. Based on these observations, the No-Observed-Adverse-Effect-Concentration (NOAEC) for local effects of sub-chronic inhalation exposure to calcium carbonate (nano) was placed at 0.212 mg/L. Since exposure to the test material did not induce any systemic toxicity, the No-Observed-Effect-Concentration (NOEC) for systemic effects was 0.399 mg/L.

[1] BAL measurements are usually a rather sensitive toxicological read-out parameter, and it is not uncommon to observe treatment-related changes at concentrations below a level at which histopathology is induced.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

0.212 mg/L

Study duration:

subchronic

Species:

rat

Quality of whole database:

OECD 413 (read-across)

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No repeated-dose toxicity studies are available for the test substance. However, oral and inhalation dose toxicity studies are available for the read-across substances nano calcium carbonate and silicic acid, calcium salt.

Oral

In a repeated dose oral study equivalent to OECD 408, all animals dosed with 250, 500 or 1000 mg/kg bw/day for 90 days survived, and no abnormal symptoms related to nano calcium carbonate administration (read-across substance) were observed through general clinical observation and eye examination. Moreover, there were no significant differences in body weight, food consumption, urine, hematological measures, blood biochemistry, and organ weight, and no lesions following gross anatomical examination and histopathological analysis. Thus, the NOAEL for nano calcium carbonate is >1000 mg/kg bw/day (Sung et al., 2014).

A 28 day repeat dose oral toxicity study in rats was performed according to OECD Guideline 422 and GLP with concentrations of the read-across substance calcium carbonate (nano) of 0, 100, 300 and 1000 mg/kg bw/day (Dunster, 2010). Treatment-related effects were observed at all dose levels. However, these effects were considered not to represent an adverse effect of treatment. Hence, the NOAEL for systemic toxicity was considered to be 1000 mg/kg bodyweight/day and calcium carbonate is not considered to be toxic following repeated oral exposure for a period of 28 days.

In a study similar to OECD 452 rats have received the test item silicic acid, calcium salt (read-across substance) via feed (1, 5, 7.5 and 10% nominal in diet). No difference in survival from the control and no gross signs of toxicity have been reported. In the highest dose group growth suppression and slightly elevated pH of the urine but no tumours have been observed. The NOEL is considered to be the 5% dietary level, which is estimated to correspond to about 2500 – 3200 mg/kg bw/day, based on reduction in body-weight gain and isolated cases of calculi and brittle in kidney and urinary bladder, respectively, as well as a few cases of cholangitis-like lesions at the higher dose levels (Columbia, 1956).

Daily oral administration of micro calcium carbonate and nano calcium carbonate (read-across substances) for 28 days did not produce any obvious symptoms of toxicity or mortality even at the highest dose administered to ICR mice via gavage. A careful and extensive necropsy revealed no gross organ changes. The NOAEL for both micro calcium carbonate and nano calcium carbonate were reported to be 1300 mg/kg bw/day (Huabg et al., 2009).

Oral administration of the read-across substance calcium carbonate (nano) to rats for a period of fourteen consecutive days at dose levels of 1000, 500 and 250 mg/kg bw day produced no toxicologically significant changes in the parameters measured. The NOAEL and a suitable high dose level for use on future toxicity studies was considered to be 1000 mg/kg bw/day (Liwska, 2010).

Data from non-guideline studies are available which support the conclusion that repeated doses of calcium carbonate do not lead to toxic effects. The European Commission Scientific Committee on Food has produced a report on the Tolerable Upper Intake Level of Calcium (2003). The report describes the results obtained in a number of studies on calcium salts and concludes the following: “The Committee decided to base the derivation of an upper intake level (UL) for calcium on the evidence of different interventional studies of long duration in adults, some of which were placebo controlled and in which total daily calcium intakes of 2500 mg from both diet and supplements were tolerated without adverse effects. Because of the abundance of data, the application of an uncertainty factor was considered unnecessary. An UL of 2500 mg of calcium per day for calcium intake from all sources is proposed.” For a 70 kg person, this UL is then equivalent to a dose of 36 mg Ca/kg bw/day or 89 mg CaCO3/kg bw/day. The toxicity of any carbonate ingested is expected to be very low given that the low pH environment of the stomach acid would convert the carbonate anion into carbon dioxide and water. In addition, calcium carbonate is considered safe and lawful for use in dietary supplements for human consumption (European Commission 2003, Opinion of the Scientific Committee on Food on the Tolerable Upper Intake Level of Calcium, SCF/CS/NUT/UPPLEV/64 Final).

Inhalation

A study was conducted according to OECD 413 to provide data on the sub-chronic (13‑week) toxicity of inhaled calcium carbonate (nano) in rats (van Triel, 2015). Five main groups of 10 male and 10 female rats each were exposed by nose-only inhalation exposure to 0 (control), 0.026 (± 0.002), 0.123 (± 0.006), 0.212 (± 0.013) or 0.399 (± 0.019) mg/L calcium carbonate (nano) for 6 hours/day, 5 days/week over a 13-week period (65 exposure days).

The dose ranges have been obtained from a 14-day inhalation study in which inhalation exposure to 0.73 mg/L calcium carbonate (nano) resulted in adverse pulmonary changes, characterized by minimal (multi)focal alveolitis, increased lung weight, and slight changes in bronchoalveolar lavage parameters indicating tissue damage and an early inflammatory response. All changes were reversible within a 14-day recovery period after exposure. Based on these observations, the NOAEC of Calcium carbonate (nano) in rats exposed for 6 hours/day, 5 days/week for a period of 14 days was considered to be 0.26 mg/L (van Triel, 2014).

Animals of the main groups in the OECD 413 study were sacrificed on the day after the last exposure. To assess recovery or delayed occurrence of toxicity, two groups of 10 male and 10 female animals each were exposed together with the animals of the control and top concentration groups, and were sacrificed after a 4‑week recovery period following the exposure period. The exposure conditions were close to their respective targets. The aerodynamic particle size distribution of the test atmospheres was highly comparable across the groups with an average mass median aerodynamic diameter (MMAD) in the range of 1.29 – 1.35 µm and a geometric standard deviation of 1.52 – 1.54. The relative contribution of nanoparticles (< 100 nm) in the various test atmospheres was determined to be very low. Scanning electron microscopy of aerosol samples confirmed that the particles were primarily present in agglomerates, which varied in size (ranging 60 nm – 30 µm, with trace amounts of primary particles) and shape, with little to no difference between the groups. The exposure to the test material was well tolerated by the animals. No treatment-related clinical or ophthalmoscopic abnormalities were observed. A transient decrease in growth (females) or food consumption (males), observed in the top concentration group shortly after the initiation of exposure, was no longer observed after a few weeks. Haematology results, clinical chemistry analysis and necropsy findings did not show any treatment-related changes. No indications for systemic toxicity of inhaled calcium carbonate (nano)were observed in this study. Exposure to the test material resulted in local changes in the lower airways. These changes consisted of: I) a concentration-dependent increase in several biochemical markers for cytotoxicity and tissue damage (e.g. ALP, GGT, LDH, total protein) in bronchoalveolar lavage (BAL) fluid of animals of the mid, high and top concentration main groups; II) slight changes in differential white blood cell numbers in BAL fluid of animals of the high and top concentration main groups, characterized by an increase in the number of neutrophils and – for females of the top concentration group only – a slight increase in the number of lymphocytes; and III) an increased lung weight in males and females of the top concentration main group. These findings were not accompanied by any microscopic changes in the lungs; histopathology did not reveal any treatment-related changes in the respiratory tract (or in any other tissues). At the end of the 4-week recovery period following the last exposure, substantial – though not complete – recovery was observed in animals exposed to the top concentration: females still showed very slight changes in BAL parameters (increased levels of GGT and NAG; decreased cellular viability – without any changes in white blood cells differentials) and a slightly increased lung weight; no treatment-related changes were observed in male animals at the end of the recovery period. Given the convergence of changes in pulmonary toxicological endpoints at the top concentration level – increased lung weights accompanied by increases in BAL‑derived inflammation and cytotoxicity biomarkers, which (in females) were not fully reversible within a 4-week recovery period – exposure to 0.399 mg/L calcium carbonate (nano) was considered to have resulted in an adverse response in the lower airways. Exposure to 0.212 mg/L calcium carbonate (nano) resulted in very limited alterations in BAL parameters only. These findings were not substantiated by any concomitant changes in lung weight or treatment-related histopathology. Therefore, the findings at the high concentration level were considered to be of no toxicological relevance and were judged as non-adverse. Under the conditions of the study, inhalation exposure to 0.399 mg/L calcium carbonate (nano) resulted in treatment-related changes in the lower airways, characterized by an increased lung weight accompanied by slight increases in BAL‑derived inflammation and cytotoxicity biomarkers. These changes were largely, but not fully, reversible within a 4‑week recovery period after the last exposure. Based on these observations, the NOAEC for local effects of sub-chronic inhalation exposure to calcium carbonate (nano) was placed at 0.212 mg/L. Since exposure to the test material did not induce any systemic toxicity, the NOEC for systemic effects was 0.399 mg/L.

Administration of the read-across substance lauric acid coated nano calcium carbonate by inhalation to Han Wistar rats for 6 hours daily, for 5 days at achieved concentrations of 73, 266 or 771 μg/L was well tolerated and did not induce any systemic toxicity (Robinson, 2016). There were no test-item related effects on clinical signs, body weight or food consumption. There were also no macroscopic changes. Microscopic observations revealed degeneration of the olfactory epithelium in the dorsal meatus and/or dorsal aspect of the nasal septum in animals exposed to 266 or 771 μg/L. These are common sites of nasal injury following exposure by the inhalation route and degeneration may be a consequence of a direct irritant effect of the test item. The degenerative changes were relatively localized and as the olfactory epithelium is capable of regeneration the changes were considered non-adverse. Eosinophilic material was present in the alveoli of all animals exposed to 771 μg/L and was generally spread diffusely throughout the lungs. An accompanying increase in alveolar macrophages was present, which may reflect attempted clearance of the eosinophilic material from the alveoli. The microscopic findings seen in the lungs correlated with the increased lung weight and were considered to be non-adverse. Analysis of bronchoalveolar lavage fluid revealed higher total and differential white counts for both sexes exposed to 771 μg/L, in the absence of any accompanying histopathological inflammatory changes in the lungs these changes were considered non-adverse. Based on the results of this study the NOAEL is considered to be greater than 771 μg/L, the NOEL was considered to be 73 μg/L.

In a review of the results of epidemiological studies of silicosis prevalence (WHO 1986) the threshold value suggested for the avoidance of silicosis is a time-weighted average concentration of respirable crystalline silica dust of 0.04 mg/m³ for an 8-hour shift, 40-hour week and 35-year working life. From the results of studies reviewed by the MAK Commission, a NOAEL for the respirable crystalline silica dust concentration can be derived, at best in the range below 0.020 mg/m³. This concentration, which is in the range of the detection limit of the currently used analytical method with personal sampling, was obtained mathematically by extrapolation from higher concentrations for shorter exposure periods to a working lifetime of 45 years. Use of a mathematical model without a threshold value predicts that reduction of the concentration of respirable crystalline silica dust to 0.05 mg/m³, measured as the average concentration during 40 years, corresponding to a cumulative exposure level of 2 mg/m³ × years, could reduce the cumulative silicosis risk below that resulting from higher level exposures. According to the "Ausschuss für Gefahrstoffe" general dust limits were determined: 4 mg/m³ (respirable fraction) and 3 mg/m³ (alveoli fraction).

Conclusion

Based on the results provided for the read-across substances (nano and micro sized) no systemic effects are expected after oral and inhalation exposure for the test substance. Systemic oral effects have only been detected at high dosages of >2000 mg/kg bw/day. Local effects after inhalation exposure have been reported. Here, treatment-related changes in the lower airways, characterized by an increased lung weight accompanied by slight increases in BAL derived inflammation and cytotoxicity biomarkers were reported. These changes were largely, but not fully, reversible within a 4 week recovery period after the last exposure.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available data and information are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No 1272/2008 as amended for the eighteenth time in Regulation (EU) 2022/692.