Reaction mass of Limestone and calcium fluoride and calcium sulfate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014 - 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

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

Remarks:

white powder, nano form

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

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

Administration / exposure

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Remarks on MMAD:

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

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:

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

Examinations

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.

Results and discussion

Results of examinations

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.

Effect levels

1

Key result

Dose descriptor:

NOAEC

Remarks:

local

Effect level:

>= 0.212 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

organ weights and organ / body weight ratios

other: Treatment-related changes in the lower airways: increased lung weight; 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.

2

Key result

Dose descriptor:

NOEC

Remarks:

systemic

Effect level:

>= 0.399 mg/L air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 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.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

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.

Executive summary:

Discussion and conclusion

The aim of the presentstudy was to provide data on the sub-chronic (13‑week) toxicity of inhaled calcium carbonate (nano) in rats. Fivemain 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 eachwere 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, thefindings 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 anincreased 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, theNo-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.