Captan

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:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

dated May 30, 2008

Deviations:

no

Test guideline 3

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

Test material information

Constituent 1

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

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 results

Test results 1

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

Test results 2

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.