[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

14 December 2009 to 15 March 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 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

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

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)
- Physical state: white powder
- Purity: 98.5% w/w
- Batch No.: GICM014427
- Storage condition of test material: room temperature in the dark

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.

Method

Target gene:

Not applicable

Species / strain

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.

Controls

Controls 1

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

Controls 2

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:

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.