[No public or meaningful name is available]

Acute Toxicity: inhalation

Administrative data

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 April 2010 to 04 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.2 (Acute Toxicity (Inhalation))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1300 (Acute inhalation toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

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: bulk

Details on test material:

Identification: Calcium carbonate (CAS: 471-34-1)
Description: White powder
Batch Number: F19928
Purity: 97.9%
Expiry Date (Retest Date): 15-Apr-2011
Storage Conditions: At room temperature (20 ± 5°C), protected from direct sunlight

Three different samples of Calcium Carbonate were supplied by the Sponsor:

1). Calcium Carbonate (nano, calcite)
Batch No.: GICM014427 – 03/08/2009

2). Calcium Carbonate (bulk, aragonite form)
Batch No.: DP 99 01 00 -01

3). Calcium carbonate (CAS: 471-34-1) - bulk, calcite
Batch No.: F19928

Technical trials were conducted with the above-mentioned samples differing in size (nano versus bulk) and crystal structure (aragonite versus calcite).
The original intention was to use the nano form of calcium carbonate because this form was anticipated to represent the worst case as it is likely to be more soluble than the bulk form due to the smaller particle size and hence greater surface area for systemic absorption and passage throughout the lungs. However, the aerosol concentration targeted in this study could only be achieved with Calcium Carbonate (CAS: 471-34-1. Aerosol generation trials with Calcium Carbonate (nano) and Calcium Carbonate (aragonite form) resulted in blockage of the aerosol generator. As a result the study was performed with calcium carbonate (bulk, calcite).

Specific details on test material used for the study:

Bulk calcium carbonate

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands
- Age at study initiation: 9 weeks
- Weight at study initiation: Males: 254.7 - 271.5 g; Females: 177.3 - 200.3 g
- Housing: Optimal Hygienic Conditions (OHC) inside a barrier system. Animals were housed in groups of 5 of the same sex in Makrolon® type-IV cages with wire mesh tops and standard softwood bedding including paper enrichment.
- Diet: Pelleted standard Harlan Teklad 2914C rat maintenance diet available ad libitum except during the period when the animals were restrained in exposure tubes.
- Water: Community tap water from Füllinsdorf available ad libitum in water bottles, except during the period when they were restrained in exposure tubes.
- Acclimation period: Seven days under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes: 10-15 per hour
- Photoperiod: 12 hour fluorescent light / 12 hour dark cycle

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Remarks:

Flow past exposure

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Inhalation exposure was performed using a flow-past, nose-only exposure system. The inhalation exposure system is located inside a ducted extraction cabinet.
- Exposure chamber volume: Not specified
- Method of holding animals in test chamber: The animals were confined separately in restraint tubes which were positioned radially around the exposure chamber.
- Source and rate of air: The flow of air at each tube was 0.97 L/min, which is sufficient to minimize re-breathing of the test aerosol as it is more than twice the respiratory minute volume of rodents. The actual airflow rate through the exposure chamber was recorded at approximately 30 minute intervals from the start of the inhalation exposure.
- Method of conditioning air: Not specified
- System of generating particulates/aerosols: A dust aerosol was generated from the test item using a CR3020 rotating brush aerosol generator connected to a micronizing jet mill. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutralizer.
- Method of particle size determination: The particle size distribution of the test aerosol was determined three times during exposure using a 7 stage Mercer cascade Impactor. The particle size distribution was measured by gravimetrically analyzing the test item deposited on each stage of the cascade impactor.
Mass Median Aerodynamic Diameters (MMAD) and Geometric Standard Deviations (GSD) were calculated on the basis of the results from the impactor, using Microsoft Excel Software. The target range was 1 to 4 μm for the MMAD and between 1.5 and 3 for the GSD.
- Treatment of exhaust air: The exhaled air is exhausted through the gap near each feed tube out of the exposure chamber.
- Temperature, humidity, pressure etc in air chamber: The oxygen concentration, airflow rate, temperature and relative humidity of the test atmosphere was measured continuously during exposure using a calibrated device. The results were recorded manually and were reported at 30 minute intervals from the start of exposure and additionally at the end of exposure. The oxygen concentration was maintained above 19% during each exposure period.
Mean oxygen concentration: 20.9 vol%, Mean temperature: 22.4 °C; Mean relative humidity: 7.3%

TEST ATMOSPHERE
- Brief description of analytical method used:
Gravimetric determinations of aerosol concentration were performed eight times during exposure. The samples were collected on a Millipore®durapore filter, Type HVLP loaded in a 47 mm inline stainless steel filter sampling device. The filters were weighed before and immediately after sampling using a calibrated balance. The test aerosol concentration was calculated from the amount of test item present on the filter and the sample volume.
Chemical determinations of aerosol concentration were performed eight times during exposure using the filters collected for gravimetric determination. The filters were transferred into appropriate labeled vials, forwarded at ambient temperature the scientist responsible for formulation analysis and stored at room temperature (20 ± 5 °C) until analysis. The samples were analyzed using an AAS method as follows:
Instrument: Perkin-Elmer Model PE 2100 (software 4100) atomic absorption spectrometer
Flame: Acetylene flame/nitrogen(I) oxide
Slit Width: 0.7 nm high
Wavelength: Calcium: 422.7 nm
- Samples taken from breathing zone: yes

TEST ATMOSPHERE
- Particle size distribution: See table; the particle size of the generated aerosol was fairly stable during the whole exposure period.
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): The Mass Median Aerodynamic Diameters (MMAD) obtained from three gravimetric measurements of particle size distribution during the exposure were similar (MMAD between 2.28 μm and 2.89 μm).The MMADs were well within the target range of 1 to 4 μm, thus deposition of the particles can be assumed to have occurred in both the upper and the lower respiratory tract. In addition, the Geometric Standard Deviations (GSD) were within the target range of 1.5 to 3. Hence, the particle size distributions obtained were considered to be appropriate for acute inhalation toxicity testing.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

Atomic absorption spectrometry

Duration of exposure:

4 h

Remarks on duration:

Exposure was interrupted three times for a total of 8 minutes for cleaning purpose. Nevertheless, the animals were exposed for a period of 4 hours as those interruptions were accounted for.

Concentrations:

Target aerosol concentration: 5 mg/L air
Nominal aerosol concentration: 9.4 mg/L air
Mean measured gravimetric aerosol concentration: 3.0 mg/L air
Mean measured chemical aerosol concentration: 3.0 mg/L air

It was not possible to achieve the target concentration of 5 mg/L air as the achieved level was at the technical limit. Therefore, some variations in the aerosol concentrations occurred. The chemical aerosol concentration compared favourably to the gravimetrically determined concentration. This was in accordance with the high purity of the test item.

No. of animals per sex per dose:

5 animals/ sex

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations for viability were recorded once before exposure on the day of exposure (test day 1), three times during exposure, immediately and 1 h after exposure on test day 1 and twice daily during the observation period.
- Necropsy of survivors performed: yes: All animals were necropsied and examined for macroscopic abnormalities.
- Other examinations performed: Clinical signs: Each animal was examined three times during exposure, immediately and 1 h after exposure on test day 1 and once daily during the observation period. Only grossly abnormal signs were detectable during exposure, as the animals were restrained in the exposure tubes; Body weight: The body weight of each animal was recorded on test days 1 (before exposure), 2, 4, 8 and 15 (before necropsy).

Statistics:

No statistical analysis was performed as only one group was allocated to the study.

Results and discussion

Effect levels

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 3 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

All animals survived the scheduled observation period.

Clinical signs:

other: Clinical signs consisted of ruffled fur only and were recorded for all animals starting one hour after exposure. This observation was noted for up to a maximum of test day 4. From test day 5 onwards all animals were free of clinical signs.

Body weight:

The body weight of animal No. 1 on test day 1 was considered to be incorrect, probably due to a weighing error. The body weight of this animal from test day 2 onwards was within the range of the other male animals. These animals showed slight body weight loss from test day 1 to test day 2. Marginal to slight body weight loss was also noted in three females from test day 1 to test day 2 and in one female up to test day 4. All these animals showed normal body weight thereafter as well as the remaining female animal.

Gross pathology:

There were no macroscopic findings during this study.

Any other information on results incl. tables

Table 1: Test atmosphere conditions

Recording time (hours:min)		O2 concentration (vol %)		Temperature (° C)		Relative humidity (% RH)
08:10		20.7		22.5		6.4
08:40		20.6		22.5		6.2
09:10		20.7		22.5		6.7
09:45		20.6		22.5		13.8
10:15		20.7		22.5		7.7
10:45		20.7		22.6		6.7
11:15		20.7		22.5		6.2
11:45		20.6		22.3		6.0
12:15		20.7		22.3		6.8
12.18		20.7		22.2		6.8
MEAN		20.6		22.4		7.3
St. Dev.		0.0		0.1		2.3
N		10		10		10

 

 

Table 2: Chemical determination of aerosol concentrations

Sampling time (hours:min)	Sampling volume (L)	Amount of test item on the filter (mg)	Chemical aerosol concentration (mg/L air)
08:25-08:30	5.1	29.49	5.8
09:25 - 09:30	5.1	10.84	2.1
09:44 - 09:48	4.0	5.799	1.4
10:03 - 10:07	4.0	7.885	2.0
10:25 - 10:29	4.0	14.14	3.5
10:56 - 11:00	4.0	11.90	2.9
11:30 - 11:34	4.0	13.11	3.2
11:55 - 11:59	4.0	12.37	3.1
Mean			3.0
St. Dev.			1.3
N			8

 

 

Table 3: Particle size distribution

Time	Total amount collected (µg)	Percentages: Stage No. Effective cut-off diameter (µm)								MMAD (µm)	GSD	Corr. Coeff. (R)	%<4 µm
		1 --	2 4.6	3 3.0	4 2.13	5 1.6	6 1.06	7 0.715	8 0.325
08:36	754	15.4	25.6	27.1	17.4	8.0	3.6	1.1	2.0	2.61	2.11	0.946	71.6
10:32	849	22.0	26.3	19.4	15.4	9.2	3.2	2.5	2.0	2.89	2.37	0.965	64.7
11:37	735	6.9	34.3	25.9	16.2	11.2	2.3	0.3	3.0	2.28	1.94	0.913	80.2

 

Table 4: Body weights

Day	Animal body weight (g)
	1	2	3	4	5
Males
1	140.7a	264.6	271.5	268.9	254.7
2	255.7	252.5	264.7	262.1	243.3
4	264.8	262.2	271.9	270.7	254.7
8	283.3	279.8	282.5	286.3	266.5
15	305.8	300.5	306.1	310.2	282.2
Females
1	200.3	194.0	199.0	177.3	194.9
2	198.6	189.8	195.6	185.6	189.5
4	203.9	197.0	194.7	193.3	197.0
8	214.3	205.7	205.9	196.5	209.5
15	214.6	213.0	221.4	209.3	226.5

a Error undetermined

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The LC50 of Calcium carbonate (CAS: 471-34-1) obtained in this study was estimated to be greater than 3.0 mg/L air (chemically determined mean aerosol concentration). This was the highest technically achievable concentration and in accordance with Regulation (EC) No. 1272/2008 (EU CLP) the substance is not considered to be classified.