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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro DNA damage and/or repair study
Remarks:
Type of genotoxicity: other: Neutral comet assay
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication which meets basic scientific principles: Tice, R.R. et al., 2000 and Burlinson, B. et al., 2007
Justification for type of information:
The solubility of aluminium lanthanum trioxide (AlLaO3) is low since dissolution of AlLaO3 in water resulted in La concentrations < 0.01 mg/L and Al concentrations < 0.03 mg/L after 34 days. The dissolution of AlLaO3 in water results in Al (3+) (=Al(H2O)6 (3+)) ions and La (3+) ions. Thus, the toxicological moieties of concern are aluminium and lanthanum ions. Thus, in the assessment of toxicity, data available for different aluminium and lanthanum substances are read-across since aluminium and lanthanum ions determine the toxicological potential of aluminium lanthanum trioxide, i.e. are the common toxicological moieties of concern. The effects of the substance aluminium lanthanum trioxide with regard to genetic toxicity are predicted to be equal to the effects of Al (3+) ions and La (3+) ions.

Data source

Reference
Reference Type:
publication
Title:
Analysis of metal-ion induced DNA damage, apoptosis and necrosis in human (Jurkat) T-cells demonstrates Ni2+ and V3+ are more toxic than other metals: Al3+, Be2+, Co2+, Cr3+, Cu2+, Fe3+, Mo5+, Nb5+, Zr2+.
Author:
Caicedo M, Jacobs JJ, Reddy A, Hallab NJ.
Year:
2008
Bibliographic source:
J Biomed Mater Res 86A; 905-913.

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The Comet Assay depends on the abaility of negatively-charged fragments of DNA to undergo electrophoresis in an agarose gel. The extent to which DNA migrates correlates directly with the amount of DNA damage. A suspension of cells is mixed with low melting point agarose, spread on a slide and lysed. DNA is then unwound and undergoes electrophoresis at a particular pH. The pH used dictates the types of DNA damage detected. For example, a neutral pH (7-8), as used in this study, detects predominantly double strand breaks and cross links while a pH>13 allows detection of single strand breaks, incomplete excision repair sites, and alkali labile sites (ALS) in addition to the lesions detected at neutral pH. Under the influence of an electric field, the damaged DNA migrates towards the anode producing a shape like a comet. The amount of migration and shape of the comet are an index of the DNA damage that can then be analysed visually or with image analysis software.
GLP compliance:
not specified
Type of assay:
comet assay

Test material

Constituent 1
Reference substance name:
AlCl3.6H2O
IUPAC Name:
AlCl3.6H2O
Details on test material:
- Name of test material (as cited in study report): AlCl3 obtained from Sigma
- Analytical purity: no data

Method

Target gene:
Not applicable.
Species / strain
Species / strain / cell type:
other: CD4+T cells
Details on mammalian cell type (if applicable):
Species/Cell Line/Source of Cells:
Human CD4+ T cells were obtained from a T-helper lymphoma Jurkat cell line from American Type Culture Collection (Manassas, VA, USA).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Test concentrations with justification for top dose:
0, 50, 100, 500, 1000 and 5000 μM AlCl3.
Vehicle / solvent:
No information.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
no
Positive control substance:
no
Details on test system and experimental conditions:
Cell culture processing & conditions:
Cells were cultures in Dulbecco’s modified Eagle medium (DMEM; GIBCO, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Hyclone Laboratories, Logan, UT) at 37 ºC in a atmosphere composed of 0.5% CO2. After they became confluent (every 3 to 4 days), the cultures were subcultured. Cells were washed twice with PBS and resuspended in 24 well-plates containing fresh medium and the test solutions.

Preparation of test solutions:
No information.

Administration of test solutions:
Volume: NR; Exposure occurred in well-plates.

Duration of exposure, schedule/duration of incubations:
The cells were challenged by exposure to metal solutions for 48 hours.

Analytical verification of dose levels:
Not carried out.

Incubations per dose/time point:
This was not stated for the Comet Assay. Apoptosis and viability tests were carried out in trplicate and prolferation measurement in quadruplicate.

Measurement of study outcomes:
Neutral Comet Assay
The comet assay (Trevigen, Gaithersburg, MD) was used. The authors report following manufacturer protocols. The metal-challenged Jurkat T-lymphocytes were cast in molten agarose (37 ºC) on glass slides (1 x 10E5 cells/mL at 1:10 ratio), lysed and unwound using alkaline (pH > 13) solutions. The gels then underwent electrophoresis (10 min at 22 mV). A stain (SYBR Green I nucleic acid gel stain) was added to show the fluorescent tails of DNA fragments that had migrated. The amount of DNA damage was measured by the comet tail length and normalized to untreated controls to give a DNA damage index (IDD).

50 different cells from two separate slides at each concentration were examined for each metal and the images analysed using a publicly available image analysis program for the Comet Assay.


Evaluation criteria:
Neutral Comet Assay
The length of the comet tail was used as an index of DNA damage and normalized to the value in the untreated control. An IDD (index of DNA damage) >75 was considered a high (“significant”) level of DNA damage. An IDD <75 but >0 was moderate damage and an IDD=0 was defined as low DNA damage.

Apoptosis (flow cytometry/caspase-9)
“Significant” apoptosis: ≥ 50% caspase-9 positive cells.

Significant detriment to viability or ‘toxicity”:
>50% propidium iodide positive cells.

Proliferation inhibition [3H-thymidine uptake]:
P<0.05 comparing metal-treated cells counts per minute with the untreated control.
Statistics:
Not described.
-Legend of figure 1 indicates the use of Student’s t-test and a significance level of p<0.05.

Results and discussion

Test results
Species / strain:
other: CD4+T cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Additional information on results:
- cytotoxicity
Cell viability was determined using propidium iodide (PI) staining and in a FACScan flow cytometer (Becton Dickinson Co.) General viability/toxicity for the different metal treatments were ranked using an LC50 index (half lethal concentration, i.e., the concentration at which 50% of the cells were viable). These tests were conducted in triplicate.

- apoptosis
For each metal, approximately 1 x 10E6 cells were incubated for 48 hours in a 24-well culture plate in 1 mL of media. Ten microlitres of a FITC-conjugated pan-caspase inhibitor (ApoStat) was added during the last 30 min. Cells were stained, harvested, washed twice with PBS, and resuspended in 400 µL of buffer for flow cytometry to quantify intracellular caspase-9 activity (FACScan flow cytometer - Becton Dickinson Co., San Diego, CA), Scatter gates were set to exclude cellular debris. All tests were conducted in triplicate.

- proliferation assays
- [3H]-thymidine incorporation
Quadruplicate proliferation assays were performed for 6 days using 96-well culture plates (Sigma), with a density of about 0.2 x 10E6 cells/ well for 6 days in 150 µL/well of complete media (DMEM, 10% FBS). The temperature was 37 ºC and the atmosphere had 0.5% CO2.. [3H]-Thymidine (1 mCi/well) was added during the last 12 h of the 6-day culture period and a cell harvester was used to collect the CD4+ T lymphocyte Jurkat cell membranes. A Beta plate counter was used to measure the [3H]-thymidine incorporation.
Remarks on result:
other: strain/cell type: CD4+T cells
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Primary Endpoint(s):

Neutral Comet Assay

Significant genotoxic effects were not observed at any of the concentrations tested (50 to 5000 µM-Al).

Apoptosis (flow cytometry/caspase-9)

Significant apoptosis was observed only at the highest concentration tested (5000 µM-Al)

 

Significant detriment to viability or ‘toxicity”:

Significant toxicity was not observed at any of the concentrations tested (50 to 5000 µM-Al).

 

Proliferation inhibition [3H-thymidine uptake]:

Significant inhibition of proliferation was not observed at any of the concentrations tested (50 to 5000 µM-Al).

Other salts:

Metal Chloride Concentrations (µM) at which Significant Harmful Effects (as defined in the article) were observed:

Metal

DNA damage

Apoptosis (Caspase-9)

Viability (PI)

Prolife-ration Inhibition

V

50

50

1000

50

Ni

50

100

5000

500

Co

5000

5000

500

100

Cu

>5000

500

5000

100

Nb

>5000

500

500

>5000

Mo

>5000

1000

>5000

500

Zr

5000

500

5000

>5000

Be

>5000

5000

1000

5000

Cr

>5000

>5000

>5000

>5000

Al

>5000

5000

>5000

>5000

Fe

>5000

5000

>5000

>5000

Applicant's summary and conclusion

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

Aluminium did not result in inhibition of cell growth (3H-thymidine uptake), significant increases in DNA double strand breaks (neutral Comet Assay), or viability (PI dye that stains nuclear DNA) at any concentration applied. A significant apoptotic effect determined through measurement of intracellular caspase-9 activity was observed only at the highest concentration of AlCl3 used (5000 µM-Al). Significant genotoxic effects were observed for vanadium, nickel and cobalt. Based on DNA damage, apoptosis, and effects on viability and prolfieration, the authors ranked the metal chloride salts as follows for harmful effects: V>Ni>Co>Cu>Nb>Mo>Zr>Be>Cr>Al>Fe.
Executive summary:

Caicedo et al. (2008) examined DNA damage in human (Jurkat) T-cells at a range of concentrations of AlCl3 (50, 100, 500, 1000 and 5000 µM-Al) using the neutral Comet Assay. Unwinding and electrophoresis of DNA at neutral pH detects fewer types of DNA damage compared with use of pH > 13. The neutral assay detects predominantly double strand breaks. Cell viability was measured using propidium iodide staining, cell proliferation using 3H-thymidine uptake and apoptosis using caspase-9 immunostaining and flow cytometry. A significant effect for DNA damage was defined as an index of DNA damage (based on relative comet tail length) of >75. More than 50% caspase-positive cells was considered significant apoptosis and >50% propidium iodide positive cells as a significant effect on viability. If the p-value from the Student’s t-test was less than 0.05 in the comparison of the counts per minute in metal-treated cells compared to untreated controls, the inhibition of proliferation based on 3H-thymidine uptake was considered significant. Aluminium did not result in significant inhibition of proliferation, significant increases in DNA double strand breaks, or a significant effect on viability at any concentration applied.

Apoptosis was significant only at 5000 µM-Al. The cells used in this study have not been extensively used with this assay. The responses observed are thus of unclear biological significance. The pH used for lysing and unwinding was also not clear from the report in the article. Overall the study appeared to follow GLP and the results were negative but of only limited relevance.