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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A full set of in-vitro tests is available for cobalt zinc aluminate blue spinel: (i) the substance was negative in (i) a bacterial reverse mutation assay (AMES test), (ii) a micronucleus assay in cultured human lymphocytes, and (iii) in a gene mutation test at the HPRT locus in V79 cells. Therefore, cobalt zinc aluminate blue spinel can be considered as void of any genotoxic potential.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-11-11 to 2015-11-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997-07-21
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2015-09-14
Type of assay:
bacterial reverse mutation assay
Target gene:
TA1537: his C 3076
TA98: his D 3052
TA1535 & TA100: his G 46
E. coli WP2 uvrA: trp-
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:
phenobarbital/β-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate (with and without metabolic activation)
Experiment II: 33, 100, 333, 1000, 2500, and 5000 µg/plate (with and without metabolic activation)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
All formulations were prepared freshly before treatment and used within two hours of preparation.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Positive control (without metabolic activation): concentration: 10 μg/plate (strains TA 1535 & TA 100)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-o-phenylene-diamine
Remarks:
Positive control (without metabolic activation): concentration: 10 μg/plate (strain TA 98) & 50 µg/plate (strain TA 1537)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Positive control (without metabolic activation): concentration: 2.0 μL/plate (strain E. coli WP2 uvrA)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
Positive control (with metabolic activation): concentration: 2.5 μg/plate (strains TA1535, TA1537, TA98 & TA100) & 10 µg/plate (strain E. coli WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Pre-experiment/Experiment I: in agar (plate incorporation); Experiment II: preincubation

DURATION
- Preincubation period: 60 minutes
- Exposure duration (Experiment I & II): at least 48 hours at 37°C

NUMBER OF REPLICATIONS: triplicate

DETERMINATION OF CYTOTOXICITY
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. This pre-experiment was conducted as plate incorporation test.
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, since the following criteria are met:
Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

ACCEPTABILITY OF THE ASSAY:
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of our historical data
- the positive control substances should produce a significant increase in mutant colony frequencies
- a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
A statistical analysis of the data is not mandatory.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
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
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
Species / strain:
E. coli WP2 uvr A
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
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
- Precipitation: the test item precipitated in the overlay agar in the test tubes at 5000 μg/plate in experiment I and from 1000 to 5000 μg/plate in experiment II. Precipitation of the test item in the overlay agar on the incubated agar plates was observed at 5000 μg/plate in both experiments. The undissolved particles had no influence on the data recording.

DOSE SELECTION:
In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment is reported as experiment I. Since no toxic effects were observed 5000 μg/plate were chosen as maximal concentration.

CYTOTOXICITY:
The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

EXPERIMENT I & EXPERIMENT II:
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with cobalt zinc aluminate blue spinel at any concentration level, neither in the presence nor absence of metabolic activation. There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Please also refer for results to the field "Attached background material" below.
Conclusions:
The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
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
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
Target gene:
not applicable
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.
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)
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
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.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-11-10 to 2015-12-10
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)
Version / remarks:
2015-07-28
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
signed 2015-09-14
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
Stocks of the V79 cell line (supplied by Laboratory for Mutagenicity Testing; Technical University, Darmstadt, Germany) are stored in liquid nitrogen. Before freezing, the level of spontaneous mutants may be reduced by treatment with HAT-medium. Each master cell stock is screened for spontaneous mutant frequency.
- Checked for mycoplasma contamination: yes
- Checked for karyotype stability: yes

Thawed stock cultures were propagated at 37 °C in 75 cm² plastic flasks. About 2 - 3 x10^6 cells were seeded into each flask with 15 mL of MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS, neomycin (5 µg/mL) and amphotericin B (1 %). The cells were sub-cultured once or twice weekly. All incubations were done at 37 °C in a 1.5 % carbon dioxide in humidified air.
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
Pre-experiment: 0.92, 2.74, 8.2, 24.7, 74.1, 222.2, 666.7, and 2000 µg/mL (with and without metabolic activation; 4 hour treatment)
Main experiment: 6.9, 13.8, 27.5, 55.0, 110.0, and 220.0 (with and without metabolic activation; 4 hour treatment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water
Immediately before treatment, the test item was dissolved in deionised water. The final concentration of deionised water in the culture medium was 10% (v/v).
- Justification for choice of solvent/vehicle: the solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Positive control without metabolic acitvation: purity: 99 %; vehicle: nutrient medium; final concentration: 0.225 mg/mL = 1.8 mM
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
deionised water
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
Remarks:
Positive control with metabolic acitvation: purity: ≥ 95 %; vehicle: dimethylsulfoxide (final concentration in nutrient medium 0.5%); final concentration: 2.2 µg/mL = 8.6 µM
Details on test system and experimental conditions:
PRE-TEST ON TOXICITY:
A pre-test was performed in order to determine the concentration range for the mutagenicity experiment. The general culture conditions and experimental conditions in this pre-test were the same as described for the mutagenicity experiment below. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the cloning efficiency.
The highest applied concentration of the pre-test on toxicity (2000 μg/mL) was chosen with regard to the current OECD Guideline 476.
In the pre-experiment no relevant cytotoxic effects, indicated by a relative cloning efficiency (survival) of 50% or below was observed up to the highest concentration with and without metabolic activation.
The test medium was checked for precipitation or phase separation at the end of treatment (4 hours) prior to removal to the test item. Precipitation occurred at 222.2 μg/mL and above after 4 hours treatment with and without metabolic activation.
The dose range of the main experiment was set according to phase separation observed in the pre-experiment. The individual concentrations were spaced by a factor of 2.0.

MAIN EXPERIMENT
NOTE: the cultures at the highest concentration with and without metabolic activation were not continued to avoid evaluation of too many precipitating concentrations.

- Culture medium:
For seeding of the cell cultures the complete culture medium will be MEM (minimal essential medium) containing Hank’s salts, neomycin (5 μg/mL), 10% FBS, and amphotericin B (1 %). During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium will be supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).
- Seeding:
Two to four days after subculturing stock cultures are trypsinised at 37 °C for approximately 5 to 10 minutes. Then the enzymatic digestion is stopped by adding complete culture medium with 10 % FBS and a single cell suspension is prepared. The trypsin concentration for all subculturing steps is 0.2 % in saline. Prior to the trypsin treatment the cells were rinsed with PBS buffer. Approximately 0.7 to 1.2×10^7 were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.
- Treatment:
After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G".
The pH was adjusted to 7.2.
Immediately after the end of treatment the cells were trypsinised and subcultivated. At least 2.0×10^6 cells per experimental point (concentration series plus controls) were subcultured in 175 cm² flasks containing 30 mL medium.
Two additional 25 cm² flasks were seeded per experimental point with approx. 500 cells each to determine the relative survival as measure of test item induced cytotoxicity. The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2. The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment as described below.
Three or four days after treatment approximately 2.0×10^6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium.
Following the expression time of approx. 7 days five 75 cm² cell culture flasks were seeded with about 4 - 5×10^5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 for about 8 days. The colonies were stained with 10% methylene blue in 0.01% KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

OTHER EXAMINATIONS:
- pH-value: pH was determined in culture medium of the solvent control and of the maximum concentration of the test item in the pre-experiment without metabolic activation.
Solvent control: 7.32
2000 µg/mL: 7.31
- Osmolarity: osmolarity was determined in culture medium of the solvent control and of the maximum concentration of the test item in the pre-experiment without metabolic activation
Solvent control: 284 mOsm
2000 µg/mL: 286 mOsm

ACCEPTABILITY OF THE ASSAY:
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the mean values of the numbers of mutant colonies per 10^6 cells found in the solvent controls of both parallel cultures remain within the 95% confidence interval of the laboratory historical control data range.
b) the positive control substances should produce a significant increase in mutant colony frequencies and remain within the historical control range of positive controls.
c) the cloning efficiency II (absolute value) of the solvent controls must exceed 50 %.
The data of this study comply with the above mentioned criteria.
Evaluation criteria:
A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls was also taken into consideration.
Statistics:
A linear regression (least squares, calculated using Sum_neu_v2.xltm, version 2.0) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
Please refer to the field "Additonal information on results" below
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
Please refer to the field "Additonal 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
- Precipitation: at the start of treatment, precipitation of the test item was observed by the naked eye at 27.5 μg/mL and above with and at 110 μg/mL and above without metabolic activation. At the end of treatment, precipitation of the test item was noted by the naked eye at 110 μg/mL and above with and without metabolic activation. Microscopical evaluation of precipitation showed non-dissolved particles down to the lowest concentration. However, the non-dissolved particles were homogeneously distributed and did not form aggregates.

MAIN EXPERIMENT:
No relevant and reproducible increase in mutant colony numbers/10^6 cells was observed in the main experiment up to the maximum concentration. The range of the historical solvent control data was not exceeded.

In the first culture without metabolic activation the mutant colonies/10^6 cells exceeded the 95% confidence interval of the corresponding solvent control at 110.0 μg/mL. The effect however, was judged as biologically irrelevant as it was not reproduced in the parallel culture under identical conditions.
A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. A significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was solely detected in the first culture with metabolic activation. This trend however, was judged as irrelevant as it was reciprocal, going down versus increasing concentrations.
In the main experiment with and without S9 mix the range of the solvent controls was from 8.5 up to 19.7 mutants per 10^6 cells; the range of the groups treated with the test item was from 9.6 up to 32.8 mutants per 10^6 cells.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effect indicated by a relative adjusted cloning efficiency I below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation.
Conclusions:
The test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore, cobalt zinc aluminate blue spinel is considered to be non-mutagenic in this HPRT assay.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Tests on the mutagenic potential of poorly soluble inorganic metal compounds in bacteria can be considered as dispensable for principal considerations, since inorganic metal compounds are frequently negative in this assay due to limited capacity for uptake of metal ions (Guidance on information requirements and chemical safety assessment, Chapter R.7a, p. 387; HERAG facts sheet mutagenicity, Chapter 2.1). Nevertheless, it is noted that a bacterial test performed with the substance yielded a negative response.

Cobalt zinc aluminate blue spinel also did not induce micronuclei in cultured human peripheral blood lymphocytes following treatments in the absence and presence of a metabolic activation system (S-9). Concentrations were tested and analysed up to and in excess of the solubility limit in culture medium.

Likewise, Cobalt zinc aluminate blue spinel did not induce mutations at the HPRT locus in V79 cells when tested under the conditions employed in this study. These conditions included treatments up to precipitating concentrations in two independent experiments, in the absence and presence of a rat liver metabolic activation system (S-9).

Further testing such as for in vivo genetic toxicity is not considered necessary in view of the above.


Justification for classification or non-classification

The references Sokolowski (2016, AMES, MN) and Wollny (2016, hprt) are considered as the key studies for in vitro genetic toxicity and will be used for classification. The overall results are as follows:

The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used (S. typhimurium TA 1535, TA 1537, TA 98 and TA 100).

Cobalt zinc aluminate blue spinel did not show a significant or dose-dependent increase in micronuclei in cultured human lymphocytes up to the maximum dose applied.

Cobalt zinc aluminate blue spinel did induce gene mutations at the HPRT locus in V79 cells up to the maximum dose applied in the test.

The classification criteria according to regulation (EC) 1272/2008 as germ cell mutagen are not met.