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EC number: 931-468-2 | CAS number: 1190625-94-5
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Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 21 August 2014 to 25 October 2014
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Study conducted to GLP in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 014
- Report date:
- 2014
Materials and methods
Test guidelineopen allclose all
- 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
- Type of assay:
- in vitro mammalian chromosome aberration test
Test material
- Reference substance name:
- C14-16-18 Alkyl phenol
- EC Number:
- 931-468-2
- Cas Number:
- 1190625-94-5
- Molecular formula:
- For substance information - see Section 1.2
- IUPAC Name:
- C14-16-18 Alkyl phenol
- Test material form:
- other: slightly viscous liquid
- Details on test material:
- - Appearance: Clear amber liquid
- Storage conditions of test material: At room temperature in the dark
Constituent 1
Method
Species / strain
- Species / strain / cell type:
- lymphocytes: Cultured peripheral human lymphocytes
- Details on mammalian cell type (if applicable):
- - Blood samples: Blood was collected from healthy adult, non-smoking, male volunteers by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Culture medium: Culture medium consisted of RPMI 1640 medium, supplemented with 20 % (v/v) heat-inactivated (56 °C; 30 minutes) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively) and 30 U/mL heparin.
- Lymphocyte cultures: Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 mL (9 mg/mL) phytohaemagglutinin was added.
- Environmental conditions: All incubations were carried out in a controlled environment, in which optimal conditions were humid atmosphere of 80 to 100 % (actual range 60 to 91 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.0 to 37.4 °C). - Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- - Range-finding test: 0.54, 1.7, 5.4, 17, 52, 164 and 512 µg/mL (without metabolic activation; 24 hour exposure time, 24 hour fixation time; 48 hour exposure time, 48 hour fixation time)
- First cytogenetic assay: 5.4, 17 and 52 µg/mL (with and without metabolic activation; 3 hour exposure time, 24 hour fixation time)
- Second cytogenetic assay: 10, 30, 40, 50, 60 and 70 µg/mL (without metabolic activation; 24 hour exposure time, 24 hour fixation time; 48 hour exposure time, 48 hour fixation time) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: ethanol absolute. The final concentration of the solvent in the exposure medium was 0.5 % (v/v).
Controls
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- mitomycin C
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium. The cytogenetic assay was carried out as described by Evans, 1984 with minor modifications.
- Cytogenetic assay 1A: Lymphocytes were cultured for 48 ± 2 hours and thereafter exposed to selected doses for 3 hours in the absence and presence of S9-mix. After 3 hours of exposure, the cells were separated from the exposure medium by centrifugation (5 minutes, 365 x g). The supernatant was removed and the cells were rinsed once with 5 mL Hank’s Balanced Salt Solution (HBSS). After a second centrifugation step, HBSS was removed and cells were re-suspended in 5 mL culture medium and incubated for another 20 to 22 hours (24 hour fixation time).
- Second cytogenetic assay: Lymphocytes were cultured for 48 ± 2 hours and thereafter exposed to selected doses for 24 and 48 hours in the absence of S9-mix. The cells were not rinsed after exposure but were fixed immediately after 24 and 48 hours (24 and 48 hour fixation time).
SPINDLE INHIBITOR: colchicine
STAIN: Giemsa
NUMBER OF REPLICATIONS: Experiments were carried out in duplicate
CHROMOSOME PREPARATION
During the last 2.5 to 3 hours of the culture period, cell division was arrested by the addition of colchicine (0.5 µg/mL medium). Thereafter the cell cultures were centrifuged for 5 minutes at 365 x g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 minute treatment with hypotonic 0.56 % (w/v) potassium chloride solution at 37 °C. After hypotonic treatment, cells were fixed with 3 changes of methanol: acetic acid fixative (3:1 v/v).
PREPARATION OF SLIDES
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96 % (v/v) ethanol/ether and cleaned with a tissue. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 to 30 minutes with 5 % (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene/pertex and mounted with a coverslip in an automated coverslipper.
NUMBER OF CELLS EVALUATED: One hundred metaphase chromosome spreads per culture were examined
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5 %). At least three analysable concentrations were used for scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analysed from those cultures with an inhibition of the mitotic index of about 50 % or above whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also, cultures treated with an intermediate dose were examined for chromosome aberrations. In case the test material was not cytotoxic and/or difficult to dissolve in aqueous solutions, the highest concentration analysed at the 3 hour exposure time was determined by the solubility in the culture medium. If dose related cytotoxicity was observed, the highest concentration analysed at the 24 and 48 hour continuous exposure times was based on toxicity irrespective of the solubility of the test material in the culture medium. However, the extent of precipitation should not interfere with the scoring of chromosome aberrations.
ANALYSIS OF SLIDES
One hundred metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was = 25 in 50 metaphases, no more metaphases were examined. Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with aberrations and the number of aberrations were calculated. - Evaluation criteria:
- A test material is considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.
A test material was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations. - Statistics:
- The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics:
¿² = [(N – 1)(ad – bc)²] / [(a+b) (c+d) (a+c) (b+d)]
where:
b = the total number of aberrant cells in the control cultures
d = the total number of non-aberrant cells in the control cultures
n0 = the total number of cells scored in the control cultures
a = the total number of aberrant cells in treated cultures to be compared with the control
c = the total number of non-aberrant cells in treated cultures to be compared with the control
n1 = the total number of cells scored in the treated cultures
N = sum of n0 and n1
If P[¿² = [(N – 1)(ad – bc)²] / [(a+b) (c+d) (a+c) (b+d)]] (one-tailed) is small (p< 0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent control group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95 % confidence level.
Biological relevance is evaluated against historical control ranges; any increase in aberrant cells remaining within the historical control data ranges is considered to be not biologically relevant.
Results and discussion
Test results
- Species / strain:
- lymphocytes: Cultured peripheral human lymphocytes
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- DOSE RANGE FINDING TEST / FIRST CYTOGENETIC ASSAY
At a concentration of 52 µg/mL, the test material precipitated in the culture medium. The pH and osmolarity at a concentration of 17 µg/mL were determined to be 7.7 and 371 mOsm/kg, respectively (compared to 7.8 and 281 mOsm/kg in the solvent control).
Table 1 shows the mitotic index of cultures.
All dose levels were selected for scoring of chromosome aberrations. Both in the absence and presence of S9-mix, the test material did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations (Table 2).
Both in the absence and presence of S9-mix, the test material did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.
SECOND CYTOGENETIC ASSAY
Table 1 shows the mitotic index of cultures. Based on these observations, the following doses were selected for scoring of chromosome aberrations:
Without S9-mix: 10, 40 and 50 µg/mL culture medium (24 hour exposure time, 24 hour fixation time); 10, 30 and 40 µg/mL culture medium (48 hour exposure time, 48 hour fixation time).
The test material did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations (Table 3).
The test material did not increase the number of polyploid cells and cells with endoreduplicated chromosomes.
CONTROLS
The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly. - Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
Any other information on results incl. tables
Table 1: Mitotic Index
Test Material Concentration (µg/mL) |
± Metabolic Activation |
Exposure Time (hours) |
Fixation Time (hours) |
Number of Metaphases* |
Percentage of Control |
|||
Absolute |
Number of Cells Scored |
|||||||
Range-finder |
||||||||
Control |
-S9 |
24 |
24 |
78 |
- |
1042 |
- |
100 |
0.54 |
52 |
- |
1008 |
- |
67 |
|||
1.7 |
69 |
- |
999 |
- |
88 |
|||
5.4 |
64 |
- |
1005 |
- |
812 |
|||
17 |
79 |
- |
1002 |
- |
101 |
|||
52¹ |
24 |
- |
1005 |
- |
31 |
|||
164² |
16 |
- |
1005 |
- |
21 |
|||
512¹ |
2 |
- |
1009 |
- |
3 |
|||
Control |
-S9 |
48 |
48 |
33 |
- |
1004 |
- |
100 |
054 |
25 |
- |
1007 |
- |
76 |
|||
1.7 |
34 |
- |
1012 |
- |
103 |
|||
5.4 |
54 |
- |
1004 |
- |
164 |
|||
17 |
46 |
- |
1020 |
- |
139 |
|||
52¹ |
17 |
- |
1002 |
- |
52 |
|||
164¹ |
2 |
- |
1007 |
- |
6 |
|||
512¹ |
1 |
- |
1003 |
- |
3 |
|||
Assay 1A |
||||||||
Control |
-S9 |
3 |
24 |
93 |
86 |
1003 |
1003 |
100 |
5.4 |
94 |
85 |
1011 |
1016 |
100 |
|||
17 |
88 |
73 |
1001 |
1013 |
90 |
|||
52¹ |
99 |
77 |
1003 |
1011 |
98 |
|||
MMC 0.5 |
62 |
61 |
1008 |
1005 |
69 |
|||
MMC0.75 |
42 |
59 |
1006 |
1002 |
56 |
|||
Control |
+S9 |
3 |
24 |
74 |
77 |
1008 |
1004 |
100 |
5.4 |
74 |
50 |
1002 |
1015 |
82 |
|||
17 |
73 |
79 |
1002 |
1003 |
101 |
|||
52¹ |
72 |
87 |
1004 |
1011 |
105 |
|||
CP 10 |
43 |
47 |
1005 |
1004 |
60 |
|||
Second Cytogenetic assay |
||||||||
Control |
-S9 |
24 |
24 |
76 |
73 |
1000 |
1000 |
100 |
10 |
71 |
72 |
1002 |
1001 |
96 |
|||
30 |
65 |
62 |
1000 |
1003 |
85 |
|||
40¹ |
51 |
58 |
1000 |
1010 |
73 |
|||
50¹ |
38 |
30 |
1004 |
1002 |
46 |
|||
60¹ |
19 |
14 |
1000 |
1000 |
22 |
|||
70¹ |
6 |
7 |
1000 |
1000 |
9 |
|||
MMC 0.2 |
36 |
39 |
1000 |
1004 |
50 |
|||
MMC 0.3 |
24 |
21 |
1000 |
1004 |
30 |
|||
Control |
-S9 |
48 |
48 |
70 |
69 |
1005 |
1041 |
100 |
10 |
60 |
69 |
1001 |
1004 |
93 |
|||
30 |
57 |
55 |
1000 |
1000 |
81 |
|||
40¹ |
31 |
37 |
1005 |
1000 |
49 |
|||
50¹ |
18 |
21 |
1002 |
1006 |
28 |
|||
60¹ |
12 |
10 |
998 |
1002 |
16 |
|||
70¹ |
5 |
4 |
1000 |
1000 |
6 |
|||
MMC 0.1 |
37 |
31 |
1000 |
1004 |
49 |
|||
MMC 0.15 |
31 |
25 |
1003 |
1000 |
40 |
*Duplicate cultures with the exception of the range-finding test
¹The test material precipitated in the culture medium
² The test material precipitated in the culture medium. Mean of 2 scorings (28 metaphases in 1004 cells and 4 metaphases in 1006 cells)
MMC = Mitomycin C
CP = Cyclophosphamide
Table 2: Chromosome Aberrations in the First Cytogenetic Assay
|
Concentration (µg/mL) |
|||||||||
3 Hour Exposure Time, 24 Hour Fixation Time (-S9) |
3 Hour Exposure Time, 24 Hour Fixation Time (+S9) |
|||||||||
Control |
5.4 |
17 |
52 |
MMC 0.5 |
Control |
5.4 |
17 |
52 |
CP 10 |
|
Culture |
A + B |
A + B |
A + B |
A + B |
A + B |
A + B |
A + B |
A + B |
A + B |
A + B |
MI (%) |
100 |
100 |
90 |
98 |
69 |
100 |
82 |
101 |
105 |
60 |
No. of cells scored |
200 |
200 |
200 |
200 |
200 |
200 |
200 |
200 |
200 |
200 |
No. of cells with aberrations (+ gaps) |
0 |
0 |
1 |
0 |
65*** |
2 |
0 |
0 |
4 |
69*** |
No. of cells with aberrations (- gaps) |
0 |
0 |
0 |
0 |
65*** |
1 |
0 |
0 |
3 |
69*** |
Total aberrations (+ gaps) |
0 |
0 |
1 |
0 |
88 |
2 |
0 |
0 |
4 |
105 |
Total aberrations (- gaps) |
0 |
0 |
0 |
0 |
86 |
1 |
0 |
0 |
3 |
105 |
MMC = Mitomycin C
CP = Cyclophosphamide
MI = Mitotic index
***Significantly different from the control group (Chi-square test), p< 0.001
Table 3: Chromosome Aberrations in the Second Cytogenetic Assay
|
Concentration (µg/mL) |
|||||||||
24 Hour Exposure Time, 24 Hour Fixation Time (-S9) |
48 Hour Exposure Time, 48 Hour Fixation Time (-S9) |
|||||||||
Control |
10 |
40 |
50 |
MMC 0.2 |
Control |
10 |
30 |
40 |
MMC 0.1 |
|
Culture |
A + B |
A + B |
A + B |
A + B |
A + B |
A + B |
A + B |
A + B |
A + B |
A + B |
MI (%) |
100 |
96 |
73 |
46 |
50 |
100 |
93 |
81 |
49 |
49 |
No. of cells scored |
200 |
200 |
200 |
200 |
200 |
200 |
200 |
200 |
200 |
200 |
No. of cells with aberrations (+ gaps) |
0 |
0 |
2 |
2 |
61*** |
1 |
2 |
3 |
1 |
66*** |
No. of cells with aberrations (- gaps) |
0 |
0 |
1 |
1 |
60*** |
1 |
2 |
2 |
1 |
66*** |
Total aberrations (+ gaps) |
0 |
0 |
2 |
2 |
73 |
1 |
2 |
3 |
1 |
81 |
Total aberrations (- gaps) |
0 |
0 |
1 |
1 |
71 |
1 |
2 |
2 |
1 |
78 |
MMC = Mitomycin C
CP = Cyclophosphamide
MI = Mitotic index
***Significantly different from the control group (Chi-square test), p< 0.001
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information):
negative with and without metabolic activation
Under the conditions of this study the test material is not clastogenic in human lymphocytes with and without metabolic activation. - Executive summary:
The ability of the test material to induce chromosome aberrations in cultured peripheral human lymphocytes was evaluated in a study conducted in accordance with the standardised guidelines OECD 473 and EU Method B.10 under GLP conditions.
The study investigates the effect of the test material on the number of chromosome aberrations in cultured peripheral human lymphocytes in the presence and absence of a metabolic activation system (phenobarbital and ß-naphthoflavone induced rat liver S9-mix).
Experiments were carried out in duplicate and concurrent solvent (ethanol) and positive controls took place.
The dose levels tested were as follows:- Range-finding test: 0.54, 1.7, 5.4, 17, 52, 164 and 512 µg/mL (without metabolic activation; 24 hour exposure time, 24 hour fixation time; 48 hour exposure time, 48 hour fixation time)
- First cytogenetic assay: 5.4, 17 and 52 µg/mL (with and without metabolic activation; 3 hour exposure time, 24 hour fixation time)
- Second cytogenetic assay: 10, 30, 40, 50, 60 and 70 µg/mL (without metabolic activation; 24 hour exposure time, 24 hour fixation time; 8 hour exposure time, 48 hour fixation time)
In the first cytogenetic assay, the test material precipitated in the culture medium at 52 µg/mL. In the second cytogenetic assay, appropriate toxicity was reached at the dose levels.
The test material did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.
No effects of the test material on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test material does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions used.
The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The positive controls both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
Under the conditions of this study the test material is not clastogenic in human lymphocytes with and without metabolic activation.
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