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EC number: 436-120-9 | CAS number: 99627-05-1
- 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
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- Flash point
- Auto flammability
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- Oxidation reduction potential
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- Stability: thermal, sunlight, metals
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- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
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- Endpoint summary
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- Environmental data
- Additional information on environmental fate and behaviour
- 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
- Toxicity to other aquatic organisms
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- Toxicological Summary
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- Acute Toxicity
- Irritation / corrosion
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- Specific investigations
- Exposure related observations in humans
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- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
The test item was not mutagenic in Salmonella typhimurium TA 98, TA 100, TA 102, TA 1535, TA 1537, and E. coli WP2 uvr A pKM 101 with or without addition of S9 mix (reference 7.6.1 -1). Furthermore, the substance is non-mutagenic in mammalian cells under conditions where the positive controls exerted potent mutagenic effects (reference 7.6.1-2).
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:
- July 26, 2000 - January 10, 2001
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- 21st July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Version / remarks:
- 29.Dec.1992
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
- Version / remarks:
- September 11, 1989
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- All these strains contain mutations in the histidine operon, thus imposing a requirement for histidine in the growth medium.
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
- Species / strain / cell type:
- E. coli WP2 uvr A pKM 101
- Metabolic activation:
- with and without
- Metabolic activation system:
- Due to migration, the value was transferred to one of the current document's attachments
- Test concentrations with justification for top dose:
- 1st series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 µg per plate
2nd series: 5.00, 15.8, 50.0, 158 and 500 µg per plate
The test material concentrations used were selected according to the EEC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO (test item and positive controls N-Ethyl-N'-nitro-N-nitrosoguanidine, cumene hydroperoxide, 2-Aminoanthracene, Benzo[a] pyrene) and Ethanol for positive control 9-Aminoanthracene
- Justification for choice of solvent/vehicle: Preferentially distilled water or dimethyl sulfoxide (DMSO), alternatively acetone or ethanol, are used as solvents. Analysis of the historical data of the laboratory and experience of other research groups (Maron et al. 1981) showed that the amounts of the selected solvents used have no influence on the number of spontaneous revertants of any strain. For this reason only the respective solvent control was used as the negative control in this study.
- Justification for percentage of solvent in the final culture medium: Since on the one hand organic solvents may have diverse effects on e.g. gene regulation and, on the other hand, high amounts of water (added as the solvent) will dilute the top agar, usually the maximum amount of solvent is limited to 100 µl per plate for water and 10 µL per plate for DMSO, ethanol, acetone or other organic solvents. Only the highest test material concentration may be plated with either 316 µL water or 31.6 µL organic solvent. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: Daunomycin
- Remarks:
- without S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- without S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- without S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- with S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cumene hydroperoxide
- Remarks:
- without S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-Aminoanthracene
- Remarks:
- with S9 mix
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
3 parallel plates were used for each concentration step of the test material and the positive controls. Twice as many solvent control plates were used for each bacterial strain.
- Number of independent experiments : 2
METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: Incubation of plates was performed at + 37 °C for 2 to 3 days.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition - Evaluation criteria:
- A test material is defined as non-mutagenic in this assay if
• "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)
A test material is defined as mutagenic in this assay if
• a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
• "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.
In all further cases, a third test series with the bacterial strain in question should be performed. If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system. - Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- 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:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation and time of the determination: Precipitation of the test material on the agar plates occurred at concentrations > 500 µg/plate in the 1st test series.
STUDY RESULTS :
- Signs of toxicity :
toxicity to the bacteria was observed at concentrations > 500 or 1580 µg/plate depending upon the experimental conditions used.
- Mean number of revertant colonies per plate and standard deviation : see tables 1-4
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- May 03, 2004 - January 26, 2005
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test) (migrated information)
- Version / remarks:
- 21st July 1997
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- 8. June 2000
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Details on mammalian cell type (if applicable):
- CELLS USED
- Type and source of cells: L5178Y TK(+/-) mouse lymphoma cells
For cell lines:
- Absence of Mycoplasma contamination: yes
- Methods for maintenance in cell culture: For the different experimental investigations, the cells were thawed rapidly, diluted in RPMI 10 and incubated in a humidified atmosphere of 5% v/v CO2 in air.
- Periodically ‘cleansed’ of spontaneous mutants: yes
MEDIA USED
- Exposure medium:
RPMI- 5 (RPMI 1640 with 5% horse serum) 470 mL RPMI 1640 25 mL horse serum (heat-inactivated) 5 mL penicillin/streptomycin
- Culture medium:
RPMI-10 (RPMI 1640 with 10% heat-inactivated horse serum) 445 mL RPMI 1640 50 mL horse serum (heat-inactivated horse serum) 5 mL penicillin/streptomycin
- Survivor- and selection medium:
RPMI-20 (RPMI 1640 with 20% heat-inactivated horse serum) 395 mL RPMI 1640
100 mL horse serum {heat-inactivated horse serum) 5 mL penicillin/streptomycin - Metabolic activation:
- with and without
- Metabolic activation system:
- Due to migration, the value was transferred to one of the current document's attachments
- Test concentrations with justification for top dose:
- without S9-Mix:
5.00, 15.8, 50.0, 158, 281 and 500 µg/ mL medium
with S9-Mix:
1.58, 5.00, 15.8,50.0, 158 and 500 µg / mL medium
The concentrations selected were modified and adapted in order to achieve a toxicity level recommended by OECD guideline 476. A dose-range finding test was performed to detect adequate concentrations. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Preferentially water (maximally 1%) or DMSO, acetone or ethanol (maximally 0.1%), are used as solvents.
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 4-nitroquinoline-N-oxide
- Remarks:
- without S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Remarks:
- with S9 mix
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration
Each treatment, in the absence or presence of S9 mix, was performed in duplicate (single cultures only used for positive control treatments).
METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10E7 cells in RPMI 5 medium
- Test substance added in medium
TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 3 hours in the presence and 24 hours (1st series) or 3 hours (2nd series) in the absence of S9 mix at 37°C.
FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): Cultures were maintained in flasks for 2 days (until day 3 of the experiment)
- Selective agent: At the end of the expression period (day 3), the cell densities in the selected cultures were adjusted to 1 x 10E4/mL. TFT (300 µg/mL) was diluted 100-fold into these suspensions to give a final concentration of 3 µg/mL. Plates were incubated until scorable (day 10 to day 14)
- Criteria for small (slow growing) and large (fast growing) colonies: At least for the negative and positive controls and, in case of a positive test material-induced effect, also for those cultures that showed the highest test material-induced effect, the mutation frequency is determined separately for small and large colonies in addition to the total mutation frequency.
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: relative total growth (RTG) - Evaluation criteria:
- The effects of the test material upon the mutation frequency are defined as
• "No effect" or "no increase" in the mutation frequency if the mean frequency of the parallel incubations of a given test material concentration is less than 2.0-fold above the mean of the actual negative controls or the mean mutation falls within the historical range of the negative controls.
• "Clear effect" or "clear increase" in the mutation frequency if the test material induces at least a 3.0-fold increase above the mean of the actual negative controls and the mean mutation frequency for a given test material concentration is at least 1.5-fold above the highest value of the historical negative controls.
• All other results are defined as a "weak effect" or a "weak increase" of the mutation frequency.
Test materials are assessed as negative or non-mutagenic in this test system if
• the assay is considered valid and
• no effect (no increase in the mutation frequency) occurs in the two experimental series performed or
• a weak effect (weak increase) occurs in one series and no effect (no increase) in the other series of experiments.
Test materials are assessed as positive or mutagenic in this test system if
• the assay is considered valid and
• a clear effect (clear increase in the mutation frequency) occurs at similar concentrations of the test material in the two experimental series performed, or
• a clear effect (clear increase) occurs in one series and a weak effect (weak increase) in the other series of experiments at identical concentrations, or
• weak effects (weak increases) occur dose-dependently (over at least two test material concentrations) and reproducibly at identical concentrations in the two experimental series performed. - 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:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: The test material did not lead to a relevant fluctuation in the pH of the cell culture medium.
- Data on osmolality: The test material did not lead to a relevant fluctuation in the osmolarity of the cell culture medium.
- Precipitation and time of the determination: No precipitation of the test material in the cell culture medium occurred
RANGE-FINDING/SCREENING STUDIES
In a preceding range finding test, the relative survival was determined after exposure to various test material concentrations ranging between 5.00 and 5000 µg/mL. A reduction in the relative survival of the cells occurred at concentrations > 158 or 1580 in the absence of S9 mix and 15.8 µg/mL in the presence of S9 mix, respectively.
Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements: Toxicity to the cells was observed at concentrations > 5.00 or 500 µg/mL, depending upon the experimental condition.
- Genotoxicity results: see table 1
HISTORICAL CONTROL DATA : see table 2
Referenceopen allclose all
Table 1: Results / Series No.: 1
Positive Controls: Individual and Mean Number of Revertant Colonies
Strain |
Positive Control |
Concentr. [µg/plate] |
+ /- # |
Mean revertant colonies per plate |
SD* |
Individual revertant colonies per plate |
||
TA 98 |
DAUN |
2 |
- |
643 |
196 |
869 |
538 |
521 |
TA 100 |
ENNG |
5 |
- |
904 |
41 |
866 |
947 |
899 |
TA 102 |
CUM |
67 |
- |
396 |
37 |
421 |
414 |
354 |
TA 1535 |
ENNG |
10 |
- |
332 |
12 |
329 |
321 |
345 |
TA 1537 |
9-AA |
50 |
- |
1683 |
331 |
1727 |
1990 |
1333 |
WP2 |
ENNG |
5 |
- |
1327 |
83 |
1264 |
1296 |
1421 |
|
|
|
|
|
|
|
|
|
TA 98 |
2-AA |
1 |
+ |
322 |
19 |
338 |
301 |
326 |
TA 100 |
2-AA |
1 |
+ |
537 |
25 |
564 |
532 |
514 |
TA 102 |
B (a) p |
10 |
+ |
806 |
3 |
804 |
805 |
809 |
TA 1535 |
2-AA |
1 |
+ |
151 |
17 |
152 |
134 |
167 |
TA 1537 |
2-AA |
2 |
+ |
92 |
34 |
95 |
124 |
56 |
WP2 |
2-AA |
10 |
+ |
1459 |
IB |
1477 |
1441 |
1459 |
# Test material incubated in the presence
(+) or absence
(-) of S9-Mix
* Standard deviation
DAUN Daunomycin
ENNG N-Ethyl-N’-nitro-N-nitroso-guanidine
CUM Cumene hydroperoxide
9-AA 9-Aminoacridine
2-AA 2-Aminoanthracene
B (a) p Benzo(a)pyrene
Table 2: Results/ Series No.: 2
Positive Controls: Individual and Mean Number of Revertant Colonies
Strain |
Positive Control |
Concentr. [µg/plate] |
+ /- # |
Mean revertant colonies per plate |
SD* |
Individual revertant colonies per plate |
||
TA 98 |
DAUN |
2 |
- |
93 |
18 |
73 |
97 |
108 |
TA 100 |
ENNG |
5 |
- |
520 |
39 |
565 |
500 |
494 |
TA 102 |
CUM |
150 |
- |
661 |
72 |
703 |
702 |
578 |
TA 1535 |
ENNG |
10 |
- |
187 |
11 |
194 |
192 |
174 |
TA 1537 |
9-AA |
50 |
- |
186 |
40 |
144 |
191 |
224 |
WP2 |
ENNG |
5 |
- |
1103 |
12 |
1117 |
1096 |
1097 |
|
|
|
|
|
|
|
|
|
TA 98 |
2-AA |
10 |
+ |
329 |
150 |
434 |
396 |
158 |
TA 100 |
2-AA |
10 |
+ |
333 |
30 |
302 |
336 |
361 |
TA 102 |
B (a) p |
10 |
+ |
775 |
120 |
790 |
887 |
649 |
TA 1535 |
2-AA |
2 |
+ |
94 |
11 |
107 |
87 |
649 |
TA 1537 |
2-AA |
20 |
+ |
42 |
10 |
17 |
49 |
30 |
WP2 |
2-AA |
10 |
+ |
933 |
81 |
861 |
1020 |
918 |
# Test material incubated in the presence
(+) or absence
(-) of S9-Mix
* Standard deviation
DAUN Daunomycin
ENNG N-Ethyl-N’-nitro-N-nitroso-guanidine
CUM Cumene hydroperoxide
9-AA 9-Aminoacridine
2-AA 2-Aminoanthracene
B (a) p Benzo(a)pyrene
Table 3: Results / Series No.: 1
Summary of the Mean Number of Revertant Colonies
Test material |
Concentr. [µg/plate] |
+ /-
|
Mean revertant colonies / plate |
|||||
TA 98 |
TA 100 |
TA 102 |
TA 1535 |
TA 1537 |
WP2 |
|||
Solvent control Test item |
|
- |
14 |
97 |
269 |
12 |
4 |
128 |
5 |
- |
14 |
91 |
264 |
12 |
3 |
119 |
|
15.8 |
- |
17 |
96 |
268 |
12 |
6 |
127 |
|
50 |
- |
15 |
99 |
249 |
15 |
4 |
122 |
|
158 |
- |
16 |
112 |
242 |
12 |
7 |
114 |
|
500 PE |
- |
8 |
73 |
189 |
11 |
2 |
53 |
|
1580 PE |
- |
0 |
3 |
2 |
2 |
0 |
2 |
|
5000 PE |
- |
0 |
3 |
0 |
2 |
0 |
2 |
|
|
|
|
|
|
|
|
|
|
Solvent control Test item |
|
+ |
20 |
120 |
271 |
13 |
7 |
170 |
5 |
+ |
25 |
121 |
277 |
20 |
9 |
164 |
|
15.8 |
+ |
27 |
132 |
288 |
14 |
8 |
180 |
|
50 |
+ |
26 |
142 |
278 |
13 |
9 |
167 |
|
158 |
+ |
27 |
143 |
342 |
17 |
8 |
130 |
|
500 PE |
+ |
22 |
130 |
214 |
12 |
5 |
81 |
|
1580 PE |
+ |
2 |
4 |
2 |
3 |
0 |
3 |
|
5000 PE |
+ |
0 |
5 |
0 |
3 |
0 |
5 |
|
|
|
|
|
|
|
|
|
|
Postive controls |
Name |
- |
DAUN |
ENNG |
CUM |
ENNG |
9-AA |
ENNG |
Conc (µg/plate) |
2 |
5 |
67 |
10 |
50 |
5 |
||
Revert. / plate |
343 |
904 |
396 |
332 |
1683 |
1327 |
||
|
|
|
|
|
|
|
|
|
Name |
+ |
2-AA |
2-AA |
B (a) p |
2-AA |
2-AA |
2-AA |
|
Conc (µg/plate) |
1 |
1 |
10 |
1 |
2 |
10 |
||
Revert. / plate |
322 |
537 |
806 |
151 |
92 |
1459 |
DAUN Daunomycin
ENNG N-Ethyl-N’-nitro-N-nitroso-guanidine
CUM Cumene hydroperoxide
2-AA 2-Aminoanthracene
B (a) p Benzo(a)pyrene
9-AA 9-Aminoacridine
Table 4 Results / Series No.: 2
Summary of the Mean Number of Revertant Colonies
Test material |
Concentr. [µg/plate] |
+ /-
|
Mean revertant colonies / plate |
|||||
TA 98 |
TA 100 |
TA 102 |
TA 1535 |
TA 1537 |
WP2 |
|||
Solvent control Test item |
|
- |
15 |
93 |
218 |
11 |
5 |
130 |
5 |
- |
13 |
87 |
219 |
7 |
7 |
130 |
|
15.8 |
- |
12 |
92 |
220 |
11 |
8 |
127 |
|
50 |
- |
13 |
88 |
222 |
12 |
4 |
122 |
|
158 |
- |
16 |
88 |
221 |
15 |
7 |
119 |
|
500 |
- |
11 |
79 |
206 |
11 |
6 |
91 |
|
|
|
|
|
|
|
|
|
|
Solvent control Test item |
|
+ |
25 |
140 |
275 |
18 |
6 |
134 |
5 |
+ |
22 |
139 |
254 |
17 |
5 |
133 |
|
15.8 |
+ |
20 |
153 |
226 |
20 |
7 |
151 |
|
50 |
+ |
20 |
156 |
259 |
16 |
3 |
144 |
|
158 |
+ |
26 |
176 |
266 |
18 |
6 |
128 |
|
500 |
+ |
30 |
153 |
261 |
12 |
6 |
115 |
|
|
|
|
|
|
|
|
|
|
Postive controls |
Name |
- |
DAUN |
ENNG |
CUM |
ENNG |
9-AA |
ENNG |
Conc (µg/plate) |
2 |
5 |
150 |
10 |
50 |
5 |
||
Revert. / plate |
93 |
520 |
661 |
187 |
186 |
1327 |
||
|
|
|
|
|
|
|
|
|
Name |
+ |
B (a) p |
B (a) p |
B (a) p |
2-AA |
B (a) p |
2-AA |
|
Conc (µg/plate) |
10 |
10 |
10 |
2 |
20 |
10 |
||
Revert. / plate |
329 |
333 |
775 |
94 |
42 |
1459 |
DAUN Daunomycin
ENNG N-Ethyl-N’-nitro-N-nitroso-guanidine
CUM Cumene hydroperoxide
B (a) p Benzo(a)pyrene
9-AA 9-Aminoacridine
2-AA 2-Aminoanthracene
Table 1: Summary of results
Without S9 mix (EZ.1571 / 1581)
1stExperimental series |
||||
Test material [µg/mL] |
RSa (%) |
RTGb (%) |
MFc |
|
Solvent |
0 |
100 |
100 |
121 |
|
|
|
|
|
Test item |
5.00 |
91.1 |
121 |
173 |
15.8 |
83.5 |
111 |
151 |
|
50.0 |
71.1 |
50.2 |
137 |
|
158 |
1.37 |
0.0896 |
144 |
|
281 |
0 |
- |
- |
|
500 |
0 |
- |
- |
|
|
|
|
|
|
NQO |
0.10 |
42.7 |
118 |
1050 |
0.20 |
21.1 |
35.2 |
1140 |
2ndExperimental series |
||||
Test material [µg/mL] |
RSa (%) |
RTGb (%) |
MFc |
|
Solvent |
0 |
100 |
100 |
89.6 |
|
|
|
|
|
Test item |
15.8 |
89.8 |
100 |
120 |
50.0 |
91.3 |
88.4 |
110 |
|
158 |
77.2 |
75.8 |
122 |
|
500 |
27.8 |
4.31 |
143 |
|
889 |
0.622 |
- |
- |
|
1580 |
- |
- |
- |
|
|
|
|
|
|
NQO |
0.10 |
86.7 |
31.2 |
792 |
0.20 |
60.4 |
33.9 |
529 |
a: Relative Survival (CE of cells after treatment)
b: Relative Total Growth
c: 5-TFT Mutant Frequency per 106viable cells
EZ: Internal study number
NQO: 4-Nitroquinoline N-oxide
DMBA: 7,12-dimethylbenz[a]anthracene
- : Lost due to toxicity of test material or not plated for selection of mutants
With S9 mix(EZ 1572/ 1582/ 1594)
1stExperimental series |
||||
Test material [µg/mL] |
RSa (%) |
RTGb (%) |
MFc |
|
Solvent |
0 |
100 |
100 |
202 |
|
|
|
|
|
Test item |
1.58 |
97.8 |
89.3 |
223 |
5.00 |
82.3 |
61.2 |
289 |
|
15.8 |
21.5 |
10.6 |
1110 |
|
50.0 |
13.5 |
9.04 |
846 |
|
158 |
5.56 |
2.09 |
1250 |
|
500 |
0 |
- |
- |
|
|
|
|
|
|
DMBA |
2.00 |
62.1 |
64.2 |
1160 |
3.00 |
78.3 |
48.8 |
2000 |
2nd. Experimental series |
||||
Test material [ug/mL] |
RSa (%) |
RTGb (%) |
MFc |
|
Solvent |
0 |
100 |
100 |
161 |
|
|
|
|
|
Test item |
1.58 |
93.0 |
76.3 |
196 |
5.00 |
91.4 |
35.6 |
344 |
|
8.89 |
49.7 |
32.6 |
199 |
|
15.8 |
23.2 |
19.8 |
256 |
|
50.0 |
11.0 |
11.6 |
203 |
|
158 |
2.02 |
0.165 |
250 |
|
|
|
|
|
|
DMBA |
1.00 |
50.5 |
38.1 |
2120 |
2.00 |
45.9 |
6.53 |
2970 |
3rd. Experimental series |
||||
Test material [ug/mL] |
RSa (%) |
RTGb (%) |
MFc |
|
Solvent |
0 |
100 |
100 |
196 |
|
|
|
|
|
Test item |
1.58 |
84.7 |
94.3 |
178 |
2.81 |
64.8 |
79.0 |
190 |
|
5.00 |
51.0 |
74.7 |
194 |
|
8.89 |
30.7 |
36.3 |
256 |
|
15.8 |
13.9 |
22.8 |
255 |
|
28.1 |
20.8 |
23.1 |
269 |
|
|
|
|
|
|
DMBA |
2.00 |
66.4 |
40.6 |
2460 |
3.00 |
6.81 |
9.07 |
6640 |
a: Relative Survival (CE of cells after treatment)
b: Relative Total Growth
c: 5-TFT Mutant Frequency per 106 viable cells
EZ: Internal study number
NQO: 4-Nitroquinoline N-oxide
DMBA: 7,12-dimethylbenz[a]anthracene
- : Lost due to toxicity of test material or not plated for selection of mutants
Table 2: Historical data
Study number |
Mean Mutation Frequency [x10-6]a |
|||||||||||
Solvent controlsb |
Positive controlsc |
|||||||||||
- S9 mix |
+ S9 mix |
- S9 mix |
+ S9 mix |
|||||||||
T14248 |
63 |
123 |
|
95 |
106 |
|
378 |
794 |
|
488 |
568 |
|
T14277 |
128 |
179 |
|
168 |
133 |
|
1440 |
755 |
|
955 |
963 |
|
T14278 |
184 |
|
|
94 |
|
|
718 |
|
|
429 |
|
|
T14325 |
118 |
158 |
|
172 |
219 |
|
498 |
684 |
|
445 |
816 |
|
T14327 |
199 |
79 |
|
148 |
86 |
|
1181 |
1490 |
|
672 |
413 |
|
T14506 |
88 |
103 |
166 |
123 |
112 |
|
746 |
843 |
571 |
673 |
675 |
|
T14646 |
178 |
246 |
289 |
233 |
|
|
1196 |
869 |
924 |
518 |
|
|
T14690 |
166 |
251 |
|
174 |
117 |
|
1920 |
1439 |
|
606 |
478 |
|
T14786 |
103 |
106 |
|
90 |
53 |
|
1263 |
1529 |
|
554 |
465 |
|
T14862 |
88 |
122 |
|
90 |
257 |
|
955 |
645 |
|
484 |
1448 |
|
T14905 |
219 |
131 |
|
143 |
246 |
|
760 |
1008 |
|
562 |
2046 |
|
T15101 |
147 |
141 |
|
121 |
120 |
233 |
541 |
857 |
|
576 |
1453 |
1230 |
T15141 |
222 |
159 |
|
157 |
152 |
|
749 |
537 |
|
1196 |
1440 |
|
T15152 |
132 |
183 |
|
186 |
114 |
|
465 |
473 |
|
696 |
632 |
|
T15358 |
156 |
153 |
|
179 |
170 |
202 |
883 |
1244 |
|
1548 |
2172 |
2152 |
T15359 |
146 |
129 |
|
121 |
215 |
137 |
443 |
683 |
|
1050 |
1741 |
2096 |
T15360 |
136 |
131 |
|
157 |
187 |
|
358 |
462 |
|
2400 |
955 |
|
T15597 |
71.8 |
84.1 |
|
104 |
96 |
|
486 |
2755 |
|
1098 |
805 |
|
T15549 |
82.5 |
91.4 |
|
102 |
88 |
|
431 |
661 |
|
463 |
608 |
|
T15575 |
179 |
97.8 |
|
87.9 |
55.6 |
|
614 |
315 |
|
836 |
669 |
|
T15630 |
93.9 |
89.3 |
|
177 |
78.7 |
|
1050 |
834 |
|
1320 |
12040 |
|
T15596 |
140 |
103 |
|
106 |
171 |
|
666 |
524 |
|
1700 |
1200 |
|
T15681 |
98 |
131 |
|
95.2 |
95.2 |
|
704 |
1110 |
|
5150 |
1560 |
|
T15682 |
113 |
136 |
|
122 |
245 |
|
957 |
471 |
|
1240 |
2470 |
|
T15766 |
219 |
157 |
|
301 |
230 |
|
970 |
369 |
|
1660 |
1530 |
|
|
|
|
|
|
|
|
|
|
|
|
|
|
Historical Controls |
||||||||||||
|
|
|
|
|
|
|
|
|
|
|
|
|
Mean±SD: |
141±50 |
146±57 |
847±445 |
1160±810 |
||||||||
Range: |
53 - 301 |
315 - 2755 |
413 - 5150 |
a: Mean values from 1 to 4 Individual cultures from 1 to 3 experimental series of studies performed in the laboratory using the described protocol
b; All solvent controls from different exposure times are pooled for each study
c: NQO (0.10 to 0.25 µg/mL) in the absence and BaP (1.25 to 5.0 µg/mL) or DMBA (1.0 to 3.0 µg/mL) in the presence of S9 mix
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Ames test
A study according OECD TG 471 was performed to test for the mutagenic potential of the substance using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535 and TA 1537, and Escherichia coli WP2 uvrA pkM101. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed. In the two series with S9 mix, 10 % and 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively.
The substance was dissolved in dimethyl sulfoxide and tested at concentrations ranging from 5.0 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred at concentrations > 500 µg/plate. Toxicity to the bacteria was observed at concentrations > 500 or 1580 µg/plate depending upon the experimental conditions used.
Daunomycin, N-ethyl-N'-nitro-N-nitrosoguanidine, 9-aminoacridine and cumene hydroperoxide served as strain specific positive control compounds in the absence of S9 mix. 2-Aminoanthracene and benzo[a]pyrene were used for testing the bacteria and the activity of the S9 mix. Each treatment with the substances used as positive controls led to a clear increase in revertant colonies, thus showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used.
With and without addition of S9 mix as the external metabolizing system, the substance was not mutagenic under the experimental conditions described (reference 7.6.1-1).
In vitro gene mutation in mammalian cells
The substance was tested for its ability to induce mutations at the TK locus (5-trifluorothymidine resistance) in mouse lymphoma cells according to OECD TG 476. The study consisted of two or three independent experimental series, conducted in the absence and presence of an exogenous metabolizing system (S9 mix from livers of rats pre-treated with Aroclor 1254). The exposure times were 3 and 24 hours in the absence and 3 hours in the presence of S9 mix, respectively.
Concentrations ranging from 1.58 to 1580 µg/mL were tested in the absence or presence of S9 mix. No Precipitation of the test material in the incubation medium was observed. Clear cytotoxic effects, i.e. a relevant decrease in either the % relative survival or % total growth of the test cells, occurred at concentrations > 5.00 or < 500 µg/mL, depending upon the experimental condition used. The doses tested were selected to determine viability and mutagenicity (5-trifluorothymidine (TFT) resistance) 2 days after treatment.
Negative (solvent) and positive control treatments were included in each mutation experiment in the absence and presence of S9 mix. Mutant frequencies in negative control cultures fell within normal ranges, and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline N-oxide (without S9 mix) and 7,12-dimethylbenz[a]anthracene (with S9 mix). Therefore, the study was accepted as valid.
No biologically relevant increases in mutant frequency were observed following treatment with the test item in the three experimental series performed.
It is therefore concluded that the substance is non-mutagenic in this test system under conditions where the positive controls exerted potent mutagenic effects (reference 7.6.1-2).
Justification for classification or non-classification
Classification, Labeling, and Packaging Regulation (EC) No 1272/2008
The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.
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