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EC number: 235-101-0 | CAS number: 12068-53-0
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The following studies on genetic toxicity are available for a
weight-of-evidence approach:
Ames Test: negative; CAS 12068-53-0 (Andres, 2012)
Chromosome aberration test: negative; CAS 12068-53-0 (Andres, 2013)
Mammalian cell gene mutation test: negative; CAS 12068-53-0 (Andres,
2013)
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- 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)
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- 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
- Species / strain / cell type:
- S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw)
- Test concentrations with justification for top dose:
- First experiment: 55, 150, 499, 1508 and 5005 µg/plate with and without metabolic activation
Second experiment: 324, 625, 1247, 2527 and 5003 µg/plate with and without metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- water and DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4-Nitro-1,2-phenylene diamine (20 µg in DMSO, -S9, TA 97a, TA 98 and TA 102); sodium azide (1 µg in water; -S9; TA 100 and TA 1535); 2-Amino-anthracene (1 µg in DMSO, +S9; TA 97a, TA 100, TA 102 and TA 1535); Benzo-a-pyrene (20 µg in DMSO; + S9, TA 98)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
First experiment: in agar (plate incorporation)
Second experiment: pre-incubation
DURATION
- Preincubation period: 20 min (second experiment)
- Exposure duration: 48 h (first and second experiment)
NUMBER OF REPLICATIONS: 4 replications each in 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: other: Cytotoxicity was evaluated at test concentrations of 5007 and 1504 µg/plate. Per tester strain, 4 plates were exposed for 48 h in a plate incorporation test. The quotient of the number of revertant colonies in the titre plates divided by the toxicity test plates was evaluated to determine cytoxicity. If the quotient is below 2, the test item is considered as non-cytotoxic. - Evaluation criteria:
- A test item is considered to have mutagenic potential, if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain is observed. A concentration-related increase over the range tested is also taken as a sign of mutagenicity.
Validity criteria
Negative control plates must demonstrate the characteristic mean number of spontaneous revertants compared to historical control data. - Statistics:
- Mean values and standard errors were calculated from the examined parameters.
- Species / strain:
- S. typhimurium, other: TA 97a, TA 98, TA 100, TA 102 and TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxicity was tested at 5007 µg/plate and 1504 µg/plate with 4 plates per strain on maximal soft agar. The quotient titre/tox (number of colonies in medium/number of colonies in test solutions) was calculated below 2 (see table 3). Thus, the test item was considered not to induce cytotoxicity. - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- 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, Kaiser-Friedrich-Straße 7, 55116 Mainz, Germany
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable
- Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- - Type and identity of media:
RPMI 1640 medium supplemented with
- 15% fetal calf serum (FCS)
- 1% penicillin/streptomycin (per mL: 10000 Units penicillin /10 mg streptomycin)
- 4.8 µg/mL phytohaemagglutinin solution in H2O
During 4 h exposure to the test substance, RPMI medium was used without FCS supplementation. - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- final test dilutions: 1:10, 1:20, 1:40 (For further details, please refer to "any other information on materials and methods incl. tables")
- Vehicle / solvent:
- - Vehicle/solvent used:
RPMI 1640 medium without FCS supplementation (for the test item and the positive control EMS)
0.9% NaCl (for the positive control CPA) - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- RPMI 1640 medium without FCS supplementation
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Remarks:
- + S9 mix: Cyclophosphamide monohydrate (CPA) in 0.9% NaCl, final concentrations 35 µg/mL; - S9 mix: ethylmethanesulphonate (EMS) in RPMI medium without FCS supplementation, final concentrations 362 and 724 µg/mL
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 h and 24 h
- Expression time (cells in growth medium): 24 h (including exposure time)
- Fixation time: minimum 30 min (cells were fixated with a mixture of methanol and glacial acetic acid (3:1))
METAPHASE-ARRESTING SUBSTANCE: 3 h before harvesting, colcemid was added to the cultures (final concentration: 0.1 µg/mL)
STAIN: 10 % solution of Giemsa
NUMBER OF REPLICATIONS: duplicates; two independent experiments
NUMBER OF CELLS EVALUATED: 100 well spread metaphases per culture
DETERMINATION OF CYTOTOXICITY
- Method: The mitotic index (number of metaphases per 1000 cell nuclei) was determined. - Evaluation criteria:
- A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is not above 5.0 % aberrant cells, excluding gaps.
- no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified 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 is observed. - Species / strain:
- lymphocytes: human
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- other: At the highest test dilution (1:10) without S9-mix, a reduction in mitotic index was observed in both experiments. (Exp. I: 65.4%; Exp. II: 52.1%)
- 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 solubility of the test substance is stated as <1 ppm.
COMPARISON WITH HISTORICAL CONTROL DATA: The solvent controls and positive controls were in the range of historical data of the test facility.
ADDITIONAL INFORMATION ON CYTOTOXICITY: At the highest test dilution (1:10) tested in the absence of metabolic activation, a reduction in mitotic index was observed in both experiments. Experiment I (exposure duration 4 h): 65.4%; Experiment II (expoure duration 24 h): 52.1% - Conclusions:
- Interpretation of results: negative
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 11 Mar - 23 Mar 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, Kaiser-Friedrich-Straße 7, 55116 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 (supplemented with hypoxanthine, aminopterin, thymidine and glycine);
growth medium: RPMI 1640 complete culture medium containing 10% horse serum (HS), 1% Pen./Strep. (per mL: 10000 Units Pen./ 10 mg Strep.) and 2% sodium pyruvate;
selection medium: RPMI 1640 culture base medium containing 15% horse serum, 1% Pen./Strep. (per mL: 10000 Units Pen./ 10 mg Strep.), 2% sodium pyruvate and 5 µg/mL trifluorothymidine;
treatment medium: RPMI 1640 culture base medium without 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:
- cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254 (500 mg/kg bw intrapertinoneally)
- Test concentrations with justification for top dose:
- final test dilutions:
pre-experiment, experiment I & II: test item suspension: 5 mg/mL (nominal concentration); 1:10, 1:20, 1:40, 1:80, 1:160, 1: 320, 1: 640 and 1: 1280 dilutions from shaked and centrifuged test item solution (For further details, please refer to "Any other information on materials and methods incl. tables") - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used:
Solvent controls:
RPMI 1640 medium without HS supplementation
0.9% NaCl (for the positive control CPA)
Vehicle control:
RPMI 1640 medium supplemented with 5% HS - Untreated negative controls:
- yes
- Remarks:
- (RPMI 1640 medium supplemented with 5% HS)
- Negative solvent / vehicle controls:
- yes
- Remarks:
- RPMI 1640 medium without HS supplementation (for the test item and the positive control MMS) and 0.9% NaCl (for the positive control CPA)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Remarks:
- + S9 mix: cyclophosphamide monohydrate (CPA) in 0.9% NaCl, final concentrations 4.5 µg/mL; - S9 mix: methylmethanesulfonate (MMS) in RPMI without HS, final concentrations 19.5 µg/mL (experiment I & II (not evaluated)) and 9.75 µg/mL (exp. II)
- 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): 48 h (incl. exposure period), 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 days (for viability determination) or 11- 12 days (for selection).
- Selection time (if incubation with a selection agent): 11 - 12 days
SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine
NUMBER OF REPLICATIONS: two replicates 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 slow colony growth leading to the formation of small colonies is often correlated to extensive DNA damage - Evaluation criteria:
- The test item is considered as mutagenic if:
- the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 1E+06 cells above the corresponding solvent control.
- the relative increase of the mutation frequency shows a dose-relationship.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures. 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.
Acceptability:
The assay is considered acceptable if the following criteria were fulfilled (International Workshop on Genotoxicity Testing (IWGT)) (Moore et al., Environ Mol Mutagen 47(1): 1-5, 2007):
- all plates, from either the viability or the mutagenicity portion of the experiment are analysable.
- the total suspension growth (TSG) of the solvent control corresponds to an increase factor of at least 4 or lies in the range of the historical data
- the solvent control mutant frequency is in the range of 50 – 170 per 1E+06 cells or in the range of the historical data
- the positive controls (MMS and CPA) should yield an absolute increase in total mutant frequency of at least 300 per 1E+06 cells. At least 40% of the induced mutation frequency (IMF) should be reflected in the small colony MF. Alternatively, the positive controls should induce at least 150 small colonies.
- the upper limit of cytotoxicity observed in the positive control cultures should be greater than 10% of the concurrent selective control group.
- the highest concentration of the test item should be 0.01 M or 5 mg/mL (resp. 5 µL/mL), unless limited by toxicity or solubility of the test item. If toxicity occurs, the highest evaluated concentration should result in approximately 10 - 20% relative total growth, if possible. - Statistics:
- Mean values were calculated from the examined parameters. Statistical analyses were performed by non-parametric χ2 test (positive controls) or linear regression (least squares, test item dilutions).
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- -S9: test item suspension (5 mg/mL nominal concentration)
- 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. To enable microscopic analyses, the test suspension was centrifuged prior to the preparation of the geometric series of dilutions.
COMPARISON WITH HISTORICAL CONTROL DATA: The solvent controls and positive controls were in the range of historical data of the test facility. - Conclusions:
- Interpretation of results: negative
Referenceopen allclose all
Acceptability of the study
Nearly all spontaneous revertants and all positive control values were within the range of historical data. Difference of revertants lying outside the range in the first experiment are marginal. In detail, the mean number of revertants for TA 97a in the water control plates counted 97 ± 3.4 which is slightly below the historical range reaching from 99 - 159 ± 11.
Additionally, the mean number of revertants for TA 100 in DMSO control plates counted 88 ± 4.6 which is slightly below the historical range from 89 - 155 ± 14.Therefore, the study was considered as valid.
Table 1. Experiment 1: Test results for D-PrisM (plate incorporation)
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate ± standard deviation |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA 1535 |
TA 97a |
TA 98 |
TA 102 |
||
– |
DMSO |
88± 4.6 |
16 ± 1.3 |
108± 2.5 |
10± 2.4 |
171± 8.8 |
– |
H2O |
105± 5.3 |
22 ± 4.1 |
97± 3.4 |
11± 2.7 |
151± 24.4 |
– |
55 |
94± 18 |
15± 3 |
105± 10 |
14± 1 |
131± 27 |
– |
150 |
96± 4 |
16± 2 |
110± 5 |
15± 2 |
125± 19 |
– |
499 |
111± 4 |
17± 3 |
112± 5 |
10± 1 |
127± 22 |
– |
1508 |
94± 15 |
15 ± 2 |
111± 6 |
10± 2 |
133± 17 |
– |
5005 |
104± 7 |
14 ± 2 |
106± 9 |
13± 3 |
140± 8 |
– |
Positive controls |
SA |
SA |
4NPD |
4NPD |
4NPD |
Mean No. of colonies/plate ± SD |
495 ± 60 |
201 ± 18 |
456 ± 18 |
240 ± 19 |
604±99 |
|
+ RLI (4%) |
DMSO |
112± 8.8 |
14± 1.9 |
115± 1.7 |
14± 3.2 |
171± 4.1 |
+ RLI (4%) |
H2O |
128 ± 18.7 |
15 ± 2.1 |
108 ± 8.0 |
13 ± 6.2 |
176 ± 16.8 |
+ RLI(4%) |
55 |
105 ± 15 |
10 ± 3 |
99 ± 6 |
15 ± 4 |
145 ± 17 |
+ RLI(4%) |
150 |
105 ± 10 |
11 ± 2 |
111 ± 5 |
14 ± 2 |
128 ± 10 |
+ RLI(4%) |
499 |
98 ± 8 |
11 ± 1 |
110 ± 5 |
14 ± 4 |
126 ± 23 |
+ RLI(4%) |
1508 |
111 ± 4 |
14 ± 3 |
114 ± 5 |
12 ± 3 |
150 ± 5 |
+ RLI(4%) |
5005 |
98 ± 16 |
16 ± 2 |
108 ± 6 |
12 ± 2 |
132 ± 18 |
+ RLI(4%) |
Positive controls |
2AA |
2AA |
2AA |
BaP |
2AA |
Mean No. of colonies/plate ± SD |
446 ± 63 |
201 ± 11 |
499 ± 22 |
202 ± 13 |
594 ± 88 |
|
SA = Sodium azide
4NPD = 4-Nitro-o-phenylenediamine
2AA = 2-Amino-anthracene
BaP = Benzo-a-pyrene
SD = standard deviation
RLI = induced male Sprague Dawley rat liver S9
Table 2. Experiment 2: Test results for D-PrisM (preincubation)
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate ± standard deviation |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA 1535 |
TA 97a |
TA 98 |
TA 102 |
||
– |
DMSO |
92± 5.1 |
12± 1.7 |
102± 4.3 |
15± 1.7 |
147± 8.3 |
– |
H2O |
100 ± 7.5 |
14± 2.9 |
118± 2.2 |
16± 2.8 |
178± 20.3 |
– |
324 |
95± 6 |
13± 2 |
117± 6 |
16± 3 |
168± 17 |
– |
625 |
81± 14 |
14± 2 |
113± 2 |
14± 1 |
180± 24 |
– |
1247 |
76± 11 |
14± 2 |
102± 5 |
13± 1 |
161± 3 |
– |
2527 |
102± 17 |
16± 2 |
116± 3 |
15± 2 |
171± 14 |
– |
5003 |
100± 16 |
16±2 |
114± 4 |
14± 3 |
147± 4 |
– |
Positive controls |
SA |
SA |
4NPD |
4NPD |
4NPD |
Mean No. of colonies/plate ± SD |
513± 38 |
230± 25 |
475± 68 |
221± 15 |
635 ± 51 |
|
+ RLI (4%) |
DMSO |
88 ± 11.8 |
11 ± 2.2 |
110 ± 2.2 |
14 ± 2.9 |
159 ± 8.7 |
+ RLI (4%) |
H2O |
86± 8.5 |
13± 2.2 |
112± 5.5 |
15± 3.9 |
155± 8.7 |
+ RLI(4%) |
324 |
83± 5 |
12± 4 |
110± 7 |
13± 1 |
154± 10 |
+ RLI(4%) |
625 |
79± 5 |
12± 2 |
111± 2 |
14± 1 |
191± 9 |
+ RLI(4%) |
1247 |
89± 17 |
9± 3 |
114± 5 |
13± 3 |
175± 5 |
+ RLI(4%) |
2527 |
91± 11 |
13± 1 |
116± 7 |
13± 3 |
147± 3 |
+ RLI(4%) |
5003 |
86± 6 |
11± 3 |
107± 4 |
13± 3 |
153± 16 |
+ RLI(4%) |
Positive controls |
2AA |
2AA |
2AA |
BaP |
2AA |
Mean No. of colonies/plate ± SD |
472 ± 144 |
217 ± 15 |
554 ± 23 |
242 ± 22 |
612 ± 72 |
|
SA = Sodium azide
4NPD = 4-Nitro-o-phenylenediamine
2AA = 2-Amino-anthracene
BaP = Benzo-a-pyrene
SD = standard deviation
RLI = induced male Sprague Dawley rat liver S9
Table 3: Toxicity control
Titre values |
TA 97a |
TA 98 |
TA 100 |
TA 102 |
TA 1535 |
||||||
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
- S9 |
+ S9 |
||
280± 8 |
316± 8 |
208± 37 |
213± 27 |
238± 11 |
271± 13 |
273± 58 |
220± 14 |
302± 99.0 |
277± 63.6 |
||
Toxicity control |
1504 µg/mL |
274± 17.0 |
267± 49.5 |
258± 5.7 |
275± 7.1 |
317± 35.4 |
276± 49.5 |
277± 52.3 |
336± 19.8 |
275± 4.2 |
247± 21.2 |
5007 µg/mL |
237± 7.1 |
248± 2.8 |
186± 5.7 |
209± 4.2 |
239± 4.2 |
250± 5.7 |
334± 11.3 |
358± 5.7 |
237± 7.1 |
223± 15.6 |
|
Titre/Tox |
1504 µg/mL |
1.02 |
0.59 |
0.81 |
0.39 |
0.75 |
0.51 |
0.99 |
0.33 |
1.10 |
0.56 |
5007 µg/mL |
1.18 |
0.64 |
1.12 |
0.51 |
1.0 |
0.54 |
0.82 |
0.31 |
1.27 |
0.62 |
|
Table 3: Results Cytotoxicity Experiment I
Concentration |
Experiment I: exposure period 4 hours without S9 mix Mitotic indices in % of solvent control |
Experiment I: exposure period 4 hours with S9 mix Mitotic indices in % of solvent control |
Solvent control Medium without FCS |
100.0 % |
100 % |
Solvent control NaCl 0.9% |
- |
100 % |
Positive control CPA 35 µg/mL |
- |
20.6 % |
Positive control EMS 362 µg/mL |
39.4 % |
- |
Positive control EMS 724 µg/mL |
34.6 % |
- |
test item: 1:10 |
65.4 % |
103.9 % |
test item: 1:20 |
98.1 % |
84.3 % |
test item: 1:40 |
129.8 % |
100.0 % |
test item: 1:80 |
145.2 % |
98.0 % |
test item: 1:160 |
119.2 % |
84.3 % |
test item: 1:320 |
106.7 % |
107.8 % |
test item: 1:640 |
129.8 % |
137.3 % |
test item: 1:1280 |
144.2 % |
160.8 % |
Table 4: Results Genotoxicity Experiment I
Concentration |
Aberrant cells in % |
||
Inclusive gaps* |
Exclusive gaps* |
with exchanges |
|
Experiment I: exposure period 4 hours without S9 mix |
|||
Solvent control medium without FCS |
2.5 |
0 |
0 |
Positive control EMS 724 µg/mL |
41 |
37.0# |
12.0 |
test item: 1:10 |
4.0 |
1.5 |
0 |
test item: 1:20 |
1.5 |
0.5 |
0 |
test item: 1:40 |
3.5 |
1.0 |
0 |
Experiment I: exposure period 4 hours with S9 mix |
|||
Solvent control medium without FCS |
0.5 |
0 |
0 |
Solvent control for the positive control NaCl 0.9 % |
2.0 |
0 |
0 |
Positive control CPA 35 µg/mL |
54 |
48.5# |
13 |
test item: 1:10 |
6.5 |
1 |
0 |
test item: 1:20 |
5.0 |
1 |
0 |
test item: 1:40 |
4.0 |
1.5 |
0 |
*Inclusive cells carrying exchanges
#Aberration frequency statistically significant higher than corresponding control values (p < 0.05)
Table 5: Results Cytotoxicity Experiment II
Concentration |
Experiment II: exposure period 24 hours without S9 mix Mitotic indices in % of solvent control |
Experiment II: exposure period 4 hours with S9 mix Mitotic indices in % of solvent control |
Solvent control Medium without FCS |
100.0 % |
100 % |
Solvent control NaCl 0.9% |
- |
100 % |
Positive control CPA 35µg/mL |
- |
23.1 % |
Positive control EMS 362 µg/mL |
16.1 % |
- |
Positive control EMS 724 µg/mL |
18.0 % |
- |
test item: 1:10 |
52.1 % |
78.6 % |
test item: 1:20 |
81.2 % |
90.6 % |
test item: 1:40 |
91.2 % |
99.1 % |
test item: 1:80 |
84.7 % |
112.8 % |
test item: 1:160 |
86.2 % |
110.3 % |
test item: 1:320 |
98.1 % |
106.8 % |
test item: 1:640 |
86.2 % |
125.6 % |
test item: 1:1280 |
82.8 % |
120.5 % |
Table 6: Results Genotoxicity Experiment II
Concentration |
Aberrant cells in % |
||
Inclusive gaps* |
Exclusive gaps* |
with exchanges |
|
Experiment II: exposure period 24 hours without S9 mix |
|||
Solvent control culture medium without FCS |
1.0 |
0 |
0 |
Positive control EMS 724 µg/mL |
44.0 |
36.0# |
15.5 |
test item: 1:10 |
0.5 |
0 |
0 |
test item: 1:20 |
2.0 |
1.0 |
0 |
test item: 1:40 |
2.5 |
1.5 |
0 |
Experiment II: exposure period 4 hours with S9 mix |
|||
Solvent control culture medium without FCS |
1.5 |
1 |
0 |
Solvent control for the positive control NaCl 0.9 % |
1.5 |
0 |
0 |
Positive control CPA 35µg/mL |
47.0 |
40.5# |
6.0 |
test item: 1:10 |
4.5 |
2.5 |
0 |
test item: 1:20 |
2.0 |
0.5 |
0 |
test item: 1:40 |
2.0 |
0 |
0 |
*Inclusive cells carrying exchanges
#Aberration frequency statistically significant higher than corresponding control values (p < 0.05)
Table 1: Experiment I - 4 h exposure - with metabolic activation (mean of two replicates) |
||||||
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 |
Vehicle control1 |
100 |
100 |
158.75 |
- |
52.0 |
106.75 |
Solvent control serum-free medium RPMI |
100 |
100 |
167.00 |
- |
55.0 |
112.0 |
Solvent control (NaCl 0.9%) |
100 |
100 |
160.75 |
- |
54.25 |
106.5 |
Positive control (CPA, 4.5 µg/mL) |
33.3 |
30.0 |
607.50* |
287 |
427.0 |
180.5 |
Test item: 1:10 dilution |
109.3 |
135.0 |
168.50 |
293 |
59.0 |
109.5 |
Test item: 1:20 dilution |
84.1 |
100.4 |
208.25 |
293 |
56.0 |
152.25 |
Test item: 1:40 dilution |
94.6 |
107.7 |
194.50 |
293 |
59.75 |
134.75 |
Test item: 1:80 dilution |
117.5 |
122.9 |
153.00 |
293 |
56.0 |
97.0 |
Test item: 1:160 dilution |
124.6 |
126.8 |
155.75 |
293 |
52.25 |
103.5 |
Test item: 1:320 dilution |
96.7 |
105.9 |
177.00 |
293 |
70.0 |
107.0 |
Test item: 1:640 dilution |
82.1 |
95.3 |
216.50 |
293 |
75.25 |
141.25 |
Test item: 1:1280 dilution |
92.5 |
92.9 |
213.25 |
293 |
68.5 |
144.75 |
Test item suspension (5 mg/mL) |
74.4 |
71.0 |
253.00 |
293 |
97.0 |
156.0 |
|
|
|
|
|
|
|
1: The vehicle control contains only RPMI 1640 medium with 5 % HS. |
|
|
||||
CPA = cyclophosphamide; *p < 0.001 |
|
|
|
|
||
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Table 2: Experiment I - 4 h exposure - without metabolic activation (mean of two replicates) |
|
|||||
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 |
Vehicle control1 |
100 |
100 |
225.00 |
- |
64.0 |
161.0 |
Solvent control serum-free medium RPMI |
100 |
100 |
186.75 |
- |
63.5 |
123.25 |
Positive control (MMS, 19.5 µg/mL) |
41.2 |
40.5 |
473.00* |
313 |
270.25 |
202.75 |
Test item: 1:10 dilution |
77.3 |
75.3 |
253.25 |
313 |
90.25 |
163.0 |
Test item: 1:20 dilution |
94.3 |
103.6 |
232.75 |
313 |
82.5 |
150.25 |
Test item: 1:40 dilution |
95.1 |
94.1 |
223.25 |
313 |
75.75 |
147.5 |
Test item: 1:80 dilution |
94.0 |
81.3 |
206.75 |
313 |
78.25 |
128.5 |
Test item: 1:160 dilution |
109.1 |
105.9 |
198.50 |
313 |
72.75 |
125.75 |
Test item: 1:320 dilution |
90.6 |
102.4 |
245.00 |
313 |
73.75 |
171.25 |
Test item: 1:640 dilution |
90.3 |
90.4 |
207.75 |
313 |
67.5 |
140.25 |
Test item: 1:1280 dilution |
72.7 |
86.2 |
295.50 |
313 |
87.0 |
208.5 |
Test item suspension (5 mg/mL) |
68 |
48.7 |
279.25 |
313 |
105.5 |
173.75 |
1: The vehicle control contains only RPMI 1640 medium with 5 % HS. |
||||||
MMS = methylmethanesulfonate; *p < 0.001 |
||||||
Table 3: Experiment II - 4 h exposure - with metabolic activation (mean of two replicates) |
||||||
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 |
Vehicle control1 |
100 |
100 |
191.75 |
- |
87.5 |
104.25 |
Solvent control serum-free medium RPMI |
100 |
100 |
166.25 |
- |
74.0 |
92.25 |
Solvent control (NaCl 0.9%) |
100 |
100 |
178.50 |
- |
81.5 |
97.0 |
Positive control (CPA, 4.5 µg/mL) |
51.2 |
41.1 |
507.00* |
305 |
367.0 |
140.0 |
Test item: 1:10 dilution |
95.3 |
100.2 |
197.75 |
292 |
84.0 |
113.75 |
Test item: 1:20 dilution |
109.1 |
116.1 |
148.00 |
292 |
74.0 |
74.0 |
Test item: 1:40 dilution |
99.1 |
104.3 |
195.00 |
292 |
85.5 |
109.5 |
Test item: 1:80 dilution |
120.5 |
120.8 |
169.50 |
292 |
71.75 |
97.75 |
Test item: 1:160 dilution |
100 |
107.1 |
180.00 |
292 |
86.25 |
93.75 |
Test item: 1:320 dilution |
95.4 |
101.2 |
200.25 |
292 |
82.0 |
118.25 |
Test item: 1:640 dilution |
100.9 |
107.4 |
199.00 |
292 |
77.25 |
121.75 |
Test item: 1:1280 dilution |
113.5 |
117.3 |
194.75 |
292 |
80.0 |
114.75 |
Test item suspension (5 mg/mL) |
78.3 |
70.7 |
286.75 |
292 |
144.5 |
142.25 |
1: The vehicle control contains only RPMI 1640 medium with 5 % HS. |
||||||
CPA = cyclophosphamide; *p < 0.001 |
||||||
Table 4: Experiment II - 24 h exposure - without metabolic activation (mean of two replicates) |
||||||
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 |
Vehicle control1 |
100 |
100 |
132.75 |
- |
108.0 |
24.75 |
Solvent control serum-free medium RPMI |
100 |
100 |
144.25 |
- |
121.25 |
23.0 |
Positive control (MMS, 9.75 µg/mL) |
65.3 |
51.9 |
462.50* |
270 |
419.5 |
43.0 |
Test item: 1:10 dilution |
106.5 |
89.9 |
145.25 |
270 |
118.0 |
27.25 |
Test item: 1:20 dilution |
102.1 |
94.4 |
146.75 |
270 |
108.0 |
38.75 |
Test item: 1:40 dilution |
97.1 |
85.7 |
155.50 |
270 |
123.25 |
32.25 |
Test item: 1:80 dilution |
99.7 |
97.2 |
156.50 |
270 |
129.75 |
26.75 |
Test item: 1:160 dilution |
95.9 |
88.2 |
172.00 |
270 |
156.5 |
15.5 |
Test item: 1:320 dilution |
108.6 |
101.1 |
135.25 |
270 |
112.5 |
22.75 |
Test item: 1:640 dilution |
106.5 |
93.0 |
184.25 |
270 |
176.5 |
7.75 |
Test item: 1:1280 dilution |
100.3 |
94.1 |
138.25 |
270 |
122.25 |
16.0 |
Test item suspension (5 mg/mL) |
84.3 |
24.5 |
164.50 |
270 |
134.0 |
30.5 |
1: The vehicle control contains only RPMI 1640 medium with 5 % HS. |
||||||
MMS = methylmethanesulfonate; *p < 0.001 |
||||||
Statistical Analysis
The linear regression analysis did not reveal a significant correlation between the tested dilutions and the mutation frequency.
Results on Mutagenicity
No substantial and reproducible dose-dependent increase in mutant colony numbers was observed in both experiments. In experiment I, no relevant shift of the ratio of small versus large colonies was observed up to the maximal tested concentration. In contrast, experiment II revealed a shift of the ratio small/large colonies about 10 - or 25 -fold for the test item dilution 1:160 and 1:640, respectively. However, as the total number of mutant colonies did not exceed the threshold and no dose-dependency was present (please refer to table 4), the biological relevance of the effect on colony size remains unclear and the result is not considered as sign of mutagenicity. All positive controls except MMS in experiment I yield an absolute increase in total mutant frequency above the spontaneous background mutant frequency of at least 300 per 106cells. In experiment I, MMS did not reach that level but still induced more than 150 colonies per 106cells thereby fulfilling the acceptance criteria.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Genetic toxicity
Mutagenic properties of Zinc aluminium oxide were investigated in a bacterial reverse mutation assay, a mammalian chromosome aberration test and a mammalian cell gene mutation assay.
Gene mutations in bacteria
Mutagenic properties of Zinc aluminium oxide in bacteria were investigated according to OECD 471 with Salmonella typhimurium tester strains TA 97a, TA 98, TA 100, TA 102 and TA 1535 (Andres, 2012). Suspensions with concentrations ranging from 55 to 5000 µg/plate were tested with and without metabolic activation in a plate incorporation assay (incubation for 48 hours, experiment I). In addition, a pre-incubation test was performed with concentrations ranging from 324 – 5000 µg/plate with and without metabolic activation (pre-incubation for 20 min followed by incubation for 48 hours, experiment II). Cytotoxicity was evaluated at test concentrations of 5007 and 1504 µg/plate in a plate incorporation test. The test substance did not increase the number of revertant colonies in any tester strain either in the absence or presence of metabolic activation. No cytotoxicity was observed in any tester strain or concentration (1504 and 5007 µg/plate). Due to the insolubilty of the test item, precipitates were visible on the plates. Under the conditions of the study, zinc aluminium oxid was non-mutagenic in bacteria.
Chromosome aberrations
The clastogenic potential of Zinc aluminium oxide was assessed in a chromosome aberration test in human lymphocytes similar to OECD 473 (Andres, 2013). The test item (50 mg/mL) was suspended in RPMI 1640 medium without FCS (the water solubility of the test substance is stated as <1 ppm). The suspension was shaken for 24 h (experiment I) or 96 h (experiment II). Afterwards, the suspension was centrifuged and the supernatant was used defined as resulting test item solution. A geometric series of dilutions was prepared from the resulting test item solution, leading to final test dilutions of 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640 and 1:280. No test concentrations were given, as the concentration of the supernatant is unknown.Only at the lowest test dilution (1:10) in the absence of metabolic activation, a reduction in mitotic index was observed in both experiments (Exp.I: 65.4%; Exp. II: 52.1%).The final test dilutions of 1:10, 1:20 and 1:40 were selected for evalution of structural chromsome aberration in both experiments with and without metabolic activation. The exposure duration in experiment I was 4 h in the presence and absence of S9 mix. In experiment II, the exposure duration with S9 mix was 4 h; in the absence of S9 mix 24 h. In both experiments neither a statistically significant nor a biologically relevant increase in the number of cells containing structural chromosome aberrations was observed with and without metabolic activation. All positive controls were valid. Due to insolubilty of the test item, the reason for cytotoxicity observed at the lowest test dilution remains unclear. Under the experimental conditions of this study, the test item did not induce structural chromosome aberrations in human lymphocytes.
Gene mutations in mammalian cells
Moreover, mutagenic properties of zinc aluminium oxide were determined in mouse lymphoma L5178 cells according to OECD 476 (Andres, 2013). Equivalent to the above described chromosome aberration test, 50 mg/mL test item were suspended in serum-free RPMI 1640 medium and the suspension was shaken for 96 h prior to centrifugation. Based on the resulting supernatant, a geometric series of dilutions was prepared leading to final test dilutions of 1:10, 1:20, 1:40, 1:80, 1:160, 1:320, 1:640 and 1:1280 (no test concentrations were given, as the concentration of the supernatant is unknown). In addition, a suspension of the shaked but not centrifuged test item solution with a nominal concentration of 5 mg/mL was used in the experiment. Cells were exposed to the test solutions and suspension for 4 h with S9 supplementation in experiment I and II and for 4 or 24 h without S9 supplementation in experiment I and experiment II, respectively.
Cytotoxicity resulting in a decreased cell growth of at least 50% was observed after treatment with the test item suspension with a nominal concentration of 5 mg/mL without metabolic activation. None of the used test dilutions showed an effect on cell growth. No substantial and reproducible dose-dependent increase in mutant colony numbers was observed in both experiments. In experiment I, no relevant shift of the ratio of small versus large colonies was observed up to the maximal tested concentration. In contrast, experiment II revealed a shift of the ratio small/large colonies about 10 - or 25 -fold for the test item dilution 1:160 and 1:640 (1:160: 156.5 and 15.5, 1:640: 176.5 and 7.75 for small versus large colonies, respectively). However, as the total number of mutant colonies did not exceed the threshold level (172 and 184.25 compared to a threshold of 270 mutants per 106 cells for 1:160 and 1:640 dilution, respectively) and no dose-dependency was present, the biological relevance of the effect on colony size remains unclear and the result is not considered as sign of mutagenicity. The solvent and vehicle control data were considered as valid according to the current recommendations of the International Workshop on Genotoxicity Testing (IWGT)) (Moore et al., 2007) as the mutant frequencies were in the range of 50 – 170 colonies or within the range of historical data. Positive controls (cyclosphosphamide (CPA, 4.5 µg/mL, +S9) and methylmethanesulfonate (MMS, 19.5 or 9.75 µg/mL, -S9) were considered as valid as a significant increase in mutant colony numbers and a shift in the ratio small versus large colonies was observed.
In conclusion, zinc aluminum oxide is not considered as mutagenic in mouse lymphoma L5178Y Tk+/- cells under the experimental conditions reported.
Conclusion
Based on the available data, Zinc aluminium oxide is considered to be non-genotoxic.
Justification for classification or non-classification
The available data on genetic toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.
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