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EC number: 203-402-6 | CAS number: 106-48-9
- Life Cycle description
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Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2019-08-14 to 2019-10-20
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- other: Guideline study performed under GLP. All relevant validity criteria were met.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian cell micronucleus test
Test material
- Reference substance name:
- 4-chlorophenol
- EC Number:
- 203-402-6
- EC Name:
- 4-chlorophenol
- Cas Number:
- 106-48-9
- Molecular formula:
- C6H5ClO
- IUPAC Name:
- 4-chlorophenol
- Test material form:
- solid
- Details on test material:
- Batch (Lot) Number: 0011968273
Expiry date: 23 December 2019
Physical Description: Colourless to beige blocks
Purity/Composition: 99.46%
Storage Conditions: In refrigerator (2-8°C)
Constituent 1
Method
Species / strain
- Species / strain / cell type:
- lymphocytes: Human
- Details on mammalian cell type (if applicable):
- - Type and identity of media: RPMI 1640 medium, supplemented with heat-inactivated fetal calf serum, L-glutamine, penicillin/streptomycin and heparin.
- Cells obtained from healthy non-smoking human volunteers who had not knowingly either been exposed to high levels of radiation or hazardous chemicals, or had a viral infection.
- The cell-cycle time was determined using BrdU (bromodeoxyuridine) and the average generation time (AGT) calculated. The AGT was 12.7-14.0h. - Additional strain / cell type characteristics:
- not applicable
- Cytokinesis block (if used):
- Cytochalasin B: prior to cell harvest at final concentration of 5 μg/mL for 24 hours
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbitone/β-Naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- - Preliminary toxicity test: The dose range of test item used was 31 to 1285 μg/mL (0.01M).
- Experiment I: 50, 250, 350, 400, 450, 500, 550 and 600 μg/mL (-S9); 50, 250, 350, 400, 450 and 500 μg/mL (+S9)
- Experiment II: 1, 10, 15, 20, 50, 60, 70 and 80 μg/mL, without metabolic activation - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test item was soluble in DMSO at all tested concentrations, solubility checks were performed in-house
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- Solvent for positive control: Hank's balanced salt solution without Ca or Mg.
- Positive control substance:
- mitomycin C
- Remarks:
- without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- Solvent for positive control: Hank's balanced salt solution without Ca or Mg.
- Positive control substance:
- cyclophosphamide
- Remarks:
- with metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- True negative controls:
- no
- Positive controls:
- yes
- Remarks:
- Solvent for positive control: Hank's balanced salt solution without Ca or Mg.
- Positive control substance:
- colchicine
- Remarks:
- without metabolic activation
- Details on test system and experimental conditions:
- ACTIVATION: The S9-mix was prepared prior to the dosing of the test cultures and contained the S9 fraction (20% (v/v)), MgCl2 (8mM), KCl (33mM), sodium orthophosphate buffer pH 7.4 (100mM), glucose-6-phosphate (5mM) and NADP (5mM). The final concentration of S9, when dosed into culture media, was 1.8% (v/v).
METHOD OF APPLICATION: in medium
DURATION
- Exposure duration:
Experiment I: 3 hours, with and without metabolic activation
Experiment II: 24 hours, without metabolic activation
- Expression time (cells in growth medium):
Experiment I: 24 hours with Cytochalasin B, with and without metabolic activation
Experiment II: 24 hours with Cytochalasin B, without metabolic activation
CYTOKINESIS BLOCK: Cytochalasin B (5 μg/mL)
STAIN (for cytogenetic assays): 6.7% Giemsa for 10-30 minutes, rinsed, dried and a cover slip applied using mounting medium
NUMBER OF REPLICATIONS: duplicate cell cultures (A and B) for each dose level
NUMBER OF CELLS EVALUATED: At least 1000 cells per culture
CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
The criteria for identifying micronuclei were that they were round or oval in shape, nonrefractile, not linked to the main nuclei and with a diameter that was approximately less than a third of the mean diameter of the main nuclei. Binucleate cells were selected for scoring if they had two nuclei of similar size with intact nuclear membranes situated in the same cytoplasmic boundary. The two nuclei could be attached by a fine nucleoplasmic bridge which was approximately no greater than one quarter of the nuclear diameter.
DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis Block Proliferation Index (CBPI) - Evaluation criteria:
- The frequency of binucleate cells with micronuclei in the vehicle control cultures will normally be within the range of the laboratory historical control data. The frequency of spontaneous background micronuclei may be slightly elevated above the normal range and the experiment still considered valid.
All the positive control chemicals must induce positive responses (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix. - Statistics:
- A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of cells with micronuclei which was reproducible.
Results and discussion
Test results
- Species / strain:
- lymphocytes: Human
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no significant change on pH was remarked.
- Effects of osmolality: osmolality did not increase by more than 50 mOsm
- Precipitation: no precipitation of the test material was observed at any of the test concentrations
RANGE-FINDING/SCREENING STUDIES:
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: See Table 1, 2, 3, 4 and 5.
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: A minimum of 1000 cells per culture were scored.
- Indication whether binucleate or mononucleate where appropriate: See table 1, 2, 3, 4 and 5. The vehicle control cultures had frequencies of binucleate cells with micronuclei within the expected range. The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: See tables 1, 2, 3, 4 and 5. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. All the positive control chemicals must induce positive responses (p≤0.01). Acceptable positive responses demonstrate the validity of the experiment and the integrity of the S9-mix.
- Negative (solvent/vehicle) historical control data: The frequency of spontaneous binucleate cells with micronuclei in the vehicle control cultures was within the range of the laboratory historical control data.
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI cytokinesis-block method (see tables 1, 2, 3, 4 and 5).
Any other information on results incl. tables
Table 1
Cytokinesis-Block Proliferation Index of Human
Lymphocytes Cultures Treated with 4-chlorophenol in the First
Cytogenetic Assay
Without metabolic activation (-S9-mix) |
|||||||
3 hours exposure time, 27 hours harvest time |
|||||||
Concentration µg/mL |
CBPI |
Mean CBPI |
% cytostasis |
||||
0 |
1.53 |
- |
1.56 |
1.55 |
0 |
||
10 |
1.53 |
- |
1.54 |
1.54 |
2 |
||
50 |
1.49 |
- |
1.52 |
1.50 |
8 |
||
200 |
1.41 |
- |
1.42 |
1.42 |
24 |
||
250 |
1.40 |
- |
1.42 |
1.41 |
25 |
||
300 |
1.36 |
- |
1.39 |
1.37 |
32 |
||
0.25 MMC-C |
1.30 |
- |
1.34 |
1.32 |
42 |
||
0.38 MMC-C |
1.26 |
- |
1.30 |
1.28 |
48 |
||
0.1 Colch |
1.14 |
- |
1.16 |
1.15 |
73 |
||
With metabolic activation (+S9-mix) |
|||||||
3 hours exposure time, 27 hours harvest time |
|||||||
Concentration µg/mL |
CBPI |
Mean CBPI |
% cytostasis |
||||
0 |
1.57 |
- |
1.60 |
1.59 |
0 |
||
50 |
1.53 |
- |
1.55 |
1.54 |
8 |
||
250 |
1.42 |
- |
1.43 |
1.43 |
28 |
||
350 |
1.37 |
- |
1.40 |
1.38 |
35 |
||
400 |
1.36 |
- |
1.37 |
1.37 |
38 |
||
450 |
1.30 |
- |
1.32 |
1.31 |
47 |
||
500 |
1.26 |
- |
1.27 |
1.27 |
54 |
||
15 CP |
1.21 |
- |
1.23 |
1.22 |
62 |
||
17.5 CP |
1.19 |
- |
1.19 |
1.19 |
67 |
Note: All calculations were performed without rounding off.
Table 2
Cytokinesis-Block Proliferation Index of Human
Lymphocytes Cultures Treated with 4-chlorophenol in the Cytogenetic
Assay 1A
Without metabolic activation (-S9-mix) |
|||||||
3 hours exposure time, 27 hours harvest time |
|||||||
Concentration µg/mL |
CBPI |
Mean CBPI |
% cytostasis |
||||
0 |
1.39 |
- |
1.43 |
1.41 |
0 |
||
50 |
1.42 |
- |
1.45 |
1.44 |
-6 |
||
250 |
1.34 |
- |
1.38 |
1.36 |
12 |
||
350 |
1.29 |
- |
1.34 |
1.31 |
24 |
||
400 |
1.24 |
- |
1.27 |
1.26 |
38 |
||
450 |
1.21 |
- |
1.23 |
1.22 |
47 |
||
500 |
1.13 |
- |
1.13 |
1.13 |
67 |
||
550 |
1.11 |
- |
1.13 |
1.12 |
70 |
||
600 |
1.00 |
- |
1.02 |
1.01 |
98 |
||
0.25 MMC-C |
1.24 |
- |
1.28 |
1.26 |
36 |
||
0.38 MMC-C |
1.20 |
- |
1.22 |
1.21 |
48 |
||
0.1 Colch |
1.01 |
- |
1.02 |
1.01 |
97 |
||
Table 3
Number of Mononucleated or Binucleated Cells with
Micronuclei of Human Lymphocyte Cultures Treated with 4-chlorophenol in
the First Cytogenetic Assay and Cytogenetic Assay 1A
Without metabolic activation (-S9-mix) |
||||||||
3 hours exposure time, 27 hours harvest time |
||||||||
Concentration (µg/mL) |
Cytostasis (%) |
Number of mononucleated cells with micronuclei1) |
Number of binucleated cells with micronuclei1) |
|||||
1000 |
1000 |
2000 |
1000 |
1000 |
2000 |
|||
A |
B |
A+B |
A |
B |
A+B |
|||
0 |
0 |
1 |
0 |
1 |
2 |
4 |
6 |
|
50 |
-6 |
2 |
0 |
2 |
0 |
3 |
3 |
|
350 |
24 |
1 |
0 |
1 |
2 |
5 |
7 |
|
450 |
47 |
4 |
2 |
6* |
7 |
4 |
11 |
|
500 |
67 |
3 |
2 |
5 |
3 |
8 |
11 |
|
0.25 MMC-C |
36 |
3 |
0 |
3 |
22 |
17 |
39*** |
|
0.1 Colch |
97 |
31 |
25 |
56*** |
202) |
22) |
22*** |
With metabolic activation (+S9-mix) |
||||||||
3 hours exposure time, 27 hours harvest time |
||||||||
Concentration (µg/mL) |
Cytostasis (%) |
Number of mononucleated cells with micronuclei 1) |
Number of binucleated cells with micronuclei 1) |
|||||
1000 |
1000 |
2000 |
1000 |
1000 |
2000 |
|||
A |
B |
A+B |
A |
B |
A+B |
|||
0 |
0 |
1 |
0 |
1 |
2 |
3 |
5 |
|
50 |
8 |
2 |
2 |
4 |
3 |
3 |
6 |
|
400 |
38 |
1 |
3 |
4 |
5 |
2 |
7 |
|
500 |
54 |
1 |
1 |
2 |
2 |
3 |
5 |
|
15 CP |
62 |
3 |
1 |
4 |
41 |
36 |
77*** |
*)Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.
1)1000 – 1010 bi- and mononucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.
2)931 and 781 binucleated cells were scored for the presence of micronuclei, respectively.
Table4
Cytokinesis-Block Proliferation Index of Human
Lymphocyte Cultures Treated with4-chlorophenol in the Second Cytogenetic
Assay
Without metabolic activation (-S9-mix) |
|||||||
24 hours exposure time, 24 hours harvest time |
|||||||
Concentration µg/mL |
CBPI |
Mean CBPI |
% cytostasis |
||||
0 |
1.57 |
- |
1.58 |
1.58 |
0 |
||
1 |
1.49 |
- |
1.50 |
1.49 |
15 |
||
10 |
1.44 |
- |
1.47 |
1.46 |
21 |
||
15 |
1.38 |
- |
1.44 |
1.41 |
28 |
||
20 |
1.42 |
- |
1.50 |
1.46 |
20 |
||
50 |
1.38 |
- |
1.40 |
1.39 |
33 |
||
60 |
1.37 |
- |
1.38 |
1.38 |
35 |
||
70 |
1.28 |
- |
1.29 |
1.29 |
50 |
||
80 |
1.31 |
- |
1.32 |
1.31 |
46 |
||
0.15 MMC-C |
1.25 |
- |
1.30 |
1.27 |
53 |
||
0.23 MMC-C |
1.21 |
- |
1.23 |
1.22 |
61 |
||
0.05 Colch |
1.01 |
- |
1.01 |
1.01 |
98 |
Note: All calculations were performed without rounding off.
Table 5
Number of Mononucleated or Binucleated Cells with
Micronuclei of Human Lymphocyte Cultures Treated with 4-chlorophenol in
the Second Cytogenetic Assay
Without metabolic activation (-S9-mix) |
||||||||
24 hours exposure time, 24 hours harvest time |
||||||||
Concentration (µg/mL) |
Cytostasis (%) |
Number of mononucleated cells with micronuclei1) |
Number of binucleated cells with micronuclei1) |
|||||
1000 |
1000 |
2000 |
1000 |
1000 |
2000 |
|||
A |
B |
A+B |
A |
B |
A+B |
|||
0 |
0 |
1 |
1 |
2 |
3 |
3 |
6 |
|
1 |
15 |
0 |
1 |
1 |
3 |
4 |
7 |
|
15 |
28 |
2 |
2 |
4 |
2 |
1 |
3 |
|
70 |
50 |
0 |
1 |
1 |
1 |
3 |
4 |
|
0.15 MMC-C |
53 |
5 |
4 |
9* |
27 |
21 |
48*** |
|
0.05 Colch |
98 |
212) |
17 |
38*** |
03) |
23) |
2** |
*)Significantly different from control group (Chi-square test), * P < 0.05, ** P < 0.01 or *** P < 0.001.
1)1000 bi- and mononucleated cells were scored for the presence of micronuclei.
Duplicate cultures are indicated by A and B.
2)917 mononucleated cells were scored for the presence of micronuclei.
3)43 and 70 binucleated cells were scored for the presence of micronuclei, respectively.
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results: negative with and without metabolic activation
Under the conditions of this study, the test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro. - Executive summary:
The study was performed to the requirements of OECD TG 487 : guidelines under GLP conditions to assess within the in vitro cell micronucleus assay the clastogenic and aneugenic potential of the test item to the nuclei of normal human lymphocytes. Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions were used for the study. Experiment 1 used a 3-hour exposure in the presence and absence of a standard metabolizing system (S9, at a 1.8% final concentration). Experiment 2, used a 24 -hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B. The dose levels used in the main experiments were selected using data from the preliminary toxicity test. The dose levels were as follows: 3 -hour without S9 -Mix: 50, 250, 350, 400, 450, 500, 550 and 600 µg/mL culture medium; 3 -hour with S9 -Mix (1.8%): 50, 250, 350, 400, 450 and 500 µg/mL culture medium. In a second experiment the dose levels were 24 -hour without S9: 1, 10, 15, 20, 50, 60, 70 and 80 µg/mL culture medium. All vehicle (dimethyl sulphoxide) controls had frequencies of binucleate cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected. The test item did not induce any statistically significant increases in the frequency of cells with micronuclei, in either of the two experiments, using a dose range that included a dose level that induced approximately a 50% reduction in CBPI in all three exposure groups. Under the conditions of this study, the test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.
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