Registration Dossier
Registration Dossier
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EC number: 692-448-4 | CAS number: 170621-28-0
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro:
Negative Ames test (OECD 471) with S. typhimurium TA 97a, TA 98, TA 100,
TA 102 and TA 1535, with and without metabolic activation.
Negative chromosome aberration test (OECD 473) with cultured human
lymphocytes, with and without metabolic activation.
Negative Gene mutation assay (OECD 476) with mouse lymphoma cells, with
and without metabolic activation
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 12 - 18 Oct 2011
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP - 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)
- Remarks:
- Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon (S. thyphimurium)
- Species / strain / cell type:
- other: TA 1535, TA97a, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9 mix) (Trinova Biochem, Gießen, Germany), prepared from the livers of male Sprague Dawley rats intraperitoneally treated with 500 mg/kg bw Aroclor 1254
- Test concentrations with justification for top dose:
- First experiment: 51, 150, 499, 1502 and 5002 µg/plate with and without metabolic activation
Second experiment: 315, 625, 1253, 2503 and 5001 µg/plate with and without metabolic activation - Vehicle / solvent:
- - Vehicle/solvent used: sterile, demineralised water (0.1 mL for the plate incorporation and the preincubation treatment)
- Justification for choice of solvent/vehicle: water was chosen as vehicle, since the test substance was not soluble in any other of the recommended vehicles and no effects on viability. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-nitro-1,2-phenylene diamine (20 µg/plate, -S9, TA 97a, TA98 and TA 102); sodium azide (1 µg/plate, -S9, TA100 and TA 1535); 2-amino-anthracene (1 µg/plate, +S9, TA 97a, TA100, TA 102 and TA 1535); benzo-a-pyrene (20 µg/plate, +S9, TA98)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION 1: in agar (plate incorporation) for first experiment
DURATION
- Exposure duration: 48 h
METHOD OF APPLICATION 2: preincubation followed by plate incorporation for the second experiment
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: quadruplicate each in both experiments
DETERMINATION OF CYTOTOXICITY
- Method: determination of titre (cytotoxicity test) and observation of background lawn (plate incorporation and preincubation experiment) - Evaluation criteria:
- The test substance was considered to have mutagenic potential, if a significant, reproducible increase in revertant colonies per plate (increase factor ≥ 2) in at least one of the test strains was observed. A concentration-dependent increase over a range of test concentrations was considered to be a sign of mutagenic activity.
- Statistics:
- Mean values and standard deviations of revertant colonies from each test group were calculated.
- Key result
- Species / strain:
- other: TA 1535, TA97a, TA 98, TA 100 and TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test substance was only suspendable in water. Thus, it had to be stirred during application. All concentrations were freshly prepared.
COMPARISON WITH HISTORICAL CONTROL DATA:
The mean numbers of spontaneous revertants in the solvent/vehicle controls of the strains used were all within the normal historical ranges.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In a preliminary study using the plate incorporation method, each strain was incubated with 1511 and 5004 µg/plate of the test substance for 48 h. Two replicates for each concentration were performed both in the presence or absence of metabolic activation. For the determination of toxicity, the titre (colonies/plate) was determined in control and treated cultures. In control culture, the titre should give a number of at least 1E+9 cells/mL, correlating to 100 colonies/plate after dilution. A substance is considered non-toxic, if the quotient control titre/treatment titre is below 2. This criterion was fulfilled after treatment with 1511 and 5004 µg/plate of the test substance. In the main study, no signs of toxicity were observed in any strain and at any concentration tested in both experiments. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 26 Mar - 31 Jul 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP-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)
- Remarks:
- Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- lymphocytes: cultured human lymphocytes
- Details on mammalian cell type (if applicable):
- - Type and identity of media: complete culture medium RPMI 1640 (Biochrom AG, Berlin, Germany): 15% (v/v) foetal calf serum (FCS), 1% (v/v) Penicillin/Streptomycin (per mL: 10000 Units Pen./ 10 mg Strep), 4.8 µg/mL phytohaemagglutinin; complete culture medium RPMI (with FCS) was used during 4-h treatment, whereas serum-free medium RPMI was used during exposure to the test substance for 22 h.
- Properly maintained: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9 mix) (Trinova Biochem, Gießen, Germany), prepared from the livers of male Sprague Dawley rats intraperitoneally treated with 500 mg/kg bw Aroclor 1254
- Test concentrations with justification for top dose:
- Pre-Experiment (Cytotoxicity test = Experiment I) and Experiment II (with and without metabolic activation): #1:10, #1:20, #1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280 dilution of a test substance solution resulting from 24-h extraction and subsequent centrifugation of a stock solution with nominal concentration of 50 mg/mL
Experiment III (with and without metabolic activation): #1:10 dilution of a test substance solution resulting from 96-h extraction and subsequent centrifugation of a stock solution with nominal concentration of 50 mg/mL
#concentrations used for chromosome analysis - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: RPMI 1640 medium without FCS supplementation (for the test item and the positive control EMS)
0.9% NaCl (for the positive control CPA)
- Justification for choice of solvent/vehicle: the test substance was not sufficiently soluble in serum-free medium, deionised water, ethanol or DMSO. Thus, the test substance at 50 mg/mL in medium was shaken for 24 h (Exp. I and II) or 96 h (Exp. III), centrifuged, and the resulting test item solution was diluted to achieve the desired treatment solutions. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- Remarks:
- RPMI 1640 medium without FCS supplementation
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- +S9: cyclophosphamide, 35 µg/mL in 0.9% NaCl; -S9: ethylmethanesulphonate, 600 µg/mL (4-h treatment) and 300 µg/mL (22-h treatment) in RPMI medium without FCS supplementation
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: experiment I: 4 h (±S9); experiment II and III: 4 h (-S9) and 22 h (+S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 h
SPINDLE INHIBITOR (cytogenetic assays): Colcemid, 10 µg/mL in PBS
STAIN (for cytogenetic assays): Giemsa, 10% in Soerensen buffer
NUMBER OF REPLICATIONS: 2 cultures
NUMBER OF CELLS EVALUATED: 100 per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of 1000 cells per culture
OTHER EXAMINATIONS:
- Determination of polyploidy: yes - Evaluation criteria:
- A test item was considered as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups was not above 5.0 % aberrant cells, excluding gaps.
- no significant increase of the number of structural chromosome aberrations was observed.
A test item was considered as mutagenic if:
- the number of induced structural chromosome aberrations is above 5.0 % aberrant cells, excluding gaps and
- either a concentration-related or a significant increase in the number of cells with structural chromosome aberrations was observed.
A test item was considered as pro-mutagenic if:
- an increase in structural chromosome aberrations (as described above) was only observed in the experimental parts with metabolic activation system (S9 mix). - Statistics:
- The number of aberrant metaphase cells in each treatment group was compared with the solvent control value using Fisher’s exact test at the five per cent level (p < 0.05).
- Key result
- Species / strain:
- lymphocytes: cultured human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects: the test substance was not soluble in the recommended solvents and was thus suspended in medium without FCS.
RANGE-FINDING/SCREENING STUDIES: in the preliminary cytotoxicity experiment (experiment I), no cytotoxicity was observed at any dilution of the resulting test item solution, neither in the presence nor in the absence of metabolic activation.
ADDITIONAL INFORMATION ON CYTOTOXICITY: in experiment II, only very slight cytotoxicity (mitotic indices of approx. 70-80% compared to control) was observed after exposure to the test substance for 4 h in the presence of S9 mix and for 22 h in the absence of S9 mix. In Experiment III, only slight cytotoxicity (mitotic indices of approx. 80% compared to control) were noted in the presence of S9 mix (4-h treatment). - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 21 Feb - 25 April 2013
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: GLP-guideline study.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- 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)
- Remarks:
- Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- TK locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- - Type and identity of media: cleansing medium: RPMI 1640-HAT medium with supplements; growth medium 1: complete culture medium RPMI 1640 containing 5 or 10% horse serum, 1% Pen./Strep. (per mL: 10000 Units Pen./ 10 mg Strep.) and 2% sodium pyruvate; selection medium: culture base medium RPMI 1640 containing 15% horse serum, 1% Pen./Strep. (per mL: 10000 Units Pen./ 10 mg Strep.), 2% sodium pyruvate and 5 µg/mL trifluorothymidine (TFT); cleansing media: RPMI 1640-HAT and RPMI 1640-HT medium with supplements
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9 mix) (Trinova Biochem, Gießen, Germany), prepared from the livers of male Sprague-Dawley rats intraperitoneally treated with 500 mg/kg bw Aroclor 1254
- Test concentrations with justification for top dose:
- Pre-Experiment (Cytotoxicity test), Experiment I and Experiment II (with and without metabolic activation): 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280 dilution of a test substance solution resulting from 96-h extraction and subsequent centrifugation of a stock solution with nominal concentration of 50 mg/mL (= resulting test item solution)as well as a 1:10 dilution of a test substance suspension after 96-h extraction of a stock solution with nominal concentration of 50 mg/mL (= test item suspension 5 mg/mL)
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: RPMI 1640 culture base medium without supplements
- Justification for choice of solvent/vehicle: the test substance was not sufficiently soluble in in serum-free medium, deionised water, ethanol
or DMSO. Thus, for test item preparation, the substance at 50 mg/mL in serum-free medium was shaken for 96 h, centrifuged, and the supernatant was used (= resulting test item solution). The resulting test item solution was diluted to achieve the desired treatment solutions. In addition, a 1:10-dilution of the corresponding test suspension after 96-h extraction (without centrifugation) was used for treatment of cells (= test item suspension 5 mg/mL). - Untreated negative controls:
- yes
- Remarks:
- RPMI 1640 medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- 0.9% NaCl (vehicle of positive control CPA)
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- -S9: methyl methanesulphonate (MMS), 19.5 µg/mL (0.18 mM) for Exp. I and 9.75 µg/mL (0.09 mM) for Exp. II in medium without supplements; +S9: cyclophosphamide (CPA) monohydrate, 4.5 µg/mL (16.0 µM) in 0.9% NaCl
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: pre-experiment (cytotoxicity): 4 h (±S9); experiment I: 4 h (±S9); experiment II: 4 h (+S9) and 24 h (-S9)
- Expression time (cells in growth medium): after 48 h incubation, cells were plated for determination of cloning efficiency and the mutation frequency in 96-well microtitre plates containing TFT selective medium. The microtitre plates were incubated 7-10 days (for viability determination) or 10-15 days (for selection).
- Selection time (if incubation with a selection agent): 10-15 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-18 days
SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine
NUMBER OF REPLICATIONS: two replicates in duplicate cultures, in two independent experiments (main experiment)
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; other: total and relative suspension growth, relative survival, cloning viability
OTHER EXAMINATIONS:
- Determination of polyploidy: small and large colonies were counted, as small colonies are capable to indicate chromosomal mutations. - Evaluation criteria:
- The test item was considered to have mutagenic effects if:
- the induced mutation frequency reproducibly exceeded a threshold of 126 colonies per 1E+06 cells (GEF = Global Evaluation Factor)* above the corresponding solvent control.
- the relative increase of the mutation frequency showed a dose-relationship.
A mutagenic response was considered to be reproducible if it occurred in both parallel cultures.
Results of test groups were generally rejected if the relative total growth was less than 10% of the solvent control.
The biological relevance of the results was always considered first. Appropriate statistical methods were used as an aid in evaluating the test results. However, the results of statistical testing were assessed with respect to dose-response relationship. Reproducibility and historical data was also taken into consideration. Statistical significance was confirmed by means of the non-parametric χ2 test. However, both biological and statistical significance were considered together.
*References.:
Moore et al. (2006). Mouse lymphoma thymidine kinase gene mutation assay: follow-up meeting of the International Workshop on Genotoxicity Testing--Aberdeen, Scotland, 2003--Assay acceptance criteria, positive controls, and data evaluation. Environ Mol Mutagen. 47(1):1-5. - Statistics:
- Mean values were calculated. Statistical analysis of the number of mutants between positive control and vehicle control was performed using the non-parametric χ2 test. Statistical significance was indicated at p < 0.01. A linear regression (least squares) was performed to assess a possible dose-dependent increase in the number of mutants in treated compared to control cells. With the assessment of regression, it was evaluated whether mutations increase with increasing dose of the test item. A p-value of 0.05 or lower (significance level 95%) was considered as statistically significance
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: the test substance was not sufficiently soluble in serum-free medium, deionised water, ethanol or DMSO. Therefore, according to OECD 23, a stock suspension in serum free medium was prepared (containing 50 mg/mL) and shaken for 96 hours (Exp. I and II). The suspension was centrifuged (10 min, 600 x g) on the day of the experiment and the supernatant was used (= resulting test item solution). A geometric series of dilutions (factor 2) was prepared from the resulting test item solution. In addition, a 1:10-dilution of the corresponding test item suspension after 96-h extraction (without centrifugation) was used for treatment of cells (= test item suspension 5 mg/mL).
- Precipitation: in all experimental parts (with and without S9 mix, incubation for 4 or 24 hours), precipitation was only observed after treatment with the test item suspension 5 mg/mL.
RANGE-FINDING/SCREENING STUDIES: in the preliminary cytotoxicity test, no significant cytotoxicity was observed in the treated compared to control cells at any test item dilution with and without metabolic activation. A slight reduction in relative survival (given as relative cloning efficiency (RCE), 72.4% compared to control) was observed after treatment with test item suspension 5 mg/mL in the presence of metabolic activation. Similarly, only a slight reduction in the RCE value (89.7 % compared to controls) was observed in the experiment without metabolic activation after treatment of cells with the test item suspension 5 mg/mL.
COMPARISON WITH HISTORICAL CONTROL DATA: the mutant frequencies for the vehicle control RPMI 1640 medium in experiment I (+S9 and -S9) were in the range of 50 - 170 colonies per 1E+06 cells (+S9: 129.50, -S9: 135.00). In experiment II, the mutant frequency exceeded the number of 170 colonies per 1E+06 cells (+S9: 189.75, -S9: 170.25), but were still in the range of the historical data (37.5 - 192.5 and 97.5 - 196.0 in the presence or absence of S9, respectively). The mutation frequencies of the solvent control NaCl 0.9 % were also in the normal range between 50-170 colonies per 1E+06 cells (Exp.I: 148.75) or in the range of the historical data (Exp. II: 201.50 compared to 107.5 - 249.0). Therefore, the study was considered as valid according to the acceptance criteria defined under “Any other information on materials and methods incl. tables”.
ADDITIONAL INFORMATION ON CYTOTOXICITY: despite no cytotoxic effect was observed in the pre-experiment, in experiment I, cytotoxicity was observed in the test item suspension 5 mg/mL, resulting in a relative suspension growth (RSG) values of 49.5 % (+S9) and 35.2 % (-S9) compared to controls. The large variation between replicate A and B in the approach without metabolic activation (replicate A: 8.1 % and replicate B: 62.3 %) is most probably incidental and does not indicate substance-specific effects as the other replicates do not show such high variations. In contrast, the dilutions of the resulting test item solution did not show significant reduction in relative suspension growth (RSG) compared to controls, neither in the presence not in the absence of metabolic activation (values ranged from 73.6% for the 1:10-dilution up to 105.2% for the 1:1280-dilution).
In experiment II, the test item suspension 5 mg/mL resulted in RSG values of 42.3 % (+S9) and 4.3 % (-S9) compared to controls, which is consistent with the observation of significant cytotoxicity (RSG values < 50% compared to controls) in the first experiment. In contrast to the first experiment, cytotoxicity was also observed at the final test dilution 1:10, resulting in RSG values of 49.3% compared to controls without S9 mix. In addition, a RSG value of 59.1% compared to controls was observed in the absence of S9 mix after treatment with the corresponding 1:20 dilution of the resulting test item solution, which is however, considered not to be a significant cytotoxic effect. Consistent with the findings in experiment I, all other dilutions of the resulting test item solution did not cause a significant reduction in RSG values compared to controls, neither in the presence not in the absence of metabolic activation (values ranged from 88.0% for the 1:40-dilution up to 90.9% for the 1:1280-dilution).
In summary, cytotoxicity was concluded for the test item suspension with a nominal concentration of 5 mg/mL in the presence and absence of metabolic activation and for the 1:10 test item dilution in experiment II without metabolic activation.
In the viability experiments following the 48-h expression period, relative total growth (RTG) values of the test item suspension 5 mg/mL were 44.3 % (+S9) and 26.7 % (-S9) compared to controls, indicating strong reduction in cell viability. In contrast, none of the tested dilutions of the resulting test item solution did show significant reduction in RTG compared to controls. In experiment II, the test item suspension 5 mg/mL also induced strong effects on cell viability, as shown by RTG values of 42.1 % (+S9) resp. 1.8 % (-S9) compared to controls. In the approach without metabolic activation, the 1:10 and 1:20 dilutions of the resulting test item solution showed a slight reduction of the RTG values to 58.7% and 64.3%, respectively. Consistent with the observations in experiment I, no significant reduction in cell viability was observed at any other dilution of resulting test item solution.
In summary, reduced cell viability was determined for the test item suspension with a nominal concentration of 5 mg/mL in the presence and absence of metabolic activation. Further, a tendency to decrease viability was observed for the 1:10 and 1:20 test item dilutions in experiment II without metabolic activation. - Conclusions:
- Interpretation of results: negative
Referenceopen allclose all
Table1. Test results of first experiment (plate incorporation)
Aluminium magnesium vanadium oxide: Bacterial Reverse Mutation Assay, mean revertant colonies/plate (n=4 ± SD) |
|||||
EXPERIMENT I (plate incorporation) |
|||||
S9-Mix |
Without
|
||||
Concentration (per plate) |
TA 97a |
TA98 |
TA100 |
TA102 |
TA1535 |
SC (water) |
110 ± 3 |
16 ± 2 |
125 ± 14 |
159 ± 12 |
14 ± 4 |
SC (DMSO) |
107 ± 5 |
12 ± 3 |
144 ± 33 |
155 ± 20 |
15 ± 3 |
Test item |
|
|
|
|
|
5002 µg |
107 ± 8 |
15 ± 2 |
101 ± 7 |
145 ± 8 |
11 ± 4 |
1502 µg |
110 ± 8 |
15 ± 2 |
94 ± 4 |
149 ± 6 |
14 ± 1 |
499 µg |
100 ± 10 |
14 ± 1 |
92 ± 5 |
148 ± 5 |
11 ± 4 |
150 µg |
106 ± 6 |
15 ± 4 |
105 ± 7 |
145 ± 5 |
9 ± 2 |
51 µg |
106 ± 4 |
14 ± 3 |
96 ± 10 |
143 ± 7 |
9 ± 1 |
PC |
|
|
|
|
|
4NOPD (20 µg) |
703 ± 26 |
275 ± 37 |
- |
622 ± 64 |
- |
NaN3 (1 µg) |
- |
- |
645 ± 69 |
- |
254 ± 25 |
S9-Mix
|
With |
||||
Concentration (per plate) |
TA 97a |
TA98 |
TA100 |
TA102 |
TA1535 |
SC (water) |
101 ± 7 |
16 ± 2 |
99 ± 7 |
195 ± 14 |
17 ± 1 |
SC (DMSO) |
102 ± 3 |
13 ± 2 |
104 ± 12 |
186 ± 23 |
16 ± 0 |
Test item |
|
|
|
|
|
5002 µg |
118 ± 7 |
14 ± 2 |
99 ± 4 |
149 ± 7 |
12 ± 2 |
1502 µg |
101 ± 2 |
16 ± 3 |
100 ± 8 |
151 ± 14 |
12 ± 2 |
499 µg |
108 ± 6 |
14 ± 1 |
92 ± 3 |
153 ± 4 |
10 ± 2 |
150 µg |
114 ± 1 |
14 ± 2 |
101 ± 8 |
148 ± 6 |
8 ± 2 |
51 µg |
117 ± 4 |
14 ± 1 |
104 ± 12 |
149 ± 14 |
11 ± 14 |
PC |
|
|
|
|
|
2AA (1 µg) |
490 ± 113 |
- |
548 ± 50 |
511 ± 90 |
143 ± 3 |
BP (20 µg) |
- |
251 ± 35 |
- |
- |
- |
SC = Solvent control; PC = Positive control substances; SD = standard deviation; NaN3 = sodium azide; 4NOPD = 4-nitro-o-phenylene-diamine; 2AA = 2-aminoanthracene; BP = benzo-a-pyrene |
|||||
Table2. Test results of second experiment (pre-incubation)
Aluminium magnesium vanadium oxide: Bacterial Reverse Mutation Assay, mean revertant colonies/plate (n=4 ± SD) |
|||||
EXPERIMENT II (pre-incubation) |
|||||
S9-Mix |
Without
|
||||
Concentration (per plate) |
TA 97a |
TA98 |
TA100 |
TA102 |
TA1535 |
SC (water) |
111 ± 3 |
16 ± 6 |
81 ± 9 |
202 ± 14 |
23 ± 5 |
SC (DMSO) |
115 ± 4 |
15 ± 4 |
130 ± 14 |
197 ± 27 |
21 ± 8 |
Test item |
|
|
|
|
|
5001 µg |
109 ± 4 |
10 ± 0 |
115 ± 6 |
209 ± 17 |
19 ± 1 |
2503 µg |
102 ± 13 |
13 ± 2 |
133 ± 58 |
196 ± 12 |
14 ± 3 |
1253 µg |
114 ± 11 |
15 ± 4 |
122 ± 13 |
181 ± 7 |
16 ± 6 |
625 µg |
111 ± 5 |
10 ± 0 |
119 ± 15 |
203 ± 13 |
21 ± 4 |
315 µg |
133 ± 19 |
10 ± 0 |
111 ± 4 |
199 ± 15 |
16 ± 4 |
PC |
|
|
|
|
|
4NOPD (20 µg) |
479 ± 70 |
229 ± 23 |
- |
499 ± 31 |
- |
NaN3 (1 µg) |
- |
- |
338 ± 52 |
- |
254 ± 66 |
S9-Mix
|
With |
||||
Concentration (per plate) |
TA 97a |
TA98 |
TA100 |
TA102 |
TA1535 |
SC (water) |
118 ± 21 |
20 ± 8 |
96 ± 16 |
210 ± 8 |
19 ± 3 |
SC (DMSO) |
115 ± 15 |
18 ± 4 |
96 ± 9 |
210 ± 21 |
20 ± 7 |
Test item |
|
|
|
|
|
5001 µg |
101 ± 13 |
11 ± 2 |
111 ± 3 |
192 ± 11 |
18 ± 3 |
2503 µg |
113 ± 6 |
15 ± 2 |
142 ± 19 |
206 ± 12 |
18 ± 6 |
1253 µg |
113 ± 6 |
13 ± 4 |
123 ± 3 |
199 ± 17 |
20 ± 1 |
625 µg |
112 ± 8 |
10 ± 0 |
119 ± 18 |
200 ± 15 |
21 ± 5 |
315 µg |
114 ± 6 |
13 ± 3 |
113 ± 8 |
182 ± 16 |
19 ± 1 |
PC |
|
|
|
|
|
2AA (1 µg) |
439 ± 47 |
- |
987 ± 169 |
485 ± 100 |
145 ± 19 |
BP (20 µg) |
- |
324 ± 75 |
- |
- |
- |
SC = Solvent control; PC = Positive control substances; SD = standard deviation; NaN3 = sodium azide; 4NOPD = 4-nitro-o-phenylene-diamine; 2AA = 2-aminoanthracene; BP = benzo-a-pyrene |
|||||
Table 1. Test results of experiment I (preliminary cytotoxicity test)
Test item |
Concentration [µg/mL] |
Mitotic Index [% of control] |
Aberrant cells in % |
|
with gaps# |
without gaps# |
|||
Exposure period 4 h, fixation time 22 h, without S9 mix |
||||
NC |
0 |
100.0 |
3.5 |
1.0 |
EMS |
600 |
36.1 |
37.0 |
34.5* |
Test item |
1:10 dilution |
101.0 |
2.5 |
0.0 |
1:20 dilution |
106.4 |
2.5 |
0.5 |
|
1:40 dilution |
107.9 |
8.0 |
1.5 |
|
Exposure period 4 h, fixation time 22 h, with S9 mix |
||||
NC |
0 |
100.0 |
2.0 |
0.5 |
SC |
0 |
100.0 |
0.5 |
0 |
CP |
35 |
46.0 |
49.5 |
42.0* |
Test item |
1:10 dilution |
101.3 |
3.5 |
1.0 |
1:20 dilution |
102.7 |
3.0 |
1.0 |
|
1:40 dilution |
92.6 |
9.0 |
2.5 |
|
EMS: ethylmethanesulphonate; CP: Cyclophosphamide (positive controls)
NC: Negative control (medium without FCS); SC for CP = 0.9% NaCl
# incl. cells carrying exchanges
* statistically significant from negative control (p ≤ 0.05)
Table 2. Test results of experiment II
Test item |
Concentration [µg/mL] |
Mitotic Index [% of control] |
Aberrant cells in % |
|
with gaps# |
without gaps# |
|||
Exposure period 22 h, fixation time 22 h, without S9 mix |
||||
NC |
0 |
100.0 |
4.5 |
2.0 |
EMS |
300 |
80.7 |
20.0 |
13.0* |
Test item |
1:10 dilution |
76.5 |
6.0 |
1.5 |
1:20 dilution |
72.3 |
4.0 |
1.0 |
|
1:40 dilution |
72.3 |
1.5 |
0.5 |
|
Exposure period 4 h, fixation time 22 h, with S9 mix |
||||
NC |
0 |
100.0 |
8.0 |
3.5 |
SC |
0 |
100.0 |
3.5 |
1.0 |
CP |
35 |
39.4 |
41.5 |
35.5* |
Test item |
1:10 dilution |
79.3 |
6.5 |
2.0 |
1:20 dilution |
77.9 |
5.5 |
1.5 |
|
1:40 dilution |
77.9 |
7.0 |
1.0 |
|
EMS: ethylmethanesulphonate; CP: Cyclophosphamide (positive controls)
NC: Negative control (medium without FCS); SC for CP = 0.9% NaCl
# incl. cells carrying exchanges
* statistically significant from negative control (p ≤ 0.05)
Table 3. Test results of experiment III
Test item |
Concentration [µg/mL] |
Mitotic Index [% of control] |
Aberrant cells in % |
|
with gaps# |
without gaps# |
|||
Exposure period 22 h, fixation time 22 h, without S9 mix |
||||
NC |
0 |
100.0 |
9.5 |
1.5 |
EMS |
300 |
39.4 |
87.5 |
71.5* |
Test item |
1:10 dilution |
90.8 |
7.5 |
1.5 |
Exposure period 4 h, fixation time 22 h, with S9 mix |
||||
NC |
0 |
100.0 |
10.5 |
1.0 |
SC |
0 |
100.0 |
7.5 |
1.0 |
CP |
35 |
45.9 |
83.0 |
62.0* |
Test item |
1:10 dilution |
79.0 |
8.5 |
0.5 |
EMS: ethylmethanesulphonate; CP: Cyclophosphamide (positive controls)
NC: Negative control (medium without FCS); SC for CP = 0.9% NaCl
# incl. cells carrying exchanges
* statistically significant from negative control (p ≤ 0.05)
POLYPLOIDY
No increase in the frequencies of polyploid metaphases was found after treatment compared to controls.
Table 1. Experiment I - 4 h exposure - With Metabolic Activation (mean of two replicates of duplicate cultures)
Cell treatment |
Mean Relative Cloning Efficiency [%] |
Mean Relative Total Growth [%] |
Mean Mutants per 1E+06 cells |
Threshold |
Mutant small colonies per 1E+06 cells |
Mutant large colonies per 1E+06 cells |
Negative control |
100.0 |
100.0 |
128.50 |
- |
53.75 |
74.75 |
Vehicle control (RPMI) |
100.0 |
100.0 |
129.50 |
- |
57.25 |
72.25 |
Vehicle control (NaCl 0.9%) |
100.0 |
100.0 |
148.75 |
- |
64.25 |
84.50 |
Positive control (CPA) |
38.6 |
27.1 |
615.00* |
275 |
499.25 |
115.75 |
Test item: 1:10 dilution |
90.2 |
77.9 |
169.75 |
256 |
39.25 |
130.50 |
Test item: 1:20 dilution |
84.1 |
71.8 |
201.25 |
256 |
47.00 |
154.25 |
Test item: 1:40 dilution |
92.9 |
89.9 |
216.25 |
256 |
49.50 |
166.75 |
Test item: 1:80 dilution |
106.8 |
107.6 |
175.75 |
256 |
28.50 |
147.25 |
Test item: 1:160 dilution |
83.8 |
87.3 |
217.75 |
256 |
49.00 |
168.75 |
Test item: 1:320 dilution |
86.0 |
97.6 |
188.25 |
256 |
45.75 |
142.50 |
Test item: 1:640 dilution |
75.9 |
83.5 |
260.75 |
256 |
55.50 |
205.25 |
Test item: 1:1280 dilution |
106.9 |
111.7 |
175.25 |
256 |
41.00 |
134.25 |
Test item suspension 5 mg/mL |
89.3 |
44.3 |
260.00 |
256 |
78.25 |
181.75 |
CPA = cyclophosphamide; *p < 0.001
Table 2. Experiment I - 4 h exposure - Without Metabolic Activation (mean of two replicates of duplicate cultures)
Cell treatment |
Mean Relative Cloning Efficiency [%] |
Mean Relative Total Growth [%] |
Mean Mutants per 1E+06 cells |
Threshold |
Mutant small colonies per 1E+06 cells |
Mutant large colonies per 1E+06 cells |
Negative control |
100.0 |
100.0 |
117.00 |
- |
42.25 |
74.75 |
Vehicle control (RPMI) |
100.0 |
100.0 |
135.00 |
- |
56.50 |
78.50 |
Positive control (MMS) |
53.0 |
49.0 |
379.25* |
261 |
286.25 |
93.00 |
Test item: 1:10 dilution |
91.4 |
67.2 |
124.50 |
261 |
35.00 |
89.50 |
Test item: 1:20 dilution |
98.8 |
83.8 |
127.50 |
261 |
38.75 |
88.75 |
Test item: 1:40 dilution |
97.8 |
99.6 |
124.50 |
261 |
39.25 |
85.25 |
Test item: 1:80 dilution |
103.4 |
108.4 |
120.75 |
261 |
29.25 |
91.50 |
Test item: 1:160 dilution |
98.0 |
106.6 |
140.75 |
261 |
41.00 |
99.75 |
Test item: 1:320 dilution |
82.8 |
94.6 |
130.50 |
261 |
43.25 |
87.25 |
Test item: 1:640 dilution |
78.9 |
88.4 |
171.75 |
261 |
56.75 |
115.00 |
Test item: 1:1280 dilution |
98.5 |
61.2 |
121.25 |
261 |
29.75 |
91.50 |
Test item suspension 5 mg/mL |
78.9 |
26.7 |
184.00 |
261 |
41.75 |
142.25 |
MMS = methyl methanesulphonate; *p < 0.001
Table 3. Experiment II - 4 h Exposure - With Metabolic Activation (mean of two replicates of duplicate cultures)
Cell treatment |
Mean Relative Cloning Efficiency [%] |
Mean Relative Total Growth [%] |
Mean Mutants per 1E+06 cells |
Threshold |
Mutant small colonies per 1E+06 cells |
Mutant large colonies per 1E+06 cells |
Negative control |
100.0 |
100.0 |
155.75 |
- |
98.25 |
17.00 |
Vehicle control (RPMI) |
100.0 |
100.0 |
189.75 |
- |
145.25 |
44.50 |
Vehicle control (NaCl 0.9%) |
100.0 |
100.0 |
201.50 |
- |
152.75 |
48.75 |
Positive control (CPA) |
41.9 |
25.3 |
597.50* |
328 |
565.25 |
32.25 |
Test item: 1:10 dilution |
109.3 |
74.9 |
173.50 |
316 |
141.75 |
31.50 |
Test item: 1:20 dilution |
107.9 |
93.0 |
162.25 |
316 |
126.25 |
36.00 |
Test item: 1:40 dilution |
100.6 |
103.1 |
209.50 |
316 |
165.50 |
44.00 |
Test item: 1:80 dilution |
97.8 |
98.0 |
187.75 |
316 |
132.50 |
55.25 |
Test item: 1:160 dilution |
103.4 |
113.2 |
170.75 |
316 |
131.75 |
39.00 |
Test item: 1:320 dilution |
93.4 |
98.4 |
217.25 |
316 |
170.50 |
46.75 |
Test item: 1:640 dilution |
98.8 |
102.0 |
184.00 |
316 |
148.50 |
35.50 |
Test item: 1:1280 dilution |
102.6 |
100.3 |
176.50 |
316 |
134.00 |
42.50 |
Test item suspension 5 mg/mL |
99.4 |
42.1 |
193.25 |
316 |
130.50 |
62.75 |
CPA = cyclophosphamide; *p < 0.001
Table 4. Experiment II - 24 h exposure - Without Metabolic Activation (mean of two replicates of duplicate cultures)
Cell treatment |
Mean Relative Cloning Efficiency [%] |
Mean Relative Total Growth [%] |
Mean Mutants per 1E+06 cells |
Threshold |
Mutant small colonies per 1E+06 cells |
Mutant large colonies per 1E+06 cells |
Negative control |
100.0 |
100.0 |
164.75 |
- |
102.00 |
62.75 |
Vehicle control (RPMI) |
100.0 |
100.0 |
170.25 |
- |
103.75 |
66.50 |
Positive control (MMS) |
69.9 |
46.8 |
449.75* |
296 |
402.75 |
47.00 |
Test item: 1:10 dilution |
119.2 |
58.7 |
154.25 |
296 |
117.00 |
37.25 |
Test item: 1:20 dilution |
108.9 |
64.3 |
170.00 |
296 |
139.25 |
30.75 |
Test item: 1:40 dilution |
114.2 |
100.5 |
169.25 |
296 |
131.25 |
38.00 |
Test item: 1:80 dilution |
114.9 |
84.5 |
181.50 |
296 |
134.50 |
47.00 |
Test item: 1:160 dilution |
99.9 |
88.5 |
162.00 |
296 |
115.00 |
47.00 |
Test item: 1:320 dilution |
123.3 |
90.2 |
151.75 |
296 |
117.00 |
34.75 |
Test item: 1:640 dilution |
99.1 |
81.8 |
183.50 |
296 |
108.50 |
75.00 |
Test item: 1:1280 dilution |
99.7 |
90.6 |
160.50 |
296 |
84.75 |
75.75 |
Test item suspension 5 mg/mL |
35.8 |
1.8 |
253.50 |
296 |
88.25 |
165.25 |
MMS = methyl methanesulphonate; *p < 0.001
RESULTS MUTAGENICITY
In experiment I in the approach with metabolic activation the test item suspension 5 mg/mL and the final test dilution 1:640 exceeded the threshold of more than 126 colonies per 106 cells of the corresponding solvent control slightly. As the test item suspension 5 mg/mL showed a cytotoxic effect, this mutation frequency is not regarded as evaluable. The mutation frequency of the final test item dilution 1:640 showed a value of 260.75 which is barely above the threshold of 256 colonies per 106 cells. As none of the other tested dilutions exceeded the threshold level and no dose-response was present, the finding is regarded as incidental and not substance-related.
In the approach without metabolic activation none of the tested dilutions or the test item suspension 5 mg/mL exceeded the threshold value.
In experiment II, no substantial and dose-dependent increase in mutant colony numbers was observed either in the presence or absence of S9. The potential mutagenicity of the final test dilution 1:640 from experiment I could not be verified. Here the mutation frequency of 184 colonies per 106 cells did not exceed the threshold of 316 colonies per 106 cells. This fact supports the assumption, that the mutagenicity observed in experiment I represents an incidental finding and not a treatment-related effect.
EVALUATION OF LARGE AND SMALL COLONIES
In both approaches of experiment II, a relevant shift of the ratio of small versus large colonies was observed. In the approach without metabolic activation this fact could be explained by the enlarged incubation time of 24 hours. However, the high number of small colonies in the approach without metabolic activation is not consistent with the results of the same approach in experiment I. Nevertheless, as none of the mutation frequencies reached or exceeded the threshold, the biological relevance of the effect on colony growth remained unclear and the result is not considered as sign of mutagenicity.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
In vivo:
Negative Micronucleus Assay (OECD 474) in mice
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- (adopted July 1997)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- mammalian erythrocyte micronucleus test
- Species:
- mouse
- Strain:
- ICR
- Details on species / strain selection:
- A total of 36 male and 8 female Hsd:ICR(CD-1) mice were ordered and
supplied by Charles River Italy S.p.A., Calco (Lecco), Italy. All animals were
received in good health conditions. They were 5 to 6 weeks old at the time
of treatment. Females were only used for the preliminary toxicity test that
was carried out in order to select the dose levels to be used for the Main Assay. - Sex:
- male
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 and 2000 mg/kg bw were tested
- Clinical signs of toxicity in test animals: all animals survived, no abnormalities detected
- Evidence of cytotoxicity in tissue analyzed: no evidence of bone marrow toxicity
- Harvest times: 24 hours after treatment
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay):
- Appropriateness of dose levels and route:
- Statistical evaluation: - Conclusions:
- It is concluded that, under the reported experimental conditions, aluminium magnesium vanadium oxide, administered by oral gavage, does not induce cytogenetic damage and/or disruption of the mitotic apparatus in mouse bone marrow, as measured by the induction of micronuclei in polychromatic erythrocytes.
Reference
Table 1: Summary
Treatment |
Dose level mg/kg bw/day |
Incidence of micronucleated PCEs |
PCEs/(PCEs+NCEs) % over the mean control value |
|||
|
|
mean |
SE |
Range |
|
|
|
|
|
|
min |
max |
|
Vehicle |
0 |
1.5 |
0.2 |
1.0 |
2.0 |
100 |
Test item |
500 |
1.8 |
0.4 |
1.0 |
3.0 |
117 |
Test item |
1000 |
0.8 |
0.2 |
0.0 |
1.0 |
100 |
Test item |
2000 |
1.4 |
0.3 |
0.5 |
2.5 |
105 |
Mitomycin C |
3.00 |
10.8*** |
1.6 |
6.0 |
16.0 |
94 |
PCE = polychromatic erythrocytes NCE = normochromatic erythrocytes *** = incidence significantly greater than control value at p < 0.001 |
||||||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In vitro
- Gene mutation in bacteria
According to OECD guideline 471, the mutagenic potential of aluminium magnesium vanadium oxide was investigated in bacteria using the Ames test (2012). The assay was conducted under GLP conditions in two independent experiments both performed in the absence and in the presence of liver microsomal activation system (S9 mix). In the first experiment, the direct plate incorporation procedure was used to study effects in the Salmonella typhimurium strains TA 97a, TA 98, TA 100, TA 102 and TA 1535 at test substance concentrations ranging from 51 to 5002 µg/plate for a period of 48 h. In the second experiment, concentrations ranging from 315 to 5001 µg/plate were analysed using the same strains, but with modification of the study design (with pre-incubation period of 20 min). No signs of cytotoxicity were observed in both experiments. The number of revertants was not increased at any concentrations tested. The positive and solvent controls included showed the expected results in each experiment. Under the experimental conditions reported, aluminium magnesium vanadium oxide did not induce mutations in the bacterial mutation assay in the absence and presence of metabolic activation in the selected strains of S. typhimurium (TA 97a, TA 98, TA 100, TA 102 and TA 1535).
- Chromosome aberrations
An in vitro mammalian chromosome aberration test with aluminium magnesium vanadium oxide was performed in cultured human lymphocytes in accordance with OECD guideline 473 and under the conditions of GLP (2013). Due to the poor solubility of the test substance, cells were treated with dilutions of the supernatants resulting from centrifugation of test substance suspension (50 mg/mL) after an extraction period of 24 h (experiment I and II) and 96 h (experiment III), respectively. All experiments were performed in the presence and absence of a metabolic activation system (S9 mix). In the first experiment, short-term treatment with 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280 dilution of the resulting test item solution after 24-h extraction for 4 h followed by an 18 h-recovery period was performed with and without S9 mix. Since no clear cytotoxicity indicated by reduced mitotic indices was observed up to the highest concentration, the same dilutions of the resulting test item solution after 24-h extraction were chosen for treatment of cells in experiment II. In the presence of S9 mix, cells were treated with the respective dilutions for 4 h (+ 18 h recovery period). In the absence of S9 mix, cells were continuously treated for a period of 22 h without recovery period. A third (repeat) experiment was conducted with 1:10 dilution of the resulting test item solution after 96-h extraction to test whether a prolonged extraction period may enhance the solubility of the test substance in medium. Since no dose-response relationship was observed for cytotoxic effects in any experiment performed, concentrations used for evaluation of chromosome aberrations were selected based on the lowest non-cytotoxic dilution tested. Thus, concentrations chosen for chromosome analysis in experiment I and II were the 1:10, 1:20 and 1:40 dilutions of the resulting test item solutions after 24-h extraction. In third experiment, only the 1:10 dilution of the resulting test item solution after 96-h extraction was used as concentration for chromosomal analysis. In all experiments, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed after treatment with the supernatants of the test substance solution in the absence and presence of S9 mix. Furthermore, no increase in the frequencies of polyploid metaphases was found after treatment compared to controls. The positive control substances yielded the expected results. Under the conditions of this chromosome aberration assay, the test substance did not induce structural chromosomal aberrations and polyploidy in cultured human lymphocytes.
- Gene mutation in mammalian cells
The potential mutagenicity of aluminium magnesium vanadium oxide on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line was assessed in a GLP-compliant study conducted according to OECD guideline 476 (2013). Due to the poor solubility of the test substance and based on a preliminary cytotoxicity test, cells were treated with 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280 dilutions of the supernatants resulting from centrifugation of test substance suspension (50 mg/mL) after an extraction period of 96 h as well as a 1:10 dilution of the corresponding test substance suspension after 96-h extraction without centrifugation (= test item suspension 5 mg/mL). All experiments were performed in the presence and absence of a metabolic activation system (S9 mix). In the first experiment, cells were exposed to the test material for 4 h in the presence and absence of S9 mix. In the second experiment, cells were treated for 4 h with metabolic activation and for 24 h without metabolic activation. In both experiments, significant cytotoxicity was noted in the test item suspension 5 mg/mL, resulting in a relative suspension growth (RSG) < 50% compared to controls with and without S9 mix. In contrast, the dilutions of the resulting test item solution after centrifugation of the 96-h extract did not show significant reduction in RSG compared to control, except for treatment with the 1:10 and 1:20-dilution of the resulting test item solution in the absence of S9 mix, which caused a reduction in RSG values of 49.3% and 59.1% compared to controls, respectively. No cytotoxicity was noted at any other dilution of the resulting test item solution. In the first experiment, the number of mutants was slightly increased over the threshold set for the controls after treatment with the test item suspension 5 mg/mL in the presence of S9 mix. However, due to the strong cytotoxicity of the test item suspension 5 mg/mL and the presence of precipitates, the mutation frequencies of this treatment were counted, but excluded from evaluation. Furthermore, in the second experiment the threshold for the indication of a significant increase in mutation number over controls was not exceeded, indicating that the increased number of mutants observed at the test item suspension 5 mg/mL in the first experiment was of no toxicological relevance. Despite that, a very slight mutagenic effect was also observed at the 1:640-dilution of the resulting test item solution in experiment I in the presence of metabolic activation. As the increase in the number of mutants was only minimal compared to controls and the result could not be verified in the second experiment, it was not considered to be toxicologically relevant. No further increases in mutant frequency were observed at any other dilution in both experiments. Precipitation was observed in all experiments after treatment with the test item suspension 5 mg/mL. The vehicle controls had acceptable mutant frequency values that were within the historical range for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequencies compared to controls. Based on these results, the test material was considered to be non-mutagenic to L5178Y cells under the conditions of this experiment.
In vivo:
The ability of aluminium magnesium vanadium oxide to cause chromosomal damage in vivo was investigated in a micronucleous test in mice according to OECD 474 (2015). The route of administration of the test item was oral gavage. The selected vehicle was corn oil. Male Hsd:ICR (CD-1) mice were dosed twice at 24 hour interval with the vehicle, aluminium magensium vanadium oxide at 2000, 1000 and 500 mg/kg bw/day. In addition, animals were dosed once with the positive control item Mitomycin-C. Each group consisted of five animals. All animals were sacrificed 24 hours after the last dosing. Following the treatment with the test item, no clinical signs nor remarkable bodyweight loss were observed.
Bone-marrow smear slides were made from the femurs of each animal and stained with May-Gruenwald and Giemsa solution. Two thousand polychromatic erythrocytes per animal were examined for the presence of micronuclei. No statistically significant increase in the incidence of micronucleated PCEs over the control value was observed at any dose level. No inhibitory effect on erythropoietic cell division was observed at any dose level. After treatment with the positive control Mitomycin-C, statistically significant increases in the incidence of micronucleated PCEs over the control values were observed, indicating the correct functioning of the test system.
It is concluded that, under the reported experimental conditions, aluminium magnesium vanadium oxide, administered by oral gavage, does not induce cytogenetic damage and/or disruption of the mitotic apparatus in mouse bone marrow, as measured by the induction of micronuclei in polychromatic erythrocytes.
Justification for classification or non-classification
The available data on genetic toxicity of the aluminium magnesium vanadium oxide are conclusive but not sufficient for classification according to the CLP (1272/2008/EC).
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