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EC number: 205-087-0 | CAS number: 133-06-2
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Genetic toxicity: in vitro
Administrative data
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 22 Juni 2017 to 22 March 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 018
- Report date:
- 2018
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
- Version / remarks:
- August 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- dated May 30, 2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Version / remarks:
- adopted 29 July, 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
Test material
- Reference substance name:
- Captan
- EC Number:
- 205-087-0
- EC Name:
- Captan
- Cas Number:
- 133-06-2
- Molecular formula:
- C9H8Cl3NO2S
- IUPAC Name:
- 2-[(trichloromethyl)sulfanyl]-2,3,3a,4,7,7a-hexahydro-1H-isoindole-1,3-dione
- Test material form:
- solid: particulate/powder
Constituent 1
- Specific details on test material used for the study:
- Batch No.: 941381319
Physical State: solid
Colour: white
Purity: 96.4%
Expiry Date: 01 January 2020
Storage Conditions: room temperature
Method
Species / strain
- Species / strain / cell type:
- lymphocytes: Human Lymphocytes
- Details on mammalian cell type (if applicable):
- Human peripheral blood lymphocytes from healthy and non-smoking donors with no known recent exposure to genotoxic chemicals and radiation were used to examine the ability of chemicals to induce cytogenetic damage and thus to identify potential carcinogens or mutagens in vitro.
For this study (in each experiment) blood was collected only from a single donor to reduce inter-individual variability.
Blood samples were drawn by venous puncture and collected in heparinized tubes. Before use the blood was stored under sterile conditions at 4 °C for a maximum of 4 h. Whole blood samples treated with an anti-coagulant (e. g. heparin) were pre-cultured in the presence of mitogen (phyto-haematogglutinin, PHA).
- Metabolic activation:
- with and without
- Metabolic activation system:
- The S9 liver microsomal fraction was prepared at Eurofins Munich.
Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and β-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.
The preparation was performed according to Ames et al.
The following quality control determinations are performed:
a) Biological activity in:
- the Salmonella typhimurium assay using 2-aminoanthracene
- the mouse lymphoma assay using benzo[a]pyrene
- the chromosome aberration assay using cyclophosphamide.
b) Sterility Test
A stock of the supernatant containing the microsomes is frozen in aliquots of 2 and 4 mL and stored at ≤ -75 °C.
The protein concentration in the S9 preparation (Lot: 030217 pre-experiment, 020617 main experiment) was 32.4 and 34.1 mg/mL. - Test concentrations with justification for top dose:
- The following concentrations were evaluated:
Without metabolic activation: 0.02, 0.04, 0.05 and 0.06 mM
and with metabolic activation: 0.01, 0.02, 0.04 and 0.06 mM - Vehicle / solvent:
- - solvent used: DMSO
Controls
- Untreated negative controls:
- yes
- Remarks:
- Culture Medium
- Negative solvent / vehicle controls:
- yes
- Remarks:
- DMSO
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
TREATMENT AND HARVEST SCHEDULE:
- Treatment intervall: 4 h without and with metabolic activation
Preparation of the Test Item
A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 10 mM, Based on the results of the solubility test DMSO was used as solvent (1% (v/v) DMSO). Different test item stock solutions were prepared and added to the samples. The solvent control and the test item concentration 200 μM showed an osmolality of 436 mOsmol (solvent control) and 432 mOsmol/kg (200 μM). The solvent was compatible with the survival of the cells and the S9 activity.
Negative and Solvent Controls
Negative controls (treatment medium) and solvent controls (DMSO, AppliChem Lot No. 0000978834 and 0000950733) were treated the same way as all dose groups.
TEST SYSTEM
Blood Collection
Human peripheral blood lymphocytes from healthy and non-smoking donors with no known recent exposure to genotoxic chemicals and radiation were used to examine the ability of chemicals to induce cytogenetic damage and thus to identify potential carcinogens or mutagens in vitro. For this study (in each experiment) blood was collected only from a single donor to reduce inter-individual variability.
Blood samples were drawn by venous puncture and collected in heparinized tubes. Before use the blood was stored under sterile conditions at 4 °C for a maximum of 4 h. Whole blood samples treated with an anti-coagulant (e. g. heparin) were pre-cultured in the presence of mitogen (phyto-haematogglutinin, PHA). - Rationale for test conditions:
- On the basis of the data and the observations from the pre-experiment and taking into account the recommendations of the guidelines, the concentrations were selected for the main experiments I and II.
The dose group selection for microscopic analyses of chromosomal aberrations was based in accordance with the recommendations of the guidelines. - Evaluation criteria:
- Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the 95% control limits of the historical negative control data.
When all of these criteria are met, the test chemical is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.
Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the 95% control limits of the historical negative control data.
The test chemical is then considered unable to induce chromosomal aberrations in cultured human peripheral blood lymphocyte cells in this test system.
Results and discussion
Test resultsopen allclose all
- Key result
- Species / strain:
- lymphocytes: Human peripheral blood lymphocytes
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- 5.3% (0.04 mM) and 7.7% (0.06 mM), highly increased above the historic control range (refer toTable 11)
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Relative Mitotic Index: at 0.04 mM (49% rel. MI) and 0.06 mM (54% rel. MI, Table 5).
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- mammalian cell line, other:
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- Relative Mitotic Index: A biologically relevant decrease of the relative mitotic index (decrease below 70% rel. mitotic index) was noted at 0.02 mM (33%) and higher (0.04 mM: 59% rel. MI and 0.06 mM: 23% rel. MI, Table 5).
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: With metabolic activation, no biologically relevant increase was noted after treatment with the test item..
Any other information on results incl. tables
Precipitation
No precipitation of the test item was noted without and with metabolic activation at the concentrations evaluated.
Table 5: Main Experiment - Summary of Cytotoxicity Data: 4 h treatment, 24 h fixation period.
| Mitotic Index | |||||
Dose Group | Concentration [mM] | Culture | Mean | Relative [%] | Precipitate (+/-) | |
1 | 2 | |||||
without metabolic activation | ||||||
C | 0 | 73 | 78 | 75.5 | 144 | - |
S | 0 | 51 | 54 | 52.5 | 100 | - |
1 | 0.01 | 26 | 58 | 42.0 | 80 | - |
2 | 0.02 | 46 | 27 | 36.5 | 70 | - |
3 | 0.04 | 19 | 32 | 25.5 | 49 | - |
4 | 0.05 | 41 | 49 | 45.0 | 86 | - |
5 | 0.06 | 16 | 41 | 28.5 | 54 | - |
EMS | 900 µg/mL | 20 | 42 | 31.0 | 59 | - |
with metabolic activation | ||||||
C | 0 | 46 | 23 | 34.5 | 61 | - |
S | 0 | 73 | 41 | 57.0 | 100 | - |
1 | 0.01 | 49 | 42 | 45.5 | 80 | - |
2 | 0.02 | 12 | 26 | 19.0 | 33 | - |
3 | 0.04 | 24 | 43 | 33.5 | 59 | - |
4 | 0.06 | 19 | 7 | 13.0 | 23 | - |
CPA | 5 µg/mL | 22 | 15 | 18.5 | 32 | - |
The mitotic index was determined in 1000 cells per culture of each test group.
The relative values of the mitotic index are related to the solvent controls.
C: Negative Control (Culture Medium)
S: Solvent Control (DMSO)
EMS: Ethylmethanesulfonate
CPA: Cyclophosphamide
Table 6: Main Experiment – Summary of Aberration Rates
Dose Group | Concentration [mM] | Treatment Time | Fixation Interval | mean % aberrant cells | ||
incl. Gaps | excl. Gaps | Precipitation | ||||
without metabolic activation | ||||||
C | 0 | 4 | 24 | 2.3 | 1.0 | - |
S | 0 | 4 | 24 | 2.5 | 1.4 | - |
2 | 0.02 | 4 | 24 | 7.1 | 2.6 | - |
3 | 0.04 | 4 | 24 | 7.0 | 5.3 | - |
4 | 0.05 | 4 | 24 | 1.3 | 1.0 | - |
5 | 0.06 | 4 | 24 | 9.0 | 7.7 | - |
EMS | 900 µg/mL | 4 | 24 | 24.0 | 24.0 | - |
with metabolic activation | ||||||
C | 0 | 4 | 24 | 1.3 | 1.0 | - |
S | 0 | 4 | 24 | 4.0 | 3.0 | - |
1 | 0.01 | 4 | 24 | 3.7 | 3.0 | - |
2 | 0.02 | 4 | 24 | 4.3 | 3.7 | - |
3 | 0.04 | 4 | 24 | 2.2 | 2.2 | - |
4 | 0.06 | 4 | 24 | 2.7 | 1.7 | - |
CPA | 5 µg/mL | 4 | 24 | 26.4 | 21.6 | - |
300 cells evaluated for each concentration, except for the positive controls (EMS: 125 cells, CPA: 150 cells) due to a clearly positive increase in chromosomal aberrations. In the experiment without metabolic activation, less than 300 cells were evaluated for chromosome aberrations in the solvent control (282 cells) and the lowest concentrations 0.02 mM (267 cells) as well as 0.04 mM (284 cells). In the experiment with metabolic activation only 227 metaphases were evaluated for the concentration 0.06 mM due to few cells on the slides because of strong cytotoxic effects.
C: Negative Control (Culture Medium)
S: Solvent Control (DMSO)
EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate)
CPA: Positive Control (with metabolic activation: Cyclophosphamide)
Table 7 Proliferation Index determined by BrdU-Labeling
Dose Group | Concentration [mM] | Treatment Time | Proliferation Index | 1. | 2. | 3. | |
Mitosis | Mitosis | Mitosis | |||||
without metabolic activation | |||||||
C | 0 | 4 | 1.51 | 49 | 51 | 0 | |
S | 0 | 4 | 1.52 | 48 | 52 | 0 | |
4 | 0.05 | 4 | 1.07 | 93 | 7 | 0 | |
5 | 0.06 | 4 | 1.05 | 20 | 1 | 0 | |
with metabolic activation | |||||||
C | 0 | 4 | 1.44 | 56 | 44 | 0 | |
S | 0 | 4 | 1.52 | 48 | 52 | 0 | |
3 | 0.04 | 4 | 1.00 | 100 | 0 | 0 | |
4 | 0.06 | 4 | 1.05 | 95 | 5 | 0 | |
C: Negative Control (Culture Medium)
S: Solvent Control (DMSO)
Table 11: Historical Laboratory Control Data of the negative control (2010 - 2016)
| NC Number of aberrant cells metabolic activation (4 h) | NC Number of aberrant cells metabolic activation (24 h) | ||||
- | + | - | ||||
+ Gaps | - Gaps | + Gaps | - Gaps | + Gaps | - Gaps | |
mean [%] | 3.5 | 1.7 | 3.2 | 1.5 | 3.2 | 1.3 |
SD [%] | 1.51 | 0.89 | 1.64 | 0.96 | 1.53 | 0.93 |
RSD [%] | 43.5 | 52.5 | 51.2 | 63.7 | 48.4 | 72.1 |
min [%] | 0.5 | 0.0 | 0.0 | 0.0 | 0.5 | 0.0 |
max [%] | 6.5 | 3.3 | 9.7 | 4.0 | 6.9 | 4.2 |
n | 44 | 44 | 72 | 72 | 40 | 40 |
LCL | 0.45 | -0.09 | -0.08 | -0.41 | 0.10 | -0.57 |
UCL | 6.48 | 3.47 | 6.49 | 3.42 | 6.22 | 3.16 |
NC: Negative Control (cell culture medium)
mean: mean number of aberrant cells
SD: Standard Deviation
RSD: relative Standard Deviation
min.: minimum number of aberrant cells
max.: maximum number of aberrant cells
n: Number of assays
LCL: Lower control limit (95%, mean-2SD)
UCL: Upper control limit (95%, mean+2SD)
Toxicity (Relative Mitotic Index)
In experiment I without metabolic activation, a biologically relevant decrease of the relative mitotic index (decrease below 70% rel. mitotic index) was noted at 0.04 mM (49% rel. MI) and 0.06 mM (54% rel. MI, Table 5).
In the experiment with metabolic activation a biologically relevant decrease of the relative mitotic index (decrease below 70% rel. mitotic index) was noted at 0.02 mM (33%) and higher (0.04 mM: 59% rel. MI and 0.06 mM: 23% rel. MI, Table 5).
Toxicity (Proliferation Index)
The BrdU-technique was used for determining the proliferation index to detect a possible effect on the proliferation rate after treatment with the test item and thus indicating cell cycle delay. In the experiment I, the values of the proliferation index of the negative and solvent controls were 1.51 and 1.52 (without metabolic activation) as well as 1.44 and 1.52 (with metabolic activation) (Table 7). The proliferation index of the highest dose groups evaluated were both 1.05 at 0.06 mM without and with metabolic activation. A biologically relevant decrease of the proliferation index was indicated.
Clastogenicity
There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in the number of cells with chromosome aberrations for at least one of the concentrations, which is higher than the laboratory negative control range.
In the experiment without metabolic activation, the aberration rates of the negative control (1.0%), the solvent control (1.4%) and the concentrations 0.02 mM (2.6%) and 0.05 mM (1.0%) were within the historical control data of the testing facility (-0.09% – 3.47%, Table 11). Two concentrations were highly increased above the historic control range with 5.3% (0.04 mM) and 7.7% (0.06 mM) mean aberrant cells. These values were also statistically significantly increased and a concentration-dependency was observed determined with the χ² Test for trend. Therefore, the number of aberrant cells found in the concentrations treated with the test item did show a biologically relevant increase compared to the corresponding solvent control.
With metabolic activation, the aberration rates of the negative control (1.0%), the solvent control (3.0%) and most of the concentrations treated with the test item (0.01 mM: 3.0%; 0.04 mM: 2.2% and 0.06 mM: 1.7%, Table 6) were within the historical control data of the testing facility (-0.41% – 3.42%, Table 11). The test item concentration 0.02 mM showed a slightly increased amount of aberrant cells to 3.7%. However, no statistical significance or a dose-response relationship was noted. Therefore, the number of aberrant cells found in the concentrations treated with the test item did not show a biologically relevant increase compared to the corresponding solvent control.
EMS (900 μg/mL) and CPA (5 μg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.
Polyploid Cells
No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.
Applicant's summary and conclusion
- Conclusions:
- In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item Captan Technical did induce structural chromosomal aberrations in human lymphocyte cells.
Therefore, Captan Technical is considered to be clastogenic in this chromosome aberration test. - Executive summary:
A chromosome aberration assay was carried out in order to investigate a possible potential of Captan Technical to induce structural chromosome aberrations in human lymphocytes.
The metaphases were prepared 24 h after start of treatment with the test item. The treatment interval was 4 h without and with metabolic activation. Duplicate cultures were set up. Per culture 150 metaphases were scored for structural chromosomal aberrations.
The following concentrations were evaluated:
Without metabolic activation: 0.02, 0.04, 0.05 and 0.06 mM
and with metabolic activation: 0.01, 0.02, 0.04 and 0.06 mM
No precipitation of the test item was noted without and with metabolic activation at the concentrations evaluated.
In the experiment without and with metabolic activation, toxic effects (decrease below 70% rel. mitotic index) were seen at a concentration of 0.04 mM and 0.02 mM, respectively.
In experiment I a biologically relevant decreases of the proliferation index was observed without and with metabolic activation.
In the experiment without metabolic activation, a biologically relevant increase of the aberration rates was noted after treatment with the test item. With metabolic activation, no biologically relevant increase was noted after treatment with the test item.
The Fisher´s exact test was performed to verify the results in the experiment. A statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted in two test item concentrations evaluated in the experiment without metabolic activation.
The χ² Test for trend was performed to test whether there is a concentration-related increase in chromosomal aberrations. A statistically significant increase was observed in the experiment without metabolic activation.
In the experiments without and with metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.
EMS (900 μg/mL) and CPA (5 μg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the efficiency of the test system to indicate potential clastogenic effects.
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