[No public or meaningful name is available]

Repeated dose toxicity: inhalation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Calcium carbonate

EC Number:

207-439-9

EC Name:

Calcium carbonate

Cas Number:

471-34-1

Molecular formula:

CH2O3.Ca

IUPAC Name:

calcium carbonate

Test material form:

solid: nanoform

Details on test material:

- Name of test material (as cited in study report): Calcium carbonate (nano)
- Substance type: Inorganic salt
- Physical state: White powder
- Analytical purity: 98/99%
- Lot/batch No.: GICM020132
- Stability under test conditions: Stable
- Storage condition of test material: ambient temperature (15-25ºC)

Test animals

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

Administration / exposure

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.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.025 mg/L air (nominal)

Doses / concentrations 2

Dose / conc.:

0.075 mg/L air (nominal)

Doses / concentrations 3

Dose / conc.:

0.25 mg/L air (nominal)

Doses / concentrations 4

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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

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

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

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.