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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-11-18 to 2016-01-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2014-09-26
Deviations:
yes
Remarks:
please refer to the field "Prinicples of method if other than guideline" below
Principles of method if other than guideline:
The following alterations from the guidelines were performed:
A series of in-house non-GLP validation experiments was performed to get distinct responses of statistical significance when using the specified positive controls. To achieve such response the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487.
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2015-09-14
Type of assay:
in vitro mammalian cell micronucleus test

Test material

Constituent 1
Chemical structure
Reference substance name:
Cobalt zinc aluminate blue spinel
EC Number:
269-049-5
EC Name:
Cobalt zinc aluminate blue spinel
Cas Number:
68186-87-8
Molecular formula:
Co(x)Zn(1-x)Al2O4 0,1≤x≤0,9
IUPAC Name:
Cobalt zinc aluminium spinel
Test material form:
solid: particulate/powder
Details on test material:
- Chemical description: Cobalt zinc aluminate blue spinel
- Physical state: Solid, blue powder, odourless
- Structure: spinel
- Storage condition of test material: Kept dry in closed containers

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
lymphocytes: human (females)
Details on mammalian cell type (if applicable):
- Type and identity of media: culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX™. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced rat liver S9 was used as the metabolic activation system.
Test concentrations with justification for top dose:
Pre-test/ Experiment I: 0.10, 0.31, 0.92, 2.74, 8.2, 24.7, 74.1, 222.2, 666.7, and 2000.0 µg/mL (with and without metabolic activation; exposure period: 4 hours)
Experiment II: 0.03, 0.09, 0.27, 0.82, 2.47, 7.40, 22.2, 66.7, and 200.0 µg/mL (without metabolic activation; exposure period: 20 hours)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
Stock formulations of the test item and serial dilutions were made in deionised water. The final concentration of deionised water in the culture medium was 10 %.
All formulations were prepared freshly before treatment and used within two hours of preparation.
- Justification for choice of solvent/vehicle: the solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Positive control (without metabolic activation): dissolved in deionised water; concentration: 1.5 µg/mL; pulse treatment
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: demecolcin
Remarks:
Positive control (without metabolic activation): dissolved in deionized water; concentration: 125.0 ng/mL; continuous treatment
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Positive control (with metabolic activation): dissolved in saline (0.9 % NaCl [w/v]); concentration: 15.0 µg/mL
Details on test system and experimental conditions:
NOTE: in the experiments the following concentrations were evaluated:
Experiment I (without metabilic activation; exposure period: 4 hours): 2.74, 8.2, and 24.7 µg/mL
Experiment I (with metabilic activation; exposure period: 4 hours): 8.2, 24.7, and 74.1 µg/mL
Experiment II (without metabilic activation; exposure period: 20 hours): 7.40, 22.2, and 66.7 µg/mL


PRE-EXPERIMENT/EXPERIMENT I
- a preliminary cytotoxicity test (concentrations: 0.10 to 2000.0 µg/mL; with and without metabolic activation; exposure period: 4 hours) was performed to determine the concentrations to be used in the main experiment.
- solvent and positive control were tested.
- cytotoxicity is characterized by the percentages of reduction in the cytokinesis-block proliferation index (CBPI) in comparison with the controls (% cytostasis) by counting 500 cells per culture.
- experimental conditions in this pre-experimental phase were identical to those required and described below for the mutagenicity assay.
- all cell cultures were set up in duplicate.
- preparation interval was 40 hours after start of the exposure
- since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

CYTOGENETIC EXPERIMENT
1) puls exposure:
- 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration.
- culture medium was replaced with serum-free medium containing the test item.
- for the treatment with metabolic activation 50 μL S9 mix per mL culture medium was added.
- after 4 hours the cells were spun down by gentle centrifugation.
- supernatant was discarded
- cells were resuspended in and washed with "saline G" (pH 7.2, containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4), which was done twice.
- cells were resuspended in complete culture medium with 10 % FBS (v/v) and cultured for a 16-hour recovery period.
- after the recovery period Cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

2) continuous exposure (without S9 mix):
- 2 blood cultures (10 mL each) were set up in parallel in 25 cm² cell culture flasks for each test item concentration.
- culture medium was replaced with complete medium (with 10 % FBS) containing the test item.
- after 20 hours the cells were spun down by centrifugation.
- supernatant was discarded
- cells were re-suspended in and washed with "saline G", which was done twice.
- cells were re-suspended in complete culture medium containing 10 % FBS (v/v).
- cytochalasin B (4 μg/mL) was added and the cells were cultured another approximately 20 hours until preparation.

PREPARATION OF CELLS
- cultures were harvested by centrifugation 40 hours after beginning of treatment.
- supernatant was discarded
- cells were re-suspended in approximately 5 mL saline G and spun down once again by centrifugation.
- cells were resuspended in 5 mL KCl solution (0.0375 M) and incubated at 37 °C for 20 minutes.
- 1 mL of ice-cold fixative mixture of methanol and glacial acetic acid (19 parts plus 1 part, respectively) was added to the hypotonic solution and the cells were resuspended.
- after removal of the solution by centrifugation the cells were resuspended for 2 x 20 minutes in fixative and kept cold.
- slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide.
- cells were stained with Giemsa.

EVALUATION OF CYTOTOXICITY AND CYTOGENETIC DAMAGE
- evaluation of the slides was performed using NIKON microscopes with 40 x objectives.
- micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976)*.
- micronuclei have to be stained in the same way as the main nucleus.
- area of the micronucleus should not extend the third part of the area of the main nucleus.
- at least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.
- frequency of micronucleated cells was reported as % micronucleated cells.
- to describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis.
- CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.

CBPI = ((MONC x 1) + (BINC x 2) + (MUNC x 3))/n

CBPI = cytokinesis-block proliferation index
n = total number of cells
MONC = mononucleate cells
BINC = binucleate cells
MUNC = multinucleate cells

Cytostasis % = 100 – 100 [(CBPI T – 1) / (CBPI C – 1)]

T = test item
C = solvent control

ACCEPTABILITY CRITERIA
The micronucleus assay will be considered acceptable if it meets the following criteria:
− the concurrent solvent control will normally be within the laboratory historical solvent control data range.
− the concurrent positive controls should induce responses that are compatible with the laboratory historical positive control data and produce a statistically significant increase.
− cell proliferation criteria in the solvent control are considered to be acceptable.
− all experimental conditions described were tested unless one exposure condition resulted in a clearly positive result.
− the quality of the slides must allow the evaluation of an adequate number of cells and concentrations.
− the criteria for the selection of top concentration are consistent with those described

*Reference:
- Countryman P.I. and Heddle J.A. (1976) The production on micronuclei from chromosome aberrations in irradiated cultures of human lymphocytes. Mutation Research, 41, 321-332.
Evaluation criteria:
Test item is considered to be clearly negative if, in all of the experimental conditions examined:
− none of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− there is no concentration-related increase
− the results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Test item is considered to be clearly positive if, in any of the experimental conditions examined:
− at least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− the increase is concentration-related in at least one experimental condition
− the results are outside the range of the laboratory historical solvent control data
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.

There is no requirement for verification of a clear positive or negative response.

In case the response is neither clearly negative nor clearly positive as described above and/or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions (e.g. narrow concentration spacing, other metabolic activation conditions, i.e. S9 concentration or S9 origin) could be useful.
However, results may remain questionable regardless of the number of times the experiment is repeated. If the data set will not allow a conclusion of positive or negative, the test item will therefore be concluded as equivocal.
Statistics:
Chi squared test (α < 0.05)

Results and discussion

Test results
Species / strain:
lymphocytes: human (females)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
please refer to the field "Additional information on results" below
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
please refer to the field "Additional information on results" below
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
1) Effects of pH: no relevant influence on pH value was observed.
2) Effects of osmolarity: no relevant influence on osmolarity was observed.
- Precipitation:
- Experiment I: precipitation of the test item in the culture medium was observed by the unaided eye at 24.7 μg/mL and above in the absence of S9 mix and at 74.1 μg/mL and above in the presence of S9 mix at the end of treatment. After preparation precipitation was observed microscopically on the slides at 74.1 μg/mL and above in the absence and presence of S9 mix.
- Experiment II: precipitation was observed by the unaided eye in the absence of S9 mix at 66.7 μg/mL and above at the end of treatment. After preparation precipitation was observed microscopically on the slides at 0.27 μg/mL and above in the absence of S9 mix.

RESULTS:
CYTOTOXICITY:
- Experiment I (with and without metabolic activation, up to the highest evaluated concentrations): no cytotoxicity
- Experiment II (without metabolic activation, up to the highest evaluated concentrations): no cytotoxicity

CYTOGENETIC:
In both independent experiments, neither a statistically significant nor a biologically relevant increase in the number of micronucleated cells was observed after treatment with the test item

Applicant's summary and conclusion

Conclusions:
The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.
Therefore, cobalt zinc aluminate blue spinel is considered to be non-clastogenic and non-aneugenic in this in vitro micronucleus test, when tested up to precipitating concentrations.