Calcium carbonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Uncoated nano calcium carbonate was negative in the following assays:

In vitro gene mutation study in bacteria (OECD TG 471) using Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and Escherichia coli WP2 uvrA with and without metabolic activation (S9).

In vitro chromosome aberration study in mammalian cells (OECD TG 473) using human lymphocytes in the presence and absence of metabolic activation.

In vitro gene mutation study in mammalian cells (OECD TG 476) using mouse lymphoma L5178Y cells in the presence and absence of metabolic activation.

The results of these studies are read across to bulk calcium carbonate.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 December 2009 - 19 January 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver homogenate metabolising system (10% liver S9 in standard co-factors))

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate

Mutation test - Experiments 1 and 2: 50, 150, 500, 1500 and 5000 μg/plate

Vehicle / solvent:

- Vehicle: Dimethyl sulphoxide (DMSO)

- Justification for choice of vehicle: The test material was insoluble in sterile distilled water and acetone at 50 mg/mL and tetrahydrofuran at 200 mg/mL but was fully soluble in dimethyl sulphoxide and dimethyl formamide at 50 mg/mL in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.

Untreated negative controls:

yes

Remarks:

Concurrent untreated controls (spontaneous mutation rates)

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Migrated to IUCLID6: 2 μg/plate for WP2uvrA-, 3 μg/plate for TA100, 5 μg/plate for TA1535 - without S9-mix

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

Migrated to IUCLID6: 80 μg/plate for TA1537 - without S9-mix

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: 0.2 μg/plate for TA98 - without S9-mix

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA) with S9-mix: 1 μg/plate for TA100; 2 μg/plate for TA1535 and TA1537; 10 μg/plate for WP2uvrA-

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

Migrated to IUCLID6: 5 μg/plate for TA98 - with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for Experiment 1 and preincubation for Experiment 2:

Preliminary Toxicity Test: In order to select appropriate dose levels for use in the main test, a preliminary test was carried out to determine the toxicity of the test material. The concentrations tested were 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate. The test was performed by mixing 0.1 mL of bacterial culture (TA100 or WP2uvrA-), 2 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of test material formulation and 0.5 mL of S9-mix or phosphate buffer and overlaying onto sterile plates of Vogel-Bonner Minimal agar (30 mL/plate). After approximately 48 hours incubation at 37 °C the plates were assessed for numbers of revertant colonies using a Domino colony counter and examined for effects on the growth of the bacterial background lawn.

Mutation Test - Experiment 1: Five concentrations of the test material (50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 2.0 mL of molten, trace histidine or tryptophan supplemented, top agar, 0.1 mL of the test material formulation, vehicle or positive control and either 0.5 mL of S9-mix or phosphate buffer. The contents of each test tube were mixed and equally distributed onto the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix.
All of the plates were incubated at 37 °C for approximately 48 hours and the frequency of revertant colonies assessed using a Domino colony counte
r.

Mutation Test - Experiment 2: The second experiment was performed using fresh bacterial cultures, test material and control solutions. The test material dose range was the same as Experiment 1 (50 to 5000 μg/plate).
The test material formulations and vehicle control were dosed using the pre-incubation method as follows:
Measured aliquots (0.1 mL) of one of the bacterial cultures were dispensed into sets of test tubes followed by 0.5 mL of S9-mix or phosphate buffer and 0.1 mL of the vehicle or test material formulation and incubated for 20 minutes at 37 °C with shaking at approximately 130 rpm prior to the addition of 2 mL of molten, trace histidine or tryptophan supplemented, top agar. The contents of the tube were then mixed and equally distributed on the surface of Vogel-Bonner Minimal agar plates (one tube per plate). This procedure was repeated, in triplicate, for each bacterial strain and for each concentration of test material both with and without S9-mix. Manual counts were required after employing the pre-incubation method at 5000 μg/plate (absence of S9-mix only) because of a particulate test material precipitation.


DURATION
- Preincubation period: 20 minutes (Experiment 2)
- Exposure duration: 48 h (Experiment 1)


NUMBER OF REPLICATIONS: Plates were prepared in triplicate


NUMBER OF CELLS EVALUATED: All tester strain cultures should be in the range of 1 to 9.9 x 10^09 bacteria per mL.

Evaluation criteria:

There were several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without metabolic activation. Biological relevance of the results were considered first, statistical methods, as recommended by the UKEMS were also used as an aid to evaluation, however, statistical significance was not the only determining factor for a positive response.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix after employing the plate incorporation method of dosing (preliminary toxicity test and Experiment 1). However, after employing the pre-incubation method (Experiment 2) a particulate test material precipitate was noted from 1500 and at 5000 μg/plate, in the absence and presence of S9-mix, respectively.


RANGE-FINDING/SCREENING STUDIES: The test material was non-toxic to the strains of bacteria used (TA100 and WP2uvrA-). The test material formulation and S9-mix used in this experiment were both shown to be sterile.


COMPARISON WITH HISTORICAL CONTROL DATA: A history profile of vehicle and positive control values for 2007 and 2008 is presented in Appendix I.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Results for the negative controls (spontaneous mutation rates) are presented in Table 1 and were considered to be acceptable. These data are for concurrent untreated control plates performed on the same day as the Mutation Test.

The individual plate counts, the mean number of revertant colonies and the standard deviations, for the test material, positive and vehicle controls, both with and without metabolic activation, are presented in Table 2 and Table 3 for Experiment 1 and Table 4 and Table 5 for Experiment 2.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level and was, therefore, tested up to the maximum recommended dose level of 5000 μg/plate.

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation or exposure method.

Conclusions:

Interpretation of results (migrated information):
negative

The test material was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 December 2009 to 15 March 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The cell-cycle time for the lymphocytes from the donors used in this study was determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The average AGT for the regular donors used in this laboratory has been determined to be approximately 17 hours under typical experimental exposure conditions.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbitone and beta-naphthoflavone induced rat liver, S9

Test concentrations with justification for top dose:

Experiment 1 - 4(20) hour:
Without S9-mix: 0*, 31.25, 62.5, 125*, 250*,500*, 1000 (* Dose levels selected for metaphase analysis)
With S9-mix: 0*, 31.25, 62.5, 125*, 250*,500*, 1000 (* Dose levels selected for metaphase analysis)
Experiment 2 - 24 hour:
Without S9-mix: 0*, 31.25, 62.5, 125*, 250*,500*, 1000 (* Dose levels selected for metaphase analysis)

This study was run in parallel with a Mouse Lymphoma Assay (MLA) using L5178Y cells (Harlan Laboratories Ltd project No. 2974/0008) which has the capability of detecting clastogenic activity. The study was performed to meet the requirements of the OECD 476 Guideline. It was therefore considered that the results of the MLA study gave adequate scientific justification for the omission of the repeat of the with metabolic activation exposure group.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of vehicle: A solubility check process was performed to determine a suitable solvent; initially an aqueous vehicle (MEM culture medium) was investigated. However, this was unsuitable and therefore the next coice was DMSO, resulting in a suitable dosable suspension.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hours
- Exposure duration: Experiment 1 - 4 hrs with and without S9. Experiment 2 - 24 hrs without S9
- Expression time (cells in growth medium): 20 hrs for 4 hrs exposure
- Selection time (if incubation with a selection agent): Not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours

SELECTION AGENT (mutation assays): No selection agent
SPINDLE INHIBITOR (cytogenetic assays): Demecolcine
STAIN (for cytogenetic assays): When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing (Appendix 1). Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.
In the 24 hours continuous exposure group, dose level 250 µg/ml, A culture, only 80 metaphases were scored due to partial loss of pellet during the experiment. However due to no aberrations being seen in the 80 metaphases scored it was considered to have no impact on the study.
In addition, cells with 69 chromosomes or more were scored as polyploid cells and the incidence of polyploid cells (%) reported.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:

Evaluation criteria:

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

Statistics:

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test material was dosed into media
- Effects of osmolality: The osmolality did not increase by more than 50 mOsm.
- Evaporation from medium: Not applicable
- Water solubility: Not applicable, the test substance was suspended in DMSO
- Precipitation:

Preliminary test:
A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 31.25 µg/ml, in the 4(20)-hour pulse exposure group in the absence of metabolic activation and at and above 62.5 µg/ml in the 4(20)-hour exposure group in the presence of metabolic activation and in the 24-hour continuous exposure group.

Main test:
The maximum dose level selected for metaphase analysis was limited by the presence of heavy precipitate on the slides at 1000 µg/ml in all three exposure groups. The precipitate was such that it was considered impractical to accurately assess the metaphases for the presence of aberrations and, therefore, the maximum dose level selected was 500 µg/ml.

RANGE-FINDING/SCREENING STUDIES:
The dose range for the Preliminary Toxicity Test was 3.91 to 1000 µg/ml. The maximum dose was based on the maximum recommended 10 mM concentration. A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 31.25 µg/ml, in the 4(20)-hour pulse exposure group in the absence of metabolic activation and at and above 62.5 µg/ml in the 4(20)-hour exposure group in the presence of metabolic activation and in the 24-hour continuous exposure group. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1000 µg/ml in all three of the exposure groups. The mitotic index data are presented in Table 1. The test material induced modest evidence of toxicity in all three of the exposure groups.
The selection for the main experiment was the maximum recommended 10 mM concentration and was 1000 µg/ml for all three exposure groups.


COMPARISON WITH HISTORICAL CONTROL DATA:
All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The qualitative assessment of the slides determined that the toxicity was less than that observed in the Preliminary Toxicity Test and that there were metaphases suitable for scoring present at the maximum dose level of test material, 1000 µg/ml in all three exposure groups. The mitotic index data are given in Tables 1, 2 and 3 (attached background information). They confirm the qualitative observations in that no clear dose-related inhibition of mitotic index was observed in any of the exposure groups.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The chromosome aberration data are given in Table 4, Table 5 and Table 6 (attached background information). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations in either the absence or presence of metabolic activation.

The polyploid cell frequency data are given in Table 7 (attached background information). The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in any of the exposure groups.

There was no evidence of a response in the presence of metabolic activation in this study or in the MLA performed on the test material (Harlan Laboratories Ltd Project No. 2974/0008). This was taken as scientific justification to confirm that the repeat of the exposure group with metabolic action was not required.

Conclusions:

Interpretation of results (migrated information):
negative

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 December 2009 to 17 May 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

yes

Remarks:

A repeat of the 4-hour exposure with metabolic activation was not performed because there was no evidence of a response in the presence of metabolic activation in this study or in the Chromosome Aberration Test performed on the same test material.

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK.
- Properly maintained: yes - The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 µg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 µg/mL) and 10% donor horse serum (giving R10 media) at 37°C with 5% CO2 in air. The cells have a generation time of approximately 12 hours and were subcultured accordingly.
- Periodically "cleansed" against high spontaneous background: yes - Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix prepared from the livers of male Sprague-Dawley rats weighing approximately 250g. 20% S9-mix was prepared by mixing S9, NADP (5 mM), G6P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0. The final concentration of S9 was 2% throughout the study.

Test concentrations with justification for top dose:

4 hour and 24 hour exposures: 0, 7.81, 15.63, 31.25, 62.5, 125 and 250 µg/mL

Vehicle / solvent:

- Solvent used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (DMSO) controls

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Migrated to IUCLID6: - the positive control in the absence of metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Solvent (DMSO) controls

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

Migrated to IUCLID6: - the positive control in the presence of metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4-hour exposure with and without metabolic activation: The treatment vessels were incubated at 37 °C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.
24-hour exposure without metabolic activation: The treatment vessels were incubated at 37 °C with continuous shaking using an orbital shaker for 24 hours.
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 5-trifluorothymidine (TFT) in 96-well microtitre plates. The microtitre plates were then incubated for 10-14 days.

STAIN: To assist the scoring of the TFT mutant colonies 0.025 mL of MTT solution (2.5 mg/mL in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours.

SELECTION AGENT (mutation assays): 5 trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: Each treatment was performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at six dose levels of the test material (7.81 to 250 µg/mL), vehicle and positive controls.

NUMBER OF CELLS EVALUATED: At the end of the treatment period the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 10^5 cells/mL. The cultures were incubated and subcultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 10^5 cells/mL.
On Day 2 of the experiment, the cells were counted, diluted to 10^4 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.

Evaluation criteria:

The daily cell counts were used to obtain a Percentage Relative Suspension Growth (%RSG) value that gives an indication of post treatment toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Microtitre plates were scored using a magnifying mirror box after ten to fourteen days incubation. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded. Colonies were scored manually by eye using qualitative judgement. Large colonies were defined as those that cover approximately ¼ to ¾ of the surface of the well and were generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies were scored as small colonies. Small colonies were normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of MTT solution (2.5 mg/mL in PBS) was added to each well of the mutation plates. The plates were incubated for approximately two hours. MTT is a vital stain that is taken up by viable cells and metabolised to give a brown/black colour, thus aiding the visualisation of the mutant colonies, particularly the small colonies.

The normal range for mutant frequency per survivor is 50-200 x 10^-6 for the TK+/- locus in L5178Y cells. For a test material to demonstrate a mutagenic response it must produce a statistically significant increase in the induced mutant frequency (IMF) over the concurrent vehicle mutant frequency value.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

The extended exposure time had no effect on the toxicity of the test material.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

4-HOUR EXPOSURE WITH AND WITHOUT METABOLIC ACTIVATION:
There was no evidence of any marked toxicity following exposure to the test material in either the absence or presence of metabolic activation, as indicated by the %RSG and RTG values. There was also no evidence of any reductions in viability (%V) in either the absence or presence of metabolic activation, therefore indicating that no residual toxicity had occurred. The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell in either the absence or presence of metabolic activation.

24-HOUR EXPOSURE WITHOUT METABOLIC ACTIVATION:
There was no evidence of any marked toxicity following exposure to the test material, as indicated by the %RSG and RTG values. There was also once again no evidence of any reductions in viability (%V), therefore indicating that no residual toxicity had occurred. The 24-hour exposure without metabolic activation (S9) treatment, demonstrated that the extended time point had no effect on the toxicity of the test material. The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10^-6 per viable cell.

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: In the 4 hour exposure experiment a precipitate of the test material was observed at and above 62.5 µg/mL in both the absence and presence of metabolic activation. However, the purpose and integrity of the study was considered unaffected.
In the 24 hour exposure experiment a precipitate of test material was observed at and above 15.63 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test was performed on cell cultures at 5 x 10^5 cells/mL, using a 4 hour exposure time both with and without metabolic activation (S9), and at 1.5 x 10^5 cells/mL using a 24-hour exposure without S9. The dose range used in the preliminary toxicity test was 3.91 to 1000 µg/mL for all three of the exposure groups.
In the 4-hour exposure groups, both in the absence and presence of metabolic activation (S9), there was no evidence of any marked dose related reductions in the relative suspension growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. In the 24-hour exposure in the absence of metabolic activation, there was evidence of very modest reductions in %RSG values of cells treated with test material at and above 500 µg/mL. However, it was considered that this was due to some of the cell pellet being lost with the precipitate during the washing phase and not true test material induced toxicity. A precipitate of the test material was observed at and above 31.25 µg/mL in the 4-hour exposure groups, and at and above 15.63 µg/mL in the 24-hour exposure group, and increased with increase in dose concentration in all three of the exposure groups. In the subsequent mutagenicity test, with no evidence of any marked test material-induced toxicity, the maximum dose was limited to 250 µg/mL due to the nature of the precipitate observed at and above 500 µg/mL that resulted in handling problems during the washing stage of the test.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: 4 hour exposure

Table 1: Preliminary toxicity test – results for the Relative Suspension Growth (%RSG)

Dose (µg/mL)	% RSG (-S9) 4-Hour Exposure	% RSG (+S9) 4-Hour Exposure	% RSG (-S9) 24-Hour Exposure
0	100	100	100
3.91	93	94	102
7.81	94	96	101
15.63	101	92	108
31.25	95	94	112
62.5	93	107	100
125	97	91	113
250	99	94	117
500	97	91	85
1000	94	92	76

Table 2: Summary of results for 4 hour exposure (main experiment)

Treatment (µg/mL)	4-Hours-S-9			Treatment (µg/mL)	4-Hours+S-9
-	%	RTG	MF§	-	%	RTG	MF§
0	100	1.00	67.20	0	100	1.00	60.28
7.81	101	1.12	70.00	7.81	106	1.25	58.93
15.63	107	1.23	71.96	15.63	110	1.12	72.14
31.25	114	1.28	56.60	31.25	111	1.21	60.34
62.5	104	1.17	61.11	62.5	100	1.15	60.31
125	99	1.30	55.11	125	109	1.13	66.41
250	113	1.38	57.94	250	105	1.16	65.04
Linear trend		-	NS	Linear trend		-	NS
EMS	-	-	-	CP	-	-	-
400	72	0.52	566.79	2	69	0.36	746.57

Table 3: Summary of results for 24 hour exposure

Treatment (µg/mL)	24-Hours-S-9
-	%	RTG	MF§
0	100	1.00	88.92
7.81	109	1.10	83.34
15.63	111	1.05	68.82
31.25	116	1.29	55.07
62.5	112	1.08	111.52
125	121	1.26	57.35
250	109	1.00	76.21
Linear trend		-	NS
EMS	-	-	-
150	62	0.44	758.03

 

Conclusions:

Interpretation of results (migrated information):
negative with and without metabolic activation

Calcium carbonate (nano) did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro gene mutation study in bacteria:

In a reliable GLP OECD guideline 471 study (Thompson, 2010), uncoated nano calcium carbonate was tested in a bacterial reverse mutation assay in Salmonella typhimurium (strains TA 98, TA 100, TA 1535 and TA 1537) and Escherichia coli WP2 uvrA with and without metabolic activation (S9). The concentrations tested were 50, 150, 500, 1,500 and 5,000 μg/plate. No mutations occurred.

In vitro chromosome aberration study in mammalian cells:

In a reliable GLP OECD guideline 473 study (Lacey and Durwood, 2010), uncoated nano calcium carbonate was tested for its ability to induce structural chromosomal aberrations in cultured mammalian cells (human lymphocytes) in the presence and absence of metabolic activation. Uncoated nano calcium carbonate did not induce any statistically significant increases in the frequency of cells with aberrations or in the numbers of polyploid cells, in either the absence or presence of metabolic activation and was therefore considered to be non mutagenic under the conditions of the test.

In vitro gene mutation study in mammalian cells:

In a reliable GLP OECD guideline 476 study (Flanders, 2010), uncoated nano calcium carbonate was tested for its ability to induce mutations in mouse lymphoma L5178Y cells in the presence and absence of metabolic activation. Uncoated nano calcium carbonate did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and was therefore considered to be non mutagenic under the conditions of the test.

The nano form of calcium carbonate was tested because this form was anticipated to represent the worst case as it is likely to be more soluble than the bulk form due to the smaller particle size and hence greater surface area. Furthermore, the smaller particle size will be more likely to penetrate the cells.However, the results are directly applicable to the bulk form of calcium carbonate.

Justification for classification or non-classification

The results of an in vitro gene mutation study in bacteria, an in vitro chromosome aberration study in mammalian cells and an in vitro gene mutation study in mammalian cells, performed using uncoated nano calcium carbonate and read across to bulk calcium carbonate, were all negative. It is concluded that bulk calcium carbonate is not genotoxic and does not warrant classification for mutagenicity under CLP.