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EC number: 700-957-0 | CAS number: 1141852-17-6
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- Endpoint summary
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Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Link to relevant study records
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 16 Jun - 07 Jul 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- no
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Not applicable
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM (Biochrom, Berlin, Germany) with Fetal Bovine Serum Standard Quality (PAA Laboratories GmbH, Cölbe, Germany)
- Properly maintained: yes - Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of 8-12 weeks old male Wistar Hsd Cpb: WU rats induced with 80 mg/kg bw phenobarbital i.p. and ß-naphthoflavone p.o. each on three consecutive days
- Test concentrations with justification for top dose:
- Experiment I: 4 h treatment with and without S9 mix
Cytotoxicity testing: 2.4, 4.9, 9.8, 19.5, 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 6950 µg/mL
Chromosome aberration analysis: 156.3, 312.5, 2500 and 6950 µg/mL (with S9); 312.5, 1250, 2500 and 6950 µg/mL (without S9)
Experiment II: 4 h treatment with S9 mix and 18 h without S9 mix
Cytotoxicity testing: 19.5#, 39.1, 78.1, 156.3, 312.5, 625, 1250, 2500 and 6950 µg/mL (#this concentration was additionally analysed in the absence of S9)
Chromosome aberration analysis: 39.1, 625#, 1250, 2500 and 6950 µg/mL (#this concentration was additionally analysed in the presence of S9) - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- tetrahydrofuran (THF)
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: -S9: ethyl methanesulfonate (EMS), 1000 (Exp. I) and 600 (Exp. II) µg/mL; +S9: cyclophosphamide (CPA), 1.4 (Exp. I) and 1.0 (Exp. II) µg/mL
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- 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
- Conclusions:
- Under the experimental conditions reported, the test substance did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line).
- Executive summary:
The test substance Y-15866 was assayed in an in vitro mammalian chromosome aberration test conducted in accordance with GLP and similar to OECD guideline 473. In two independent experiments, V79 Chinese hamster cells were treated with the test substance at concentrations up to 6950 µg/mL, with and without metabolic activation system (S9 mix). In the first experiment, continuous treatment with the test substance for 4 h followed by a 14 h-recovery period was performed. Since no clear cytotoxicity indicated by reduced mitotic indices or reduced cell numbers was observed up to the highest concentration, 6950 µg/mL was chosen as top treatment concentration in Experiment II. In this experiment, cells were analogously treated with the test substance in the presence of S9 (4 h + 14 h recovery period). In contrast, cells were continuously treated for a period of 18 h without recovery period in the absence of S9 mix. Since no dose-response relationship was observed for cytotoxic effects in both experiments, concentrations used for evaluation of chromosome aberrations were selected based on the highest non-cytotoxic concentration tested. Thus, concentrations chosen for chromosome analysis ranged from 156.3 to 6950 µg/mL in Experiment I and 39.1 to 6950 µg/mL in Experiment II. In both experiments, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed after treatment with the test substance in the absence and presence of S9 mix. The positive control substances yielded the expected results. Under the conditions of this chromosome aberration assay, it was concluded that Y-15866 did not show clastogenic activity in V79 Chinese hamster cells.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 16 Jun - 05 Jul 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- his operon (S. thyphimurium) and trp operon (E. coli)
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of male Wistar rats induced with 80 mg/kg bw phenobarbital i.p. and ß-naphthoflavone p.o. each on three consecutive days
- Test concentrations with justification for top dose:
- Pre-experiment (Experiment I): 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation
Experiment II: 33, 100, 333, 1000, 2500 and 5000 µg/plate with and without metabolic activation - Vehicle / solvent:
- - Vehicle/solvent used: tetrahydrofuran
- Justification for choice of solvent/vehicle: the solvent was chosen because of its solubility properties and its relative non-toxicity to the bacteria. - Untreated negative controls:
- yes
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 4NOPD (-S9; 10 µg/plate (TA 98) + 50 µg/plate (TA 1537)); NaN3 (-S9; 10 µg/plate; TA 1535 + TA 100); MMS (-S9; 3 µL/plate; WP2 uvrA); 2AA (+S9; 2.5 µg/plate (TA 1535, TA 1537, TA 98, TA 100) + 10 µg/plate (WP2 uvrA))
- Remarks:
- 4NOPD: 4-nitro-o-phenylene diamine; NaN3 = sodium azide; MMS = methyl methane sulfonate; 2AA: 2-amino-anthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION 1: in agar (plate incorporation) for pre-experiment (Experiment I)
DURATION
- Exposure duration: 48 h
METHOD OF APPLICATION 2: preincubation followed by plate incorporation for Experiment II
DURATION
- Preincubation period: 60 min
- Exposure duration: 48 h
NUMBER OF REPLICATIONS: triplicate each in both experiments
DETERMINATION OF CYTOTOXICITY
- Method: determination of the number of revertants per plate and inspection of the bacterial background lawn - Evaluation criteria:
- The test substance was considered mutagenic if:
- a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control was observed,
- the threshold is exceeded at more than one concentration (dose-dependency),
- an increase exceeding the threshold at only one concentration was reproduced in an independent second experiment,
- a dose dependent increase in the number of revertant colonies below the threshold was reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
The assay was considered acceptable if the following criteria were met:
- regular background growth in the negative and solvent control,
- the spontaneous reversion rates in the negative and solvent control were in the range of the historical data,
- the positive control substances produced a significant increase in mutant colony frequencies. - Statistics:
- Mean values and standard deviations were calculated for revertant colonies.
- 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, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity, but tested up to precipitating concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: the test item precipitated in the overlay agar in the test tubes from 2500 µg/plate up to 5000 µg/plate in Experiment I and II. Precipitation on the incubated agar plate was observed from 2500 µg/plate up to 5000 µg/plate in both experiments with and without S9 mix. The undissolved particles of the test item had no influence on the data recording.
RANGE-FINDING/SCREENING STUDIES: in a pre-experiment (Experiment I) using the plate incorporation method, each strain was incubated with concentrations ranging from 3 to 5000 µg/plate of the test substance. Three replicates for each concentration were performed both in the presence and absence of metabolic activation. Since no toxic effects were observed at any concentration, 5000 µg/plate was chosen as maximal concentration.
COMPARISON WITH HISTORICAL CONTROL DATA: in Experiment I, the number of revertant colonies of the 5 tester strains remained in the range of the historical control values for these strains. In Experiment II, without metabolic activation, the number of colonies did not quite reach the lower limit of the historical control data in strain TA 100 (untreated control). Since this deviation was rather small, this effect was judged to be based upon statistical fluctuations and had no detrimental impact on the outcome of the study. - Conclusions:
- Under the experimental conditions reported, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of metabolic activation in the selected strains of S. typhimurium (TA 98, TA 100, TA 1535 and TA 1537) and E. coli WP2 uvrA.
- Executive summary:
The bacterial gene mutation assay (Ames test) was conducted in compliance with OECD guideline 471 and under GLP conditions. The assay was performed in two independent experiments both performed in the absence and in the presence of liver microsomal activation system (S9 mix). In the first experiment (pre-experiment), the direct plate incorporation procedure was conducted with the Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and the Escherichia coli strain WP2 uvrA at concentrations ranging from 3 to 5000 µg/plate for a period of 48 h. In the second experiment, concentrations ranging from 33 to 5000 µg/plate were analysed using the same strains, but with modification of the study design (with pre-incubation period of 60 min). No signs of cytotoxicity, evident as a reduction in the number of revertants, were observed in both experiments. Precipitation of the test substance occurred at doses = 2500 µg/plate, but did not influence data evaluation. The number of revertants was not increased at any concentrations tested. The positive and negative controls included showed the expected results in each experiment. Under the experimental conditions reported, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of metabolic activation in the selected strains of S. typhimurium (TA 98, TA 100, TA 1535 and TA 1537) and E. coli WP2 uvrA.
Referenceopen allclose all
Table 1. Test results of pre-experiment/Experiment I (plate incorporation)
Y-15866: Bacterial Reverse Mutation Assay, mean revertant colonies/plate (mutation factor) (n=3 ± SD) |
|||||
EXPERIMENT I (plate incorporation) |
|||||
S9-Mix |
Without
|
||||
Concentration (per plate) |
TA 1535 |
TA1537 |
TA98 |
TA100 |
E.coli WP2 uvrA |
SC (THF) |
11 ± 3 |
12 ± 3 |
23 ± 6 |
116 ± 4 |
47 ± 3 |
NC |
10 ± 3 |
13 ± 1 |
25 ± 5 |
133 ± 4 |
46 ± 2 |
Y-15866 |
|
|
|
|
|
5000 µg |
11 ± 4P |
15 ± 2P |
22 ± 6P |
132 ± 5P |
61 ± 4P |
2500 µg |
12 ± 2P |
12 ± 3P |
30 ± 1P |
119 ± 5P |
57 ± 1P |
1000 µg |
10 ± 1 |
14 ± 2 |
25 ± 3 |
117 ± 12 |
52 ± 2 |
333 µg |
10 ± 1 |
13 ± 1 |
23 ± 6 |
126 ± 11 |
58 ± 5 |
100 µg |
10 ± 3 |
12 ± 2 |
26 ± 4 |
117 ± 20 |
51 ± 1 |
33 µg |
11 ± 5 |
11 ± 2 |
21 ± 5 |
116 ± 6 |
56 ± 11 |
10 µg |
12 ± 3 |
12 ± 5 |
26 ± 8 |
124 ± 6 |
55 ± 5 |
3 µg |
11 ± 4 |
12 ± 3 |
20 ± 8 |
123 ± 12 |
47 ± 3 |
PC |
|
|
|
|
|
NaN3 (10 µg) |
1666 ± 29 |
- |
- |
1875 ± 97 |
- |
4NOPD (10 µg) |
- |
- |
325 ± 15 |
- |
- |
4NOPD (50 µg) |
- |
72 ± 14 |
- |
- |
- |
MMS (3 µL) |
- |
- |
- |
- |
942 ± 29 |
S9-Mix
|
With |
||||
Concentration (per plate) |
TA 1535 |
TA1537 |
TA98 |
TA100 |
E.coli WP2 uvrA |
SC (THF) |
19 ± 4 |
18 ± 3 |
38 ± 8 |
138 ± 3 |
71 ± 4 |
NC |
23 ± 7 |
20 ± 3 |
40 ± 4 |
136 ± 9 |
70 ± 9 |
Y-15866 |
|
|
|
|
|
5000 µg |
18 ± 4P |
20 ± 8P |
39 ± 4P |
149 ± 11P |
92 ± 8 |
2500 µg |
20 ± 4P |
16 ± 3P |
43 ± 3P |
143 ± 16P |
60 ± 9 |
1000 µg |
21 ± 1 |
19 ± 7 |
33 ± 8 |
146 ± 12 |
70 ± 4 |
333 µg |
22 ± 4 |
20 ± 6 |
38 ± 6 |
128 ± 5 |
59 ± 7 |
100 µg |
16 ± 1 |
16 ± 6 |
33 ± 4 |
135 ± 13 |
62 ± 8 |
33 µg |
18 ± 2 |
17 ± 5 |
29 ± 3 |
152 ± 3 |
74 ± 15 |
10 µg |
17 ± 4 |
19 ± 5 |
40 ± 2 |
145 ± 6 |
66 ± 9 |
3 µg |
20 ± 3 |
19 ± 3 |
38 ± 4 |
133 ± 10 |
79 ± 6 |
PC |
|
|
|
|
|
2AA (2.5 µg) |
196 ± 10 |
457 ± 42 |
2442 ± 291 |
3058 ± 180 |
- |
2AA (10 µg) |
- |
- |
- |
- |
316 ± 25 |
NC = Negative Control; SC = Solvent control; PC = Positive control substances; SD = standard deviation;NaN3 = sodium azide; 4NOPD = 4-nitro-o-phenylene-diamine; MMS = methyl methane sulfonate; 2AA = 2-aminoan-thracene P = Precipitate |
Table 2. Test results of Experiment II (pre-incubation)
Y-15866: Bacterial Reverse Mutation Assay, mean revertant colonies/plate (mutation factor) (n=3 ± SD) |
|||||
EXPERIMENT II (pre-incubation) |
|||||
S9-Mix |
Without
|
||||
Concentration (per plate) |
TA 1535 |
TA1537 |
TA98 |
TA100 |
E.coli WP2 uvrA |
SC |
16 ± 2 |
10 ± 2 |
30 ± 8 |
100 ± 8 |
40 ± 2 |
NC |
17 ± 5 |
16 ± 4 |
27 ± 7 |
76 ± 13 |
39 ± 7 |
Y-15866 |
|
|
|
|
|
5000 µg |
18 ± 5P |
10 ± 2P |
22 ± 3P |
101 ± 11P |
27 ± 3P |
2500 µg |
17 ± 1P |
11 ± 1P |
29 ± 7P |
93 ± 13P |
24 ± 3P |
1000 µg |
15 ± 2 |
12 ± 3 |
23 ± 2 |
103 ± 10 |
32 ± 2 |
333 µg |
13 ± 1 |
12 ± 4 |
26 ± 5 |
86 ± 14 |
39 ± 5 |
100 µg |
18 ± 4 |
13 ± 3 |
24 ± 12 |
84 ± 8 |
39 ± 4 |
33 µg |
14 ± 1 |
9 ± 3 |
26 ± 9 |
103 ± 8 |
34 ± 9 |
PC |
|
|
|
|
|
NaN3 (10 µg) |
1864 ± 62 |
- |
- |
702 ± 148 |
- |
4NOPD (10 µg) |
- |
- |
435 ± 56 |
- |
- |
4NOPD (50 µg) |
- |
107 ± 7 |
- |
- |
- |
MMS (3 µL) |
- |
- |
- |
- |
2539 ± 40 |
S9-Mix
|
With |
||||
Concentration (per plate) |
TA 1535 |
TA1537 |
TA98 |
TA100 |
E.coli WP2 uvrA |
SC |
17 ± 2 |
17 ± 4 |
44 ± 2 |
110 ± 5 |
48 ± 5 |
NC |
22 ± 7 |
22 ± 9 |
39 ± 6 |
112 ± 7 |
58 ± 16 |
Y-15866 |
|
|
|
|
|
5000 µg |
22 ± 3P |
17 ± 3P |
38 ± 9P |
111 ± 37P |
50 ± 6P |
2500 µg |
15 ± 2P |
19 ± 8P |
27 ± 1P |
129 ± 13P |
54 ± 6P |
1000 µg |
16 ± 5 |
16 ± 4 |
40 ± 10 |
136 ± 12 |
54 ± 11 |
333 µg |
19 ± 6 |
15 ± 1 |
41 ± 7 |
113 ± 18 |
54 ± 4 |
100 µg |
18 ± 3 |
16 ± 3 |
40 ± 4 |
121 ± 13 |
51 ± 3 |
33 µg |
20 ± 1 |
18 ± 2 |
55 ± 13 |
116 ± 18 |
56 ± 1 |
PC |
|
|
|
|
|
2AA (2.5 µg) |
302 ± 24 |
271 ± 15 |
2094 ± 279 |
1687 ± 280 |
- |
2AA (10 µg) |
- |
- |
- |
- |
349 ± 17 |
NC = Negative Control; SC = Solvent control; PC = Positive control substances; SD = standard deviation; NaN3 = sodium azide; 4NOPD = 4-nitro-o-phenylene-diamine; MMS = methyl methane sulfonate; 2AA = 2-aminoan-thracene P = Precipitate |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In vitro
- Gene mutation in bacteria
The bacterial gene mutation assay (Ames test) was conducted in compliance with OECD guideline 471 and under GLP conditions (Sokolowski, 2010). The assay was performed in two independent experiments both performed in the absence and in the presence of liver microsomal activation system (S9 mix). In the first experiment (pre-experiment), the direct plate incorporation procedure was conducted with the Salmonella typhimurium strains TA 98, TA 100, TA 1535 and TA 1537 and the Escherichia coli strain WP2 uvrA at concentrations ranging from 3 to 5000 µg/plate for a period of 48 h. In the second experiment, concentrations ranging from 33 to 5000 µg/plate were analysed using the same strains, but with modification of the study design (with pre-incubation period of 60 min). No signs of cytotoxicity, evident as a reduction in the number of revertants, were observed in both experiments. Precipitation of the test substance occurred at doses = 2500 µg/plate, but did not influence data evaluation. The number of revertants was not increased at any concentrations tested. The positive and negative controls included showed the expected results in each experiment. Under the experimental conditions reported, the test substance did not induce mutations in the bacterial mutation assay in the absence and presence of metabolic activation in the selected strains of S. typhimurium (TA 98, TA 100, TA 1535 and TA 1537) and E. coli WP2 uvrA.
- Chromosome aberrations
The test substance Y-15866 was assayed in an in vitro mammalian chromosome aberration test conducted in accordance with GLP and similar to OECD guideline 473 (Hall, 2010). In two independent experiments, V79 Chinese hamster cells were treated with the test substance at concentrations up to 6950 µg/mL, with and without metabolic activation system (S9 mix). In the first experiment, continuous treatment with the test substance for 4 h followed by a 14 h-recovery period was performed. Since no clear cytotoxicity indicated by reduced mitotic indices or reduced cell numbers was observed up to the highest concentration, 6950 µg/mL was chosen as top treatment concentration in Experiment II. In this experiment, cells were analogously treated with the test substance in the presence of S9 (4 h + 14 h recovery period). In the absence of S9, cells were continuously treated for a period of 18 h without recovery period. Since no dose-response relationship was observed for cytotoxic effects in both experiments, concentrations used for evaluation of chromosome aberrations were selected based on the highest non-cytotoxic concentration tested. Thus, concentrations chosen for chromosome analysis ranged from 156.3 to 6950 µg/mL in Experiment I and 39.1 to 6950 µg/mL in Experiment II. In both experiments, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed after treatment with the test substance in the absence and presence of S9 mix. The positive control substances yielded the expected results. Under the conditions of this chromosome aberration assay, it was concluded that Y-15866 did not show clastogenic activity in V79 Chinese hamster cells.
Justification for classification or non-classification
The available data on genetic toxicity of Y-15866 are conclusive but not sufficient for classification according to the CLP (1272/2008/EC) and DSD (67/548/EEC) criteria.
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