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EC number: 203-881-1 | CAS number: 111-55-7
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
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- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
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- Environmental data
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- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
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- Toxicological Summary
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
Gene mutation in bacteria (OECD 471, Ames test): S. typhimurium TA 1535, TA 1537, TA 98, and TA 100 and E. coli WP2uvrA: negative with and without metabolic activation
Cytogenicity in mammalian cells (OECD 473, Chromosome aberration test): Chinese hamster ovaries (CHO) cells: negative with and without metabolic activation
Gene mutation in mammalian cells (OECD 476, HGPRT Test): Mouse lymphoma L5178Y cells: negative with and without metabolic activation
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 22 Nov - 21 Dec 1999
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- no test substance purity specified
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- adopted 21 Jul 1997
- Deviations:
- yes
- Remarks:
- analytical purity of test substance not specified
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- 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:
- Experiment I:
Salmonella strains: 10, 33.3, 100, 333, 1000, 5000 µg/plate (with and without S9 mix)
E.coli strain: 33.3, 100, 333, 1000, 3330, 5000 µg/plate (with and without S9 mix)
Experiment II:
all strains: 33.3, 100, 333, 1000, 3330, 5000 µg/plate (with and without S9 mix) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- 2-nitrofluorene
- sodium azide
- other: 2-aminoanthracene (2-AA, all, 2.5 + 5.0 µg/plate, +S9); ICR-191 (TA1537, 2.0 µg/plate, -S9); sodium azide (SA, TA100 + 1535, 2.0 µg/plate, -S9); 2-nitrofluorene (2NF, TA98, 1.0 µg/plate, -S9); 4-nitroquinoline-N-oxide (4NQO, WP2, 2.0 µg/plate, -S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
DURATION
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments
DETERMINATION OF CYTOTOXICITY
- Method: density of bacterial background lawn - Evaluation criteria:
- 1) Tester strains TA98, TA100 and WP2 uvrA
For a test article to be considered positive, it had to produce at least a 2-fold increase in the mean revertants per plate of at least one of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.
2) Tester strain TA1535 and TA1537
For a test article to be considered positive, it had to produce at least a 3-fold increase in the mean revertants per plate of at least on of these tester strains over the mean revertants per plate of the appropriate vehicle control. This increase in the mean number of revertants per plate had to be accompanied by a dose-response to increasing concentrations of the test article. - Key result
- Species / strain:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- 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
- Key result
- Species / strain:
- E. coli WP2 uvr A pKM 101
- 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:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation was observed
RANGE-FINDING/SCREENING STUDIES:
The cytotoxicity of the test item was determined in TA100 and WP2 uvrA in order to allow selection of appropriate doses to be tested in the mutagenicity assay. The test article was tested up to a maximum concentration of 5 mg/plate (dose range 6.67 - 5000 µg/plate). Cytotoxicity was observed with tester strain TA100 at 667 µg/plate and above in the presence of S9 mix and 333 µg/plate and above in the absence of S9 mix as evidenced by thinning of the bacterial background lawn. No cytotoxicity was observed for tester strain WP2 uvrA. In general no test article precipitate was observed. The highest dose chosen for the main study gave a reduction of revertants per plate and/or thinning or disappearance of the bacterial background lawn.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main study no changes of the background lawn were observed. - 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
- Study period:
- 22 Nov 1999 - 21 Jan 2000
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
- Remarks:
- no analytical purity of test substance specified
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted 21 July, 1997
- Deviations:
- yes
- Remarks:
- no analytical purity of test substance specified
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- not applicable
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: McCoy's 5a culture medium supplemented with 10% fetal bovine serum (FBS), L-glutamine (2mM), penicillin G (100 units/mL and streptomycin (100 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: 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
- Test concentrations with justification for top dose:
- Experiment I:
3 hours with and without metabolic activation: 515, 735, 1050, 1500 µg/mL
Experiment II:
18 hours without and 3 hours with metabolic activation: 500, 750 1000, 1500 µg/mL - Vehicle / solvent:
- - Vehicle/solvent used: water
- Untreated negative controls:
- yes
- Remarks:
- culture medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- cell culture grade water
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- other: 2 concentrations used for both, one was evaluated; Cyclophosphamide (CP, 5 + 10 µg/mL, +S9); Mitomycin C (MMC, I: 0.75 + 1.5 µg/mL, II: 0.2 + 0.4 µg/mL, -S9)
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 3 and 18 h
- Fixation time (start of exposure up to fixation or harvest of cells): 20 h
SPINDLE INHIBITOR (cytogenetic assays): 0.1 µg/mL Colcemid
STAIN (for cytogenetic assays): 5% Giemsa solution
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: 100 per replicate
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative total growth
OTHER EXAMINATIONS:
- Determination of polyploidy
- Determination of endoreplication - Evaluation criteria:
- Evaluation of a positive response:
A test substance was considered positive for inducing chromosomal aberrations if a significant increase (the difference was considered significant when p<0.01) in the number of cells with chromosomal aberrations is observed at one or more concentrations. Statistical evaluation of the percentage of cells with more than one aberration provided an indication of the severity of the positive response observed. The linear trend test evaluated the dose responsiveness. If a significant increase was seen at one or more concentrations, a dose-response should be observed.
Evaluation of a negative response:
A test substance as considered negative for inducing chromosomal aberrations if no significant increase was observed in the number of cell with chromosomal aberrations at any of the concentrations.
Equivocal evaluation:
Although most assays give clear positive or negative results, in rare cases the data set would preclude making a definitive judgement about the activity of the test substance. Results might remain equivocal or questionable regardless of the number of times the assay is repeated. - Statistics:
- Statistical Analysis employed a Cochran-Armitage test for linear trend and Fisher's Exact Test to compare the percentage of cells with aberrations (and, if applicable, the percentage of cells with more than one aberration) in treated cells to the results obtained for the vehicle control. Statistical analysis was also performed for cells exhibiting polyploidy and/or endoreduplication in order to indicate significant (p<0.01) increases in these events as indicators of possible induction of nummerical aberrations; however, the test substance was evaluated only for structural aberrations not for numerical aberrations by this protocol.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES: an initial chromosomal aberration test without metabolic activation was done (Experiment I without S9 mix), based on these results the other 3 experiments were set up
COMPARISON WITH HISTORICAL CONTROL DATA:
the values of the controls are in range with historical control data
ADDITIONAL INFORMATION ON CYTOTOXICITY: no visual signs of toxicity could be observed for all cultures but reductions of mitotic index were seen for all experiments but for varying concentrations (see any other information on results including tables) - 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:
- 13 Apr - 31 Jul 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
- Version / remarks:
- adopted 29 Jul 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Government of India, Department of Science and Technology, New Delhi, India
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Expiration date of the batch: 20 Oct 2019
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: 15 to 25 °C
- Solubility and stability of the test substance in the vehicle were confirmed by analytical methods - Target gene:
- TK locus
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: The Dow Chemical Company, Michigan, USA
- Cell cycle length, doubling time or proliferation index: 10 - 12 h
- Modal number of chromosomes: 40
MEDIA USED
- Type and identity of media: RPMI 1640 medium containing penicillin/streptomycin
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- cofactor supplemented post-mitochondrrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254
- Test concentrations with justification for top dose:
- Experiment I
With S9 mix: 54, 162, 487 amd 1462 µg/mL (3 h)
Experiment II
Without S9 mix: 54, 162, 487 amd 1462 µg/mL (3 h)
Experiment III
Without S9 mix: 54, 162, 487 amd 1462 µg/mL (24 h)
The selection of the concentrations used in the main experiments was based on data from the range-finding experiment. No cytotoxocicity was observed up to 1462 µg/mL (10 mM), therefore this concentration was chosen as top dose for the main study. - Vehicle / solvent:
- sterile water (Milli-Q)
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- methylmethanesulfonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
- Cell density at seeding: 6 x 10E6 cells/tube
DURATION
1st experiment: 3 h exposure with and without S9 mix
2nd experiment: 3 h exposure with S9 mix
3rd experiment: 24 h exposure without S9 mix
- Expression time (cells in growth medium): 2 days after the end of treatment, cells were plated for determination of the cloning efficiency and the mutation frequency in 96-well microtiter plates containing trifluorothymidine (TFT) selective medium. The microtiter plates were incubated for 12 days.
- Selection time (if incubation with a selection agent): 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 14 days
SELECTION AGENT (mutation assays): 1-4 µg/mL TFT
NUMBER OF REPLICATIONS: single samples each in two independent experiments in 96-well microtiter plates
DETERMINATION OF CYTOTOXICITY
- Method: relative cloning efficiency and relative total growth
- OTHER:
Small and large colonies were differentiated, as small colonies are capable to indicate chromosomal mutations. - Evaluation criteria:
- Increase of mutation frequency in a dose-dependent manner im comparison with the concurrent vehicle controls.
- Statistics:
- X2 test, one-sided, p ≤ 0.05
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- 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:
The test item did not precipitate in medium and did not cause any appreciable change in the pH and osmolarity of the test medium. It was found stable in water for 24 h at room temperature at concentrations of 2500 to 150000 µg/mL. Therefore a maximum concentration of 1462 µg/mL (10 mM) was tested.
RANGE-FINDING/SCREENING STUDIES:
The test item did not show evidence of significant growth inhibition (80 - 90% inhibition over the control) as relative total growth in any of the tested concentrations (25 - 1462 µg/mL) either in the presence or absence of S9 mix.
HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minumum and maximum value of the historical control data range and within the acceptability criteria of this assay. - Conclusions:
- Under the tested conditions, the test compound was not mutagenic in mouse lymphoma L517Y TK+/- cells with and without metabolic activation up to 1462 µg/mL (10 mM).
Referenceopen allclose all
Table 1. Test results of experiment 1 (plate incorporation).
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA1535 |
WP2 uvrA |
TA98 |
TA1537 |
||
– |
vehicle |
97 ± 6 |
11 ± 4 |
184 ± 28 |
23 ± 4 |
9 ± 3 |
– |
10 |
101 ± 1 |
13 ± 3 |
-- |
18 ± 3 |
5 ± 1 |
– |
33.3 |
100 ± 8 |
14 ± 5 |
175 ± 6 |
16 ± 3 |
5 ± 3 |
– |
100 |
117 ± 12 |
13 ± 2 |
191 ± 8 |
20 ± 4 |
3 ± 1 |
– |
333 |
94 ± 5 |
12 ± 2 |
183 ± 10 |
19 ± 6 |
6 ± 3 |
– |
1000 |
101 ± 10 |
9 ± 1 |
197 ± 16 |
17 ± 6 |
5 ± 2 |
– |
3330 |
-- |
-- |
161 ± 17 |
-- |
-- |
– |
5000 |
100 ± 21 |
14 ± 4 |
188 ± 7 |
22 ± 3 |
6 ± 3 |
Positive controls, –S9 |
Name |
SA |
SA |
4NQO |
2NF |
ICR-191 |
Concentrations (μg/plate) |
2 |
2 |
2 |
1 |
2 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
751 ± 35 |
590 ± 44 |
1319 ± 59 |
298 ± 19 |
427 ± 36 |
|
+ |
vehicle |
98 ± 3 |
13 3 |
197 ± 39 |
30 ± 5 |
4 ± 2 |
+ |
10 |
130 ± 10 |
16 3 |
-- |
33 ± 4 |
7 ± 2 |
+ |
33.3 |
133 ± 6 |
12 5 |
186 ± 12 |
34 ± 5 |
8 ± 0 |
+ |
100 |
124 ± 5 |
13 4 |
198 ± 20 |
34 ± 4 |
7 ± 0 |
+ |
333 |
143 ± 8 |
16 2 |
203 ± 9 |
31 ± 2 |
6 ± 1 |
+ |
1000 |
138 ± 8 |
15 5 |
226 ± 64 |
30 ± 2 |
9 ± 1 |
+ |
3330 |
-- |
-- |
178 ± 12 |
-- |
-- |
+ |
5000 |
130 ± 8 |
14 3 |
204 ± 15 |
25 ± 3 |
4 ± 2 |
Positive controls, +S9 |
Name |
2AA |
2AA |
2AA |
2AA |
2AA |
Concentrations (μg/plate) |
2.5 |
2.5 |
5 |
2.5 |
2.5 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
774 ± 19 |
134 ± 6 |
840 ± 191 |
804 ± 36 |
128 ± 9 |
4NQO = 4-nitroquinoline-N-oxide
2AA = 2-Aminoanthracene
SA = Sodium Azide
2NF = 2-Nitrofluorene
Table 2. Test results of experiment 2 (plate incorporation).
With or without S9-Mix |
Test substance concentration (μg/plate) |
Mean number of revertant colonies per plate (average of 3 plates ± Standard deviation) |
||||
Base-pair substitution type |
Frameshift type |
|||||
TA 100 |
TA1535 |
WP2 uvrA |
TA98 |
TA1537 |
||
– |
vehicle |
96 ± 17 |
17 ± 3 |
159 ± 28 |
12 ± 1 |
6 ± 3 |
– |
33.3 |
99 ± 7 |
15 ± 5 |
135 ± 10 |
15 ± 4 |
6 ± 2 |
– |
100 |
90 ± 4 |
20 ± 5 |
142 ± 12 |
12 ± 3 |
4 ± 1 |
– |
333 |
103 ± 9 |
15 ± 6 |
120 ± 19 |
14 ± 4 |
4 ± 1 |
– |
1000 |
109 ± 11 |
13 ± 5 |
138 ± 27 |
19 ± 3 |
8 ± 1 |
– |
3330 |
108 ± 10 |
11 ± 5 |
150 ± 19 |
17 ± 3 |
5 ± 1 |
– |
5000 |
95 ± 11 |
13 ± 4 |
150 23 |
15 ± 2 |
5 ± 2 |
Positive controls, –S9 |
Name |
SA |
SA |
4NQO |
2NF |
ICR-191 |
Concentrations (μg/plate) |
2 |
2 |
2 |
1 |
2 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
581 ± 33 |
523 ± 13 |
1355 ± 87 |
268 ± 42 |
411 ± 64 |
|
+ |
vehicle |
85 ± 9 |
16 ± 5 |
178 ± 18 |
18 ± 4 |
6 ± 1 |
+ |
33.3 |
93 ± 25 |
10 ± 1 |
131 ± 14 |
15 ± 4 |
7 ± 2 |
+ |
100 |
97 ± 18 |
11 ± 2 |
141 ± 19 |
17 ± 3 |
7 ± 2 |
+ |
333 |
88 ± 10 |
14 ± 5 |
139 ± 6 |
24 ± 6 |
7 ± 1 |
+ |
1000 |
104 ± 15 |
12 ± 1 |
156 ± 14 |
21 ± 3 |
9 ± 4 |
+ |
3330 |
91 ± 15 |
8 ± 3 |
138 ± 6 |
27 ± 3 |
9 ± 2 |
+ |
5000 |
89 ± 8 |
8 ± 3 |
127 ± 6 |
24 ± 10 |
8 ± 2 |
Positive controls, +S9 |
Name |
2AA |
2AA |
2AA |
2AA |
2AA |
Concentrations (μg/plate) |
2.5 |
2.5 |
5 |
2.5 |
2.5 |
|
Mean No. of colonies/plate (average of 3 ± SD) |
834 ± 16 |
105 ± 17 |
1019 ± 50 |
842 ± 65 |
127 ± 11 |
4NQO = 4-nitroquinoline-N-oxide
2AA = 2-Aminoanthracene
SA = Sodium Azide
2NF = 2-Nitrofluorene
Table 1. Test results of experiment I.
Test item |
Concentration |
Mitotic Index |
Aberrant cells in % |
|
in µg/mL |
in % |
without gaps |
Exposure period 3h, fixation time 20h, without S9 mix |
|||
Vehicle control |
10 µL/mL |
100 |
0 |
MMC |
1.5 µg/mL |
-- |
74* |
Negative control |
0 |
76 |
0 |
Test substance |
515 |
106 |
0.5 |
735 |
133 |
0 |
|
1050 |
126 |
0 |
|
1500 |
104 |
0.5 |
|
Exposure period 3h, fixation time 24h, with S9 mix |
|||
Vehicle control |
10 µL/mL |
100 |
2.0 |
CP |
5 µg/mL |
-- |
36 |
Negative control |
0 |
66 |
0 |
Test substance |
515 |
134 |
1.5 |
735 |
137 |
2.5 |
|
1050 |
95 |
2.5 |
|
1500 |
85 |
2.0 |
MMC: Mitomycin C; CP: Cyclophosphamide (positive controls)
Table 2. Test results of experiment II.
Test item |
Concentration |
Mitotic Index |
Aberrant cells in % |
|
in µg/mL |
in % |
without gaps |
Exposure period 18h, fixation time 20h, without S9 mix |
|||
Vehicle control |
10 µL/mL |
100 |
0 |
MMC |
1.5 µg/mL |
-- |
50* |
Negative control |
0 |
50 |
1.5 |
Test substance |
500 |
78 |
0.5 |
750 |
74 |
1.0 |
|
1000 |
83 |
0 |
|
1500 |
74 |
0 |
|
Exposure period 3h, fixation time 24h, with S9 mix |
|||
Vehicle control |
10 µL/mL |
100 |
1.0 |
CP |
5 µg/mL |
-- |
32 |
Negative control |
0 |
112 |
0 |
Test substance |
500 |
74 |
1.5 |
750 |
96 |
0.5 |
|
1000 |
95 |
0.5 |
|
1500 |
101 |
0 |
MMC: Mitomycin C; CP: Cyclophosphamide (positive controls)
Table 1: Summary Results of In Vitro Mammalian Cell Gene Mutation Test in the Presence of Metabolic Activation (Experiment 1)
Treatment (µg/mL) |
Non-selective medium |
Selective medium No. of Empty Wells |
Cem | MF (x 10-6) |
Induced MF |
% RTG | |||
P(0) | Cev | R1* | R2* | Total | |||||
sterile water | 0.1875 | 104.62 | 88.5 | 87 | 175.5 | 0.008622 | 0.0000824 | 100 | |
54 | 0.1979 | 101.25 | 88 | 88.5 | 176.5 | 0.008078 | 0.0000798 | -0.0000026 | 76 |
162 | 0.2031 | 99.63 | 88.5 | 88.5 | 177 | 0.007807 | 0.0000784 | -0.000004 | 67 |
487 | 0.2135 | 96.51 | 88 | 87 | 175 | 0.008896 | 0.0000922 | 0.0000098 | 54 |
1462 | 0.2396 | 89.3 | 88 | 86 | 174 | 0.009445 | 0.0001058 | 0.0000232 | 46 |
CPA 12µg/mL | 0.3281 | 69.75 | 22 | 23 | 45 | 0.13927 | 0.0019996+ | 0.0019172 | 31 |
MF: Mutant Frequency
CE: Cloning Efficiency
CPA: Cyclophosphamide
+: Significantly higher than control (p ≤0.05)
*: Mean of two replicates
RTG: Relative Total Growth = RCE x RSG x 100
Table 2: Summary Results of In Vitro Mammalian Cell Gene Mutation Test in the Absence of Metabolic Activation (Experiment 2)
Treatment (µg/mL) |
Non-selective medium |
Selective medium No. of Empty Wells |
Cem | MF (x 10-6) |
Induced MF |
% RTG | |||
P(0) | Cev | R1* | R2* | Total | |||||
sterile water | 0.1771 | 108.19 | 86.5 | 87.5 | 174 | 0.009445 | 0.0000873 | 100 | |
54 | 0.1875 | 104.62 | 88 | 86.5 | 174.5 | 0.00917 | 0.0000877 | 0.0000004 | 78 |
162 | 0.1979 | 101.25 | 87 | 88 | 175 | 0.008896 | 0.0000879 | 0.0000006 | 71 |
487 | 0.2031 | 99.63 | 86.5 | 87.5 | 174 | 0.009445 | 0.0000948 | 0.00000948 | 60 |
1462 | 0.2135 | 96.51 | 88.5 | 86.5 | 175 | 0.008896 | 0.0000922 | 0.0000922 | 57 |
MF: Mutant Frequency
CE: Cloning Efficiency
+: Significantly higher than control (p≤0.05)
*: Mean of two replicates
RTG: Relative Total Growth = RCE x RSG x 100
Table 3: Summary Results of In Vitro Mammalian Cell Gene Mutation Test in the Absence of Metabolic Activation (Experiment 3)
Treatment (µg/mL) |
Non-selective medium |
Selective medium No. of Empty Wells |
Cem | MF (x 10-6) |
Induced MF |
% RTG | |||
P(0) | Cev | R1* | R2* | Total | |||||
sterile water | 0.2031 | 99.63 | 88.5 | 88 | 176.5 | 0.008078 | 0.0000811 | 100 | |
54 | 0.2135 | 96.51 | 86.5 | 88.5 | 175 | 0.008896 | 0.0000922 | 0.0000111 | 79 |
162 | 0.2188 | 94.97 | 86.5 | 87 | 173.5 | 0.009731 | 0.0001025 | 0.0000214 | 75 |
487 | 0.2448 | 87.96 | 86 | 87 | 173 | 0.010008 | 0.0001138 | 0.0000327 | 56 |
1462 | 0.2708 | 81.65 | 87.5 | 87.5 | 175 | 0.008896 | 0.000109 | 0.0000279 | 43 |
MMS 10µg/mL | 0.401 | 57.11 | 25.5 | 24.5 | 50 | 0.129171 | 0.0022618+ | 0.0021807 | 23 |
MF: Mutant Frequency
CE: Cloning Efficiency
MMS: Methylmethanesulfonate
+: Significantly higher than control (p≤0.05)
*: Mean of two replicates
RTG: Relative Total Growth = RCE x RSG x 100
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Gene mutation in bacteria
One study investigating the induction of gene mutation in bacteria by ethylene diacetate (CAS 111 -55 -7) is available (Covance, 2000a). The study was conducted according to OECD guideline 471 under GLP conditions. The tester strains, S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and the E. coli strain WP2 uvr A pKM 101 were used.
The experiments were performed according to the plate incorporation procedure at concentrations from 10-5000 µg/plate in the first experiment and from 33.3-5000 µg/plate in the second experiment with and without a metabolic activation system (S9-mix from rats treated with Aroclor 1245). The included positive and negative controls in the experiments showed the expected results and were therefore considered as valid. No increase in the number of revertant colonies was observed in any of the bacterial strains, with and without metabolic activation system. No cytotoxicity was observed in the main study up to the limit concentration of 5000 µg/plate. Under the conditions of this study, ethylene diacetate (CAS 111 -55 -7) did not induce mutations in the bacterial mutation tests in the absence and presence of a metabolic activation system in any of the strains tested.
In vitro cytogenicity in mammalian cells
A study investigating the cytogenicity in mammalian cells in vitro with ethylene diacetate (CAS 111 -55 -7) was performed in accordance with OECD guideline 473 under GLP conditions (Covance, 2000b). The induction of structural chromosome aberrations was evaluated in vitro in Chinese hamster ovary (CHO) cells, incubated for 3 h with and without and for 18 h without a metabolic activation system (S9-mix from rats treated with Aroclor 1245). Concentrations of 515-1500 µg/mL (3 h incubation, experiment I) and 500-1500 µg/mL (18 h and 3 h incubation, experiment II) of the test substance in the vehicle (water) were applied. The negative as well as the positive controls showed the expected results and were within the range of historical control data. The frequency of polyploid cells with and without metabolic activation was within the expected range. In the experiments without metabolic activation and 18 h incubation, an influence of the test substance was observed which led to a reduction in the mitotic index. No statistically or biologically significant increase in the incidence of chromosome aberrations was observed. Therefore, under the conditions of the study, the test substance did not show clastogenic activity in the chromosomal aberration test with and without metabolic activation performed in CHO cells in vitro.
In vitro gene mutation in mammalian cells
Ethylene diacetate (CAS 111 -55 -7) was tested for its mutagenic potential in mammalian cells according to OECD guideline 490 and in compliance with GLP (Eurofins, 2018b). Two independent experimental runs were performed with L5178Y TK+/- c-3.7.2C mouse lymphoma cells (heterozygous thymidine kinase locus). The cells were treated with the test item at four concentrations (range 54 – 1462 µg/mL) for three hours with and without metabolic activation (1st and 2nd experiment), and for 24 hours without metabolic activation (3rd experiment). In all experiments, the reduction in cell growth as relative suspension growth, as well as the reduction in cell growth as relative total growth were analysed and compared to those of vehicle controls.
A preliminary cytotoxicity test revealed that a concentration of 1462 µg/mL (10 mM) with and without metabolic activation was not cytotoxic, therefore this concentration was selected as maximum dose in the main study. Concurrent vehicle controls (sterile water) and positive control substances were included. Mutant frequencies of solvent controls were within the range of historical control data. The positive control substances cyclophosphamide (+ S9 mix) and methylmethanesulfonate (–S9 mix) markedly increased the number of mutants for L5178Y cells, thus demonstrating the sensitivity and validity of the test system.
Under the conditions of the test, the test item did not induce a significant, dose-related increase in mutant frequencies in any of the tested concentrations, either in the presence or absence of metabolic activation. Thus, the test item was not considered to be mutagenic in mammalian cells.
Conclusion for genetic toxicity
The studies with ethylene diacetate (CAS 111 -55 -7) investigating genetic mutations in bacteria in vitro, cytogenicity in mammalian cells in vitro and gene mutation in mammalian cells in vitro provided negative results. Therefore, the available data do not provide any indications for a potential regarding genetic toxicity of ethylene diacetate.
Justification for classification or non-classification
The available data on genetic toxicity do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.
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