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EC number: 205-581-6 | CAS number: 143-06-6
- 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
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
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- Nanomaterial surface chemistry
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- Endpoint summary
- Stability
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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
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro cytogenicity / micronucleus study
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 2016-12-19 to 2017-03-15
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell micronucleus test
Test material
- Reference substance name:
- (6-aminohexyl)carbamic acid
- EC Number:
- 205-581-6
- EC Name:
- (6-aminohexyl)carbamic acid
- Cas Number:
- 143-06-6
- Molecular formula:
- C7H16N2O2
- IUPAC Name:
- (6-aminohexyl)carbamic acid
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: INTERBUSINESS S.r.l. (Via Spartaco, 2520135 Milano, Italy); batch no. 6061
- Expiration date of the lot/batch: 2018-03-01
- Purity test date: 2016-03-17
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: prepared immediately before use in culture medium. No assay of test item stability, nor its concentration and homogeneity in solvent were under-taken, as not requested by the Sponsor
OTHER SPECIFICS:
Identity of the test material: INTERCURE®1
Method
Species / strain
- Species / strain / cell type:
- lymphocytes: human
- Details on mammalian cell type (if applicable):
- CELLS USED
- Source of cells: For the first Main Experiment, two batches of human whole blood, provided by Biopredic International (France), were pooled. For the second Main Experiment, one batch of human whole blood, provided by Biopredic International (France) was used
- Suitability of cells: Not specified
- Sex, age and number of blood donors if applicable: First Experiment: 1) Male (n=1); Age = 23 years; 2) Female (n = 1); Age: 27 Years. Second Experiment: 1) Female (n = 1); Age: 22 Years
- Whether whole blood or separated lymphocytes were used if applicable: separated lymphocytes
MEDIA USED
- Type and identity of media including CO2 concentration if applicable: culture medium (composition shown below) was used for lymphocytes:
RPMI 1640 1x (Dutch modification) 500 mL
Foetal Calf Serum 100 mL
L-Glutamine (200 mM) 6.25 mL
Antibiotic solution 1.25 mL
The foetal calf serum was heat-inactivated at 56°C for 20 minutes before use. For the initiationof the cultures, medium with the addition of phytohaemagglutin (PHA) was used in thefollowing proportion: 10 mL of PHA was added to 500 mL of medium.
- Properly maintained: yes - Additional strain / cell type characteristics:
- not specified
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital – 5,6-Benzoflavone induced Rat liver
- Test concentrations with justification for top dose:
- General dose selection procedure:
Cytotoxicity was determined for each of the treatment levels and the highest dose level for genotoxicity assessment was selected as a dose which produces approximately 55 ± 5% cytotoxicity compared to solvent control. If the test item does not induce toxicity at any dose level, then the highest treatment level is selected as the highest dose level for scoring.
Two lower dose levels were also selected for the scoring of micronuclei. The lowest dose level for scoring should be on the borderline of cytotoxicity. The intermediate dose level are evenly spaced between the two.
The test item was found to be soluble in culture medium at the concentration of 16.0 mg/mL after few minutes of vortex stirring. This concentration was chosen since, when added to the treatment medium in the ratio 1:10, it gave a maximum dose level of 1600 µg/mL corresponding to 10.0 mMwhich is lower than 2000 µg/mL.
Two main experiments were performed. Based on the preliminary solubility assay, dose levels of 1600, 1070, 711, 474, 316, 211, 140, 93.6, 62.4 and 41.6 µg/mL were used for the first main experiment. Based on these results, the dose for the main experiment were 1600, 1070 and 711 µg/mL. - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: Test material was solubilized in the culture medium
- Justification for choice of solvent/vehicle: not specified
Controls
- Untreated negative controls:
- yes
- Remarks:
- Untreated cultures were included in the experimental scheme and acted as reference contro
- Negative solvent / vehicle controls:
- yes
- Remarks:
- culture medium (RPMI 1640, Dutch modification, ob-tained from Gibco, supplemented with fetal calf serum, antiobiotic solution and L-glutamine)
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- other: Colchicine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 48 hours
- Exposure duration: 3 hours followed by further incubation for 28 hours (recovery period)
- Expression time (cells in growth medium): 31 hours
SPINDLE INHIBITOR (cytogenetic assays): Cytochalasin-B
STAIN (for cytogenetic assays): Acridine Orange in PBS
NUMBER OF REPLICATIONS: 2
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The lymphocyte cultures were centrifuged for 10 minutes at 1000 rpm and the supernatant was removed up to approximately 5 mm from the pellet. The cells were resuspended in hypotonic solution. Fresh methanol/acetic acid fixative was then added. After centrifugation and removal of this solution, the fixative was changed several times by centrifugation and resuspension. A few drops of the cell suspension obtained in this way were dropped onto clean, wet, grease-free glass slides. Three slides were prepared for each test point and each was labelled with the identity of the culture. The slides were allowed to air dry and kept at room temperature prior to staining with a solution of Acridine Orange in PBS.
NUMBER OF CELLS EVALUATED: 1000 binucleated cells per cell culture (solvent; negative; and positive controls); For cultures treated with Colchicine 1000 mononucleated cells per cell culturewere scored
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The criteria for identifying micronuclei were as follows:
1. The micronucleus diameter was less than 1/3 of the nucleus diameter
2. The micronucleus diameter was greater than 1/16 of the nucleus diameter
3. No overlapping with the nucleus was observed
4. The aspect was the same as the chromatin
DETERMINATION OF CYTOTOXICITY
- Method: cytotoxicity of the test item treatments calculated by the cytokinesis-block proliferation index (CBPI) - Rationale for test conditions:
- According to OECD Guideline 487.
- Evaluation criteria:
- Acceptance criteria:
The experiment is considered valid when:
- The incidence of micronucleated cells of the negative control is within the distribution range of our historical control values.
- Concurrent positive controls induce responses that are compatible with those generated in our historical positive control database and produce a statistically significant increase compared with the concurrent negative control.
- Adequate cell proliferation is observed in solvent control cultures.
- The appropriate number of doses and cells are analysed
Criterion for outcome:
The assay is considered as clearly positive if:
- Significant increases in the proportion of micronucleated cells over the concurrent controls occur at one or more concentrations.
- The proportion of micronucleated cells at such data points exceeds the normal range based on historical control values.
- There is a significant dose effect relationship.
The assay is considered as clearly negative if:
- None of the dose levels shows a statistically significant increase in the incidence of micronucleated cells.
- There is no concentration related increase when evaluated with the Cochran-Armitage trend test.
- All the results are inside the distribution of the historical control data. - Statistics:
- A modified X^2 test was used to compare the number of cells with micronuclei in control and treated cultures. Cochran-Armitage Trend Test (one-sided) was performed to aid determination of concentration response relationship.
Results and discussion
Test results
- Key result
- Species / strain:
- lymphocytes: human
- 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:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Following treatment with the test item, a slight dose related increase of the pH was observed at the highest dose levels in the absence and presence of S9 metabolism
- Effects of osmolality: No remarkable variation of osmolality was observed at any dose level
- Precipitation: Following treatment with the test item, no precipitation or opacity of the medium was observed at beginning or end of treatment, in the absence or presence of S9 metabolism.
CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: See Tables 1-3.
NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells.
- Indication whether binucleate or mononucleate where appropriate: Yes, See Tables 1-3.
HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: Data were included in the statistical analysis
- Negative (solvent/vehicle) historical control data: Data were included in the statistical analysis
ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI
- Other observations when applicable: frequency of mononucleated, binucleated cells, polynucleated cells
Any other information on results incl. tables
Table 1. Main Experiment 1 – Proliferation Index Treatment: 3 hours - without S9 Sampling Time: 32 hours |
||||||||
Treatment (µg/mL) |
Culture No. |
MonoN |
BiN |
PoliN |
Total Cells |
CBPI |
Mean Value |
Cytotoxicity |
Untreated |
1 |
160 |
260 |
80 |
500 |
1.840 |
|
|
2 |
166 |
270 |
64 |
500 |
1.796 |
1.818 |
0 |
|
|
||||||||
Test Item 316 |
11 |
162 |
258 |
80 |
500 |
1.836 |
|
|
12 |
186 |
253 |
61 |
500 |
1.750 |
1.793 |
3 |
|
Test Item 474 |
9 |
180 |
223 |
97 |
500 |
1.834 |
|
|
10 |
168 |
240 |
92 |
500 |
1848 |
1.841 |
-3 |
|
Test Item 711 |
7 |
193 |
220 |
87 |
500 |
1.788 |
|
|
8 |
207 |
233 |
60 |
500 |
1.706 |
1.747 |
9 |
|
Test Item 1070 |
5 |
182 |
251 |
67 |
500 |
1.770 |
|
|
6 |
182 |
244 |
74 |
500 |
1.784 |
1.777 |
5 |
|
Test Item 1600 |
3 |
166 |
238 |
96 |
500 |
1.860 |
|
|
4 |
182 |
237 |
81 |
500 |
1.798 |
1.829 |
-1 |
MonoN: Mononucleated cells
BiN: Binucleated cells
PoliN: Polinucleated cells
CBPI: Cytokinesis block proliferation index
% Cytotoxicity: Relative to vehicle/solvent control cultures
Table 2. Main Experiment 1 – Proliferation Index Treatment: 3 hours - with S9 Sampling Time: 32 hours |
||||||||
Treatment (µg/mL) |
Culture No. |
MonoN |
BiN |
PoliN |
Total Cells |
CBPI |
Mean Value |
Cytotoxicity |
Untreated |
23 |
160 |
244 |
96 |
500 |
1.872 |
|
|
24 |
178 |
233 |
89 |
500 |
1.822 |
1.847 |
0 |
|
|
||||||||
Test Item 316 |
33 |
128 |
259 |
113 |
500 |
1.970 |
|
|
34 |
151 |
240 |
109 |
500 |
1.916 |
1.943 |
-11 |
|
Test Item 474 |
31 |
137 |
258 |
105 |
500 |
1.936 |
|
|
32 |
135 |
256 |
109 |
500 |
1.948 |
1.942 |
-11 |
|
Test Item 711 |
29 |
166 |
223 |
111 |
500 |
1.890 |
|
|
30 |
136 |
240 |
124 |
500 |
1.976 |
1.933 |
-10 |
|
Test Item 1070 |
27 |
176 |
236 |
88 |
500 |
1.824 |
|
|
28 |
146 |
232 |
122 |
500 |
1.952 |
1.888 |
-5 |
|
Test Item 1600 |
25 |
132 |
258 |
110 |
500 |
1.956 |
|
|
26 |
129 |
262 |
109 |
500 |
1.960 |
1.958 |
-13 |
|
|
||||||||
Cyclophosphamide 20.0 |
45 |
350 |
143 |
7 |
500 |
1.314 |
|
|
46 |
320 |
167 |
13 |
500 |
1.386 |
1.350 |
59 |
|
Cyclophosphamide 15.0 |
47 |
338 |
155 |
7 |
500 |
1.338 |
|
|
48 |
311 |
168 |
21 |
500 |
1.420 |
1.379 |
55 |
MonoN: Mononucleated cells
BiN: Binucleated cells
PoliN: Polinucleated cells
CBPI: Cytokinesis block proliferation index
% Cytotoxicity: Relative to vehicle/solvent control culure
Table 3. Main Experiment 2 – Proliferation Index Treatment: 31 hours - without S9 Sampling Time: 31 hours |
||||||||
Treatment (µg/mL) |
Culture No. |
MonoN |
BiN |
PoliN |
Total Cells |
CBPI |
Mean Value |
Cytotoxicity |
Untreated |
49 |
188 |
229 |
83 |
500 |
1.790 |
|
|
50 |
137 |
285 |
78 |
500 |
1.882 |
1.836 |
0 |
|
|
||||||||
Test Item 316 |
59 |
136 |
278 |
86 |
500 |
1.900 |
|
|
60 |
153 |
265 |
82 |
500 |
1.858 |
1.879 |
-5 |
|
Test Item 474 |
57 |
138 |
265 |
97 |
500 |
1.918 |
|
|
58 |
147 |
277 |
76 |
500 |
1.858 |
1.888 |
-6 |
|
Test Item 711 |
55 |
146 |
270 |
84 |
500 |
1.876 |
|
|
56 |
157 |
263 |
80 |
500 |
1.846 |
1.861 |
-3 |
|
Test Item 1070 |
53 |
203 |
248 |
49 |
500 |
1.692 |
|
|
54 |
178 |
258 |
64 |
500 |
1.772 |
1.732 |
12 |
|
Test Item 1600 |
51 |
261 |
202 |
37 |
500 |
1.552 |
|
|
52 |
263 |
220 |
17 |
500 |
1.508 |
1.530 |
37 |
|
|
||||||||
Colchicine 0.080 |
71 |
449 |
41 |
10 |
500 |
1.122 |
|
|
72 |
468 |
27 |
5 |
500 |
1.074 |
1.098 |
88 |
|
Colchicine 0.040 |
73 |
477 |
20 |
3 |
500 |
1.052 |
|
|
74 |
490 |
10 |
0 |
500 |
1.020 |
1.036 |
96 |
|
Colchicine 0.020 |
75 |
355 |
122 |
23 |
500 |
1.336 |
|
|
76 |
387 |
93 |
20 |
500 |
1.266 |
1.301 |
64 |
MonoN: Mononucleated cells
BiN: Binucleated cells
PoliN: Polinucleated cells
CBPI: Cytokinesis block proliferation index
% Cytotoxicity: Relative to vehicle/solvent control cultures
Table 4. Main Experiment 1 – Scoring of Micronuclei Treatment: 3 hours - without S9 Sampling Time: 32 hours |
||||||
Treatment (µg/mL) |
Culture No. |
Cells scored (BiN cells) |
BiN cells with 1 Mn |
BiN cells with 2 Mn |
BiN cells with >2 Mn |
BiN cells with Mn |
Untreated |
1 |
1000 |
2 |
0 |
0 |
2 |
2 |
1000 |
2 |
1 |
0 |
3 |
|
|
||||||
Test Item 711 |
7 |
1000 |
3 |
0 |
0 |
3 |
8 |
1000 |
1 |
1 |
0 |
2 |
|
Test Item 1070 |
5 |
1000 |
5 |
0 |
0 |
5 |
6 |
1000 |
2 |
0 |
0 |
2 |
|
Test Item 1600 |
3 |
1000 |
0 |
0 |
0 |
0 |
4 |
1000 |
4 |
0 |
0 |
4 |
BiN: Binucleated cells
Mn: Micronucleus/micronuclei
Table 5. Main Experiment 1 – Scoring of Micronuclei Treatment: 3 hours - with S9 Sampling Time: 32 hours |
||||||
Treatment (µg/mL) |
Culture No. |
Cells scored (BiN cells) |
BiN cells with 1 Mn |
BiN cells with 2 Mn |
BiN cells with >2 Mn |
BiN cells with Mn |
Untreated |
23 |
1000 |
3 |
0 |
0 |
3 |
24 |
1000 |
4 |
0 |
0 |
4 |
|
|
||||||
Test Item 711 |
29 |
1000 |
2 |
0 |
0 |
2 |
30 |
1000 |
5 |
0 |
0 |
5 |
|
Test Item 1070 |
27 |
1000 |
2 |
0 |
0 |
2 |
28 |
1000 |
2 |
0 |
0 |
2 |
|
Test Item 1600 |
25 |
1000 |
1 |
0 |
0 |
1 |
26 |
1000 |
3 |
0 |
0 |
3 |
|
|
||||||
Cyclophosphamide 20.0 |
45 |
1000 |
22 |
2 |
0 |
24 |
46 |
1000 |
24 |
1 |
1 |
26 |
BiN: Binucleated cells
Mn: Micronucleus/micronuclei
Table 6. Main Experiment 2 – Scoring of Micronuclei Treatment: 31 hours - without S9 Sampling Time: 31 hours |
||||||
Treatment (µg/mL) |
Culture No. |
Cells scored (BiN cells) |
BiN cells with 1 Mn |
BiN cells with 2 Mn |
BiN cells with >2 Mn |
BiN cells with Mn |
Untreated |
49 |
1000 |
3 |
0 |
0 |
3 |
50 |
1000 |
2 |
0 |
0 |
2 |
|
|
||||||
Test Item 711 |
55 |
1000 |
2 |
0 |
0 |
2 |
56 |
1000 |
3 |
0 |
0 |
3 |
|
Test Item 1070 |
53 |
1000 |
3 |
0 |
0 |
3 |
54 |
1000 |
3 |
0 |
0 |
3 |
|
Test Item 1600 |
51 |
1000 |
5 |
0 |
0 |
5 |
52 |
1000 |
4 |
0 |
0 |
4 |
|
|
||||||
Treatment (µg/mL) |
Culture No. |
Cells scored (MonoN cells) |
MonoN cells with 1 Mn |
MonoN cells with 2 Mn |
MonoN cells with >2 Mn |
MonoN cells with Mn |
Colchicine 0.040 |
73 |
1000 |
17 |
4 |
0 |
21 |
74 |
1000 |
22 |
2 |
0 |
24 |
BiN: Binucleated cells
Mn: Micronucleus/micronuclei
Table 7. Main Experiment 1 – Statistical Analysis Treatment: 3 hours - without S9 Sampling Time: 32 hours |
||||
Treatment (µg/mL) |
Cells scored (BiN cells) |
BiN cells with Micronuclei |
χ2 |
Significance |
Untreated |
2000 |
5 |
|
|
711 |
2000 |
5 |
0.100 |
N.S. |
1070 |
2000 |
7 |
0.084 |
N.S. |
1600 |
2000 |
4 |
0.000 |
N.S. |
BiN: Binucleated cells
N.S. Not significant
Table 8. Main Experiment 1 – Statistical Analysis Treatment: 3 hours - with S9 Sampling Time: 32 hours |
||||
Treatment (µg/mL) |
Cells scored (BiN cells) |
BiN cells with Micronuclei |
χ2 |
Significance |
Untreated |
2000 |
7 |
|
|
711 |
2000 |
7 |
0.072 |
N.S. |
1070 |
2000 |
4 |
0.365 |
N.S. |
1600 |
2000 |
4 |
0.365 |
N.S. |
Cyclophosphamide 20.0 |
2000 |
50 |
31.395 |
*** |
BiN: Binucleated cells
N.S. Not significant
*** Significant atp<0.001
Table 9. Main Experiment 2 – Statistical Analysis Treatment: 31 hours - without S9 Sampling Time: 31 hours |
||||
Treatment (µg/mL) |
Cells scored (BiN cells) |
BiN cells with Micronuclei |
χ2 |
Significance |
Solvent 1% |
2000 |
5 |
|
|
711 |
2000 |
5 |
0.100 |
N.S. |
1070 |
2000 |
6 |
0.000 |
N.S. |
1600 |
2000 |
9 |
0.645 |
N.S. |
Treatment (µg/mL) |
Cells scored (MonoN cells) |
MonoN cells with Micronuclei |
χ2 |
Significance |
Colchicine 0.040 |
2000 |
45 |
30.805 |
*** |
BiN: Binucleated cells
N.S. Not significant
*** Significant atp<0.001
Table 10. Main Experiment 1 – Summary Table Treatment: 3 hours Sampling Time: 32 hours |
||||||||
Treatment
|
Presence of S9 Metabolism |
Absence of S9 Metabolism |
||||||
Dose Level (µg/mL) |
% Mn cells |
Sig. |
% Cytotoxicity |
Dose Level (µg/mL) |
% Mn cells |
Sig. |
% Cytotoxicity |
|
Untreated |
0.00 |
0.35 |
|
0 |
0.00 |
0.25 |
|
0 |
Test Item |
711 |
0.35 |
N.S. |
-10 |
711 |
0.25 |
N.S. |
9 |
Test Item |
1070 |
0.20 |
N.S. |
-5 |
1070 |
0.35 |
N.S. |
5 |
Test Item |
1600 |
0.20 |
N.S. |
-13 |
1600 |
0.20 |
N.S. |
-1 |
Cyclophosphamide |
20.0 |
2.50 |
*** |
59 |
|
- |
|
- |
%Mn cells Percentage of cells bearing micronuclei
N.S. Not significant
*** Significant atp<0.001
Table 11. Main Experiment 2 – Summary Table Treatment: 31 hours Sampling Time: 31 hours |
||||
Treatment
|
Absence of S9 Metabolism |
|||
Dose Level (µg/mL) |
% Mn cells |
Sig. |
% Cytotoxicity |
|
Untreated |
0.00 |
0.25 |
|
0 |
Test Item |
711 |
0.25 |
N.S. |
-3 |
Test Item |
1070 |
0.30 |
N.S. |
12 |
Test Item |
1600 |
0.45 |
N.S. |
37 |
Colchicine |
0.040 |
2.25 |
*** |
96 |
%Mn cells Percentage of cells bearing micronuclei
N.S. Not significant
*** Significant atp<0.001
Applicant's summary and conclusion
- Conclusions:
- Based on the results observed, the test material did not induce micronuclei in human lymphocytes after in vitro treatment.
- Executive summary:
In a key Guideline (OECD 487) in vitro micronucleus test, the test material was assayed to evaluate its ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation.
Two main experiments were performed in this study. In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively. The harvest time of 32 hours corresponding to approximately 2.0 cell cycles was used. As negative results were obtained, a second experiment was performed in the absence of S9 metabolism using a continuous treatment until harvest at 31 hours. Solutions of the test material were prepared in culture medium. For both experiments, the highest dose level of 1600 μg/mL was used (corresponding to10 mM), the upper limit to testing indicated in the Study Protocol. The following lower concentrations were assayed: 1070, 711, 474, 316, 211, 140, 93.6, 62.4 and 41.6 μg/mL. Each experiment included appropriate negative and positive controls and two cell cultures were prepared at each test point. The actin polymerisation inhibitor cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells. Dose levels were selected for the scoring of micronuclei on the basis of the cytotoxicity of the test material treatments calculated by the cytokinesis-block proliferation index (CBPI). For both experiments, the following dose levels were selected for scoring: 1600, 1070 and711 μg/mL. One thousand binucleated cells per culture were scored to assess the frequency of micronucleated cells.
Following treatment with the test material, no statistically significant increase in the incidence of micronucleated cells over the control value was observed at any dose level, in any treatment series. Statistically significant increases in the incidence of micronucleated cells were observed following treatments with the positive controls Cyclophosphamide and Colchicin and all results were within the distribution of historical negative control data.
Based on the results observed, the test material did not induce micronuclei in human lymphocytes after in vitro treatment.
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