Captan

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Short description of key information:

1) Genetic toxicity in vitro (similar to OECD 490): Captan technical was tested for mutagenic potential in an in vitro mammalian mutation assay. In the absence of exogenous metabolic activation, captan demonstrated mutagenic activity in vitro under the conditions of the test system. In the presence of exogenous metabolic activation, no reproducible evidence of mutagenic activity was observed. (In-house method according to the procedures described by Clive and Spector (1975) similar to OECD 490). Result: Positive (without S-9); Negative (with S-9)

2) Genetic toxicity in vitro (OECD 476): Captan Technical was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. Toxicity: A biologically relevant growth inhibition (relative survival < 70%) was observed in the main experiment with and without metabolic activation. Mutagenicity: A biologically relevant increase of mutants was found in the experiment after treatment with the test item (without metabolic activation). In the described mutagenicity test under the experimental conditions reported, the test item Captan Technical is considered to be mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.

3) Genetic toxicity in vitro (OECD 471): Reverse Mutation Assay using Bacteria (Salmonella typhimurium and Escherichia coli). During the described mutagenicity test and under the experimental conditions reported, Captan Technical caused gene mutations by base pair changes and frameshifts in the genome of the tester strains used. Therefore, Captan Technical is considered to be mutagenic in this bacterial reverse mutation assay. 

4) Genetic toxicity in vitro (OECD 473): Mammalian Chromosome Aberration Test in Human Lymphocytes with Captan Technical. 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. 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 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. Captan Technical is considered to be clastogenic in this chromosome aberration test.

Endpoint Conclusions in vitro: Adverse effects observed (positive)

Link to relevant study records

Reference

Reference 1

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

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

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

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

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.

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..

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.

 

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 April 2017 to 24 July 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 st July, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch No.: 941381319
Physical State: solid
Coiour: off-white
Purity: 96.5%
Expiry Date: 01 January 2020
Storage Conditions: room temperature

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: The S9 liver microsomal fraction was prepared at Eurofins Munich and obtained from Trinova Biochem GmbH, Gießen, Germany.
- method of preparation of S9 mix: Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route (Eurofins Munich) and male Sprague Dawley rats were induced with phenobarbital / ß-naphthoflavone (Trinova).

The S9 mix preparation was performed according to Arnes et al.
100 mM of ice-cold sodium-ortho-phosphate-buffer, pH 7.4, was added to the following pre-weighed sterilised reagents to give final concentrations in the S9 mix of:
- 8 mM MgCl2
- 33 mM KCl
- 5 mM glucose-6-phosphate
- 4 mM NADP
This Solution was mixed with the liver 9000 x g supernatant fluid in the following Proportion:
- co-factor solution: 9.5 parts
- liver preparation: 0.5 parts
During the experiment the S9 mix is stored on ice.
Mix Substitution Buffer: The S9 mix Substitution buffer was used in the study as a replacement for S9 mix, without metabolic activation (-S9).
Phosphate-buffer (0.2 M) contains per litre:
- 0.2 M NaH2P04 x H2O; 120 mL
- 0.2 M Na2HP04; 880 mL
The two Solutions were mixed and the pH was adjusted to 7.4. Sterilisation was performed for 20 min at 121 °C in an autoclave.
This 0.2 M phosphate-buffer was mixed with 0.15 M KCl solution (sterile) in the following proportlon:
- 0.2 M phosphate-buffer; 9.5 parts
- 0.15 M KCl solution; 0.5 parts
This S9 mix Substitution buffer was stored at 4 °C.
- concentration or volume of S9 mix and S9 in the final culture medium: A stock of the supernatant containing the microsomes was frozen in aliquots of 2 and 4 mL and stored at <-75 ''C.. The protein concentration in the S9 preparation (Lot: 030217) was 32.4 mg/mL.
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): The following quality control determinations were performed by Trinova Biochem GmbH:
a) Alkoxyresorufin-O-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity In the Salmonella
typhimurium assay using 2-aminoanthracene and benzo[a]pyrene)
A Stock of the supernatant containing the microsomes is frozen in aliquots of 5 mL and stored at -75 °C.
The protein concentration in the S9 preparation (Lot: 3727) was 33.7 mg/mL.

Vehicle / solvent:

- solvent used: DMSO

Untreated negative controls:

yes

Remarks:

A. dest, Eurofins Munich Lot No.170228, 170503

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, AppliChem Lot No. 0000978834

Positive controls:

yes

Remarks:

With metabolic activation

Positive control substance:

other: 2-AA; 2-aminoanthracene

Remarks:

With metabolic activation

Untreated negative controls:

yes

Remarks:

A. dest, Eurofins Munich Lot No.170228, 170503

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, AppliChem Lot No. 0000978834

Positive controls:

yes

Remarks:

Without metabolic activation

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-NOPD; 4-nitro-o-phenylene-dlamine

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS & CONCENTRATIONS:
The test item was tested in the pre-experiment at the following concentrations with and without metabolic activation:
3.16,10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate.
The main experiment was performed with the following concentrations with and without metabolic activation:
0.050, 0.158, 0.500,1.58, 5.00, 15.8, 50.0,158 and 500 µg/plate.
- Number of cultures per concentration (triplicate): For each strain and dose level, including the Controls, three plates were used.

Evaluation criteria:

Cytotoxicity can be detected by a Clearing or rather diminution of the background lawn (indicated "N" or “B", respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean
values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.

A biologically relevant Increase is described as follows:
- if In tester strains TA 98, TA 100 and E. coli WP2 uvrA the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this System.

Statistics:

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Concentrations of 1.58 to 50.0 µg/plate (without metabolic activation) and of 15.8 to 158 µg/ plate (with metabolic activation). Refer to "Any other information on results" Table 1.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Concentrations of 50.0 µg/plate and higher (without metabolic activation) and of 158 µg/plate and higher (with metabolic activation). Refer to "Any other information on results" Table 1.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Concentrations of 0.5 to 15.8 µg/plate (without metabolic activation) and of 15.8 to 50.0 µg/plate (with metabolic activation). Refer to "Any other information on results" Table 1.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Concentrations of 50.0 µg/plate and higher (without metabolic activation) and of 158 µg/plate and higher (with metabolic activation). Refer to "Any other information on results" Table 1.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Concentrations of 1.58 to 15.8 µg/plate (without metabolic activation) and of 15.8 µg/plate (with metabolic activation). Refer to "Any other information on results" Table 1.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Concentrations of 50.0 µg/piate and higher (with and without metabolic activation). Refer to "Any other information on results" Table 1.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Concentrations of 0.5 to 15.8 µg/plate (without metabolic activation) and of 15.8 to 50.0 µg/plate (with metabolic activation). Refer to "Any other information on results" Table 1.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Concentrations of 50.0 µg/plate and higher (without metabolic activation) and of 158 µg/plate and higher (with metabolic activation). Refer to "Any other information on results" Table 1.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Concentrations of 1.58 to 50.0 µg/plate (without metabolic activation) and 15.8 to 50.0 µg/plate (with metabolic activation). Refer to "Any other information on results" Table 1.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Concentrations of 158 µg/plate and higher (with and without metabolic activation). Refer to "Any other information on results" Table 1.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1 Results	Without metabolic activation:	With metabolic activation:
Precipitation of the test item:
Pre-experlment (plate incorporation):	≥ 2500 µg/plate	≥ 2500 µg/plate
Main experiment:
Experiment I (plate incorporation):	None	None
Experiment II (preincubation):	Not applicable	Not applicable
Toxicity of the test item (tester strains in which observed):
Pre-experiment (plate incorporation):	≥ 100 µg/plate (TA 98, TA 100)	≥ 31.6 µg/plate (TA 98, TA 100)
Main experiment:
Experiment I (plate incorporation):	≥ 50.0 µg/plate (TA 100, TA 1535, TA 1537, E. coli WP2 uvrA).	≥ 50.0 µg/plate (TA 98, TA 100, TA 1535, E. coli WP2 uvrA).
	≥ 158.0 µg/plate (TA 98)	≥ 50.0 µg/plate (TA 1537)
Experiment II (pre incubation):	not applicable	not applicable
Mutagenicity (tester strains in which observed):
Pre-experiment (plate incorporation):	10-100.0 µg/plate (TA 98)	3.16-31.6 µg/plate (TA 98)
	31.6 µg/plate (TA 100)	3.16-10.0 µg/plate (TA 100)
Main experiment:
Experiment I (plate incorporation):	1.58-50.0 µg/plate (TA 98, E. coli. WP2 uvrA)	15.8-50.0µg/plate (TA 98, TA 100, TA 1535)
	0.5-15.8 µg/plate(TA 100, TA 1535)	15.8 µg/plate(TA 1537)
	1.58-15.8 µg/plate(TA 1537)	5.8-158.0 µg/plate (E. coli. WP2 uvrA)
Experiment II (pre incubation):	not applicable	not applicable

Conclusions:

ln conclusion, it can be stated that during the described mutagenicity test and under the experimental conditlons reported, Captan Technical caused gene mutations by base pair changes and frameshifts in the genome of all tester strains.
Therefore, Captan Technical is considered to be mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item Captan Technical was investigated for its potential to induce gene mutations according to the plate incorporation test with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. co//WP2 uvrA.

Several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the Controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiment:

0.050, 0.158, 0.500, 1.58, 5.00, 15.8, 50.0,158 and 500 µg/plate

No precipitation of the test item was observed in any tester strain used with and without metabolic activation).

Toxic effects of the test item were noted in all tester strains used.

In the experiment toxic effects of the test item were observed in tester strains TA 100, TA 1535 and E coli WP2 uvrA at concentrations of 50.0 µg /plate and higher (without metabolic activation) and at concentrations of 158 µg /plate and higher (with metabolic activation). In tester strain TA 98 toxic effects of the test Item were noted at concentrations of 158 µg /plate and higher (with and without metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at concentrations of 50.0 µg /plate and higher (with and without metabolic activation).

Biologically relevant increases of revertant colony numbers were observed in tester strain TA 98 at concentrations of 1.58 to 50.0 µg /plate (without metabolic activation) and at concentrations of 15.8 to 50.0 µg /plate (with metabolic activation). The threshold value of 2.0 was exceeded and a maximum mutation factor of 15.3 was reached at a concentration of 50.0 µg /plate (with metabolic activation).

Biologically relevant increases of revertant colony numbers were also observed in tester strains TA 100 and TA 1535 at concentrations of 0.5 to 15.8 µg /plate (without metabolic activation) and at concentrations of 15.8 to 50.0 µg /plate (with metabolic activation). The threshold value of 2.0 was exceeded and a maximum mutation factor of 6.9 was reached at a concentration of 15.8 µg /plate (without metabolic activation) in tester strain TA 100. The threshold value of 2.0 was exceeded and a maximum mutation factor of 14.2 was reached at a concentration of 50.0 µg /plate (with metabolic activation) in tester strain TA 1535 . In tester strain TA 1537 biologically relevant increases of revertant colony numbers were observed at concentrations of 1.58 to 15.8 µg /plate (without metabolic activation) and at a concentration of 15.8 µg /plate (with metabolic activation). The threshold value of 3.0 was exceeded and a maximum mutation factor of 17.2 was reached at a concentration of 15.8 µg /plate (without metabolic activation) in tester strain 1537.

In tester strain E. coli WP2 uvrA biologically relevant increases of revertant colony numbers were observed at concentrations of 1.58 to 50.0 µg /plate (without metabolic activation) and at concentration of 15.8 to 158 µg / plate (with metabolic activation). The threshold value of 2.0 was exceeded and a maximum mutation factor of 18.4 was reached at a concentration of 50.0 µg /plate (with metabolic activation).

Moreover, a dose-response relationship was found in all tester strains (with and without metabolic activation).

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May 2017 to 08 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Version / remarks:

adopted July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Specific details on test material used for the study:

- Physical state: solid
- Colour: white
- Purity: 96.5 %
- Batch number: batch no: 941381319
- Expiry Date: 01 January 2020
- Storage conditions: room temperature

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79 (Chinese Hamster cells)
The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins BioPharma Product Testing Munich GmbH.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9:
The S9 liver microsomal fraction was obtained from Trinova Biochem GmbH, Giessen, Germany. Male Sprague Dawley rats (5 – 6 weeks old) were induced with phenobarbital / β-naphthoflavone.
The protein concentration in the S9 preparation (Lot: 3727) was 33.7 mg/mL.
- method of preparation of S9 mix:
An appropriate quantity of the S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. Cofactors were added to the S9 mix to reach the concentrations below:
8 mM MgCl2
33 mM KCl
5 mM Glucose-6-phosphate
5 mM NADP
in 100 mM sodium-phosphate-buffer pH 7.4.
During the experiment the S9 mix was stored on ice.
The percentage of S9 mix in the final treatment medium was 5% (v/v).

Test concentrations with justification for top dose:

Pre-Test for Toxicity:
The toxicity of the test item was measured in a pre-experiment up to a concentration of 5 mM due to precipitation determined in the solubility test. As strong cytotoxic effects were observed during the testing, the pre-experiment was repeated with lower concentration. Eight concentrations [0.0002, 0.0005, 0.001, 0.002, 0.005, 0.01, 0.02, 0.05 mM] were tested with and without metabolic activation for the 4 h short-term exposure assay.
Toxicity of the test item was evaluated using the relative survival (RS). A cytotoxic effect is observed as soon as the relative survival decreased below 70%.

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: 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.
- Justification for percentage of solvent in the final culture medium: Due to strong precipitation in the concentration 10 mM testing was performed only up to 5 mM. For the concentrations 2 and 5 mM the test item was suspended in DMSO. For concentrations of 1.25 mM and lower the test item could be dissolved in DMSO. For the experiments reported, the highest concentration to be tested was 0.05 mM due to cytotoxicity. This concentration was used to prepare separate dosing solutions of the test item by serial dilution. From the DMSO dosing solutions 1% v/v was added to the cells in MEM cell culture medium.

Untreated negative controls:

yes

Remarks:

Culture medium without solvent and with and without metabolic activation

Negative solvent / vehicle controls:

yes

Remarks:

Culture medium with solvent DMSO and with and without metabolic activation

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

EXPOSURE CONCENTRATIONS
The test item was tested at the following concentrations:
- without metabolic activation:
0.0002, 0.0005, 0.0007, 0.001, 0.002, 0.004, 0.007, 0.01, 0.02, 0.05 mM
- with metabolic activation:
0.001, 0.002, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05 mM

TIME OF EXPOSURE
The experiment with and without metabolic activation were performed as 4 h short-term exposure assay.
Due to positive results in the first experiment no second experiment was performed.

NUMBER OF REPLICATIONS:
Number of cultures per concentration: two

METHOD OF TREATMENT/ EXPOSURE:
- 5.0 x 106 cells per concentration, solvent/negative and positive control, were seeded in complete culture medium (MEM supplemented with 10% (v/v) FBS) in a culture flask, respectively.
- Approx. 24 h after seeding the cells were exposed to designated concentrations of the test item either in the presence or absence of metabolic activation in the mutation experiment. After 4 h (short-term exposure) the cultures were checked for precipitation and the treatment medium containing the test item was removed. The cells were washed twice with PBS, trypsinised and counted with a cell counter.
During the following expression period most of the cells were subcultured in complete culture medium (MEM supplemented with 10% (v/v) FBS) in a sufficient number of cells (at least 2 x 106 cells per treatment group).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
For determination of the relative survival (RS) two 25 cm2 flasks were seeded with approx. 200 cells in complete culture medium for each treatment group. After incubation for an appropriate time (6-7 days) colonies were fixed with methanol, stained with Giemsa and counted [16]. Cytotoxicity (relative survival) was calculated based on the cloning efficiency of cells plated immediately after treatment adjusted by any loss of cells during treatment (formula for calculation see.
Cytotoxicity was evaluated by relative survival (RS). The cloning efficiency (CE) of cells plated immediately after treatment was adjusted by any loss of cells during treatment as compared with adjusted cloning efficiency in negative / solvent controls (assigned a survival of 100%).

SELECTION
At the end of the expression period (after 7 to 9 days) for selection the mutants, about 4 x 105 cells for each treatment group were seeded in cell culture petri dishes (diameter 90 mm) with selection medium containing 11 μg/mL thioguanine (TG) for further incubation.
The cloning efficiencies (CE) were determined in parallel to the selection of mutants. For each treatment group two 25 cm2 flasks were seeded with approx. 200 cells in complete culture medium to determine the cloning efficiencies.
After incubation for an appropriate time (7-12 days) colonies were fixed with methanol, stained with Giemsa and counted. The mutant frequency was calculated based on the number of mutant colonies corrected by the cloning efficiency at the time of mutant selection.

Statistics:

The statistical analysis was done with the software GraphPad Prism 6. The majority of the data set was distributed normally according to Kolmogorov-Smirnov-test. Moreover, the standard deviations were not statistically different from each other according to the Browne and Forsythe test. The statistical significances of each test item concentration evaluated with one-way ANOVA (Dunnett’s test with multiple comparisons) and the positive controls evaluated with the two-tailed unpaired t-test at the 5% level (p<0.05) were listed in the following tables. The p value was used as a limit in judging for significance levels in comparison with the corresponding solvent control.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Refer to "Any other information on results" Table 1 and 2

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Refer to "Any other information on results" Table 1 and 2

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1: Summary without metabolic activation

Dose Group	Concentration [mM]	Relative Survival (RS) [%]	Mutant Frequency per 10^6 cells	Statistical Significance
NC1	0	96	31.5	-
NC2		103	26.2
S1	0	100	25.9	/
S2			31.7
1	0.0002	108	28.9	-
2	0.0005	79	42.6	-
3	0.0007	97	45.8	-
4	0.001	73	55.6	+
5	0.002	66	59.6	+
6	0.004	18	53.2	+
EMS	300 μg/ml	122	315.1	+

NC: negative control

S: solvent control

CE: cloning efficiency

RS: relative survival

EMS: Ethylmethanesulfonate

 

Table 2: Summary with metabolic activation

Dose Group	Concentration [mM]	Relative Survival (RS) [%]	Mutant Frequency per 10^6 cells	Statistical Significance
NC1	0	109	26.3	-
NC2		98	30.5
S1	0	100	32.0	/
S2			26.5
2	0.01	102	35.7	-
3	0.01	112	42.0	+
4	0.02	112	31.8	-
5	0.03	115	35.4	-
6	0.04	93	46.6	+
7		15	38.1	-
DMBA	300 μg/ml	141	117.1	+

NC: negative control

S: solvent control (DMSO)

CE: cloning efficiency

RS: relative survival

DMBA: 7,12-dimethylbenz(a)anthracene

Conclusions:

In the described in vitro cell gene mutagenicity test under the experimental conditions reported, the test item Captan Technical is considered to be mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

The test item Captan Technical was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster.

The main experiment was carried out without and with metabolic activation. The experiment with metabolic activation was performed by including liver microsomes and NADP for efficient detection of a wide variety of carcinogens requiring metabolic activation.

The selection of the concentrations used in the main experiment was based on data from the pre-experiments according to the OECD guideline 476.

In the main experiment 0.004 mM (without metabolic activation) and 0.04 mM (with metabolic activation) were selected as the highest concentrations. The experiment with and without metabolic activation were performed as a 4 h short-term exposure assay.

The pH-value detected with the test item was within the physiological range (pH 7.0 ± 0.4).

The test item was investigated at the following concentrations:

without metabolic activation:

0.0002, 0.0005, 0.0007, 0.001, 0.002, 0.004 mM

with metabolic activation:

0.002, 0.005, 0.01, 0.02, 0.03, 0.04 mM

Precipitation:

No precipitation of the test item was noted in the experiments reported. However, precipitation was observed in the solubility test in higher concentrations (2.5 mM, 5 mM and 10 mM).

Toxicity:

A biologically relevant growth inhibition (reduction of relative survival below 70%) was observed after the treatment with the test item in the experiment with and without metabolic activation.

Without metabolic activation the relative survival was 18% for the highest concentration (0.004 mM) evaluated (Table 5). The highest biologically relevant concentration evaluated with metabolic activation was 0.04 mM with a relative survival of 15% (Table 7).

Mutagenicity:

In the experiment without and with metabolic activation all validity criteria were met. The mutant values of the negative controls fall within the historical data range of the test facility and the cloning efficiencies of the negative and solvent controls are > 50%.

The positive controls, DMBA (1 μg/mL) and EMS (300 μg/mL) showed statistically significant increases in mutant frequency, thereby demonstrating both the sensitivity and validity of the test systems.

In the experiment without metabolic activation the mutant values of the negative controls and the solvent controls were within the historical control data of the test facility Eurofins Munich (about 7.8 - 39.7 mutants per 106 cells) with 31.5 and 26.2 mutants per 106 cells for the negative controls and 25.9 and 31.7 mutants per 106.cells for the solvent controls.

The positive control EMS induced a distinct increase in mutant frequency with 315.1 mutants/106 cells.

The mutant values of the test item are exceeding the historical control range at concentrations of 0.0005 mM and higher (42.6. to 59.6 mutants per 106 cells). A dose-dependent increase was noted. The highest mutant frequency was observed at a concentration of 0.002 mM (59.6 mutants per 106 cells) with a relative survival of 66%. The decrease in mutation frequency at the concentration of 0.004 mM 53.2 mutants per 106 cells can be explained by strong cytotoxicity in this concentration.

A statistical analysis displayed that the mutant frequencies were significantly increased over those of the solvent controls for the test item concentrations of 0.001 mM and higher.

Due to the exceeded mutant frequencies, the dose effect relationship and the statistical significance obtained in this experiment, this effect is regarded as biologically relevant.

In the experiment with metabolic activation the mutant values of the negative controls, the solvent controls and four of six mutant values of the test item concentrations found were within the historical control data of the test facility Eurofins Munich (about 9.2 - 38.8 mutants per 106 cells). The mutant frequencies of the negative controls were 26.3 and 30.5 mutants per 106 cells and 32.0 and 26.5 mutants per 106.cells for the solvent controls.

The positive control DMBA induced a distinct increase in mutant frequency with 117.1 mutants/106 cells.

The mutant frequencies of the test item were in the range of 31.8 to 46.6 mutants per 106 cells. The mutant values of the test item exceeded the historical control range at concentrations of 0.005 and 0.03 mM, which were also statistically significant compared to the solvent control. The highest mutant frequency was observed at a concentration of 0.03 mM (46.6 mutants per 106 cells) with a relative survival of 93%. However these increases were regarded as biologically not relevant as no concentration-related increase could be determined.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro cytogenicity study in mammalian cells or in vitro micronucleus study does not need to be conducted because adequate data from an in vivo cytogenicity test are available

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14. August 1985 to 18. August 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Mouse lymphoma L5178Y cells were obtained from Dr. J. Cole, Sussex University. These cells are heterozygous at the thymidine kinase lokus (TK +/-). Cells were stored in polypropylene ampoules in a freezing mixture consisting of heat-inactivated horse serum (Sera-Lab) containing 10% dimethylsulphoxide (DMSO), at -196 °C. Spontaneous TK-/- mutants were eliminated from the cultures by a 24 hour incubation in the presence of methotrexate (0.3 µg/ml), thymidine (9 µg/ml), hypoxanthine (15 µg/ml) and glycine (22.5 µg/ml) followed by 24 hours incubation in similar medium without methotrexate.

Metabolic activation:

with and without

Metabolic activation system:

Arochlor induced rat liver S-9

Test concentrations with justification for top dose:

Captan dissolved in DMSO was tested
without metabolic activation at concentrations of 0.025 to 0.4 μg/mL,
with metabolic activation at concentrations of 3.125 to 100 μg/L.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

in the absence of metabolic activation with ethyl methane sulphonate (EMS) dissolved in DMSO at a final concentration of 500 μg/mL in the presence of metabolic activation with 20-methylcholanthrene at a final concentration of 7 μg/mL.

Positive control substance:

ethylmethanesulphonate

other: 20-methylcholanthrene

Details on test system and experimental conditions:

Captan (purity not specified, batch no. 610541) was tested for its mutagenic potential in an in vitro mammalian cell mutation assay using the mouse lymphoma L5178Y cell mutation test.
In the preliminary toxicity test L5178Y cells were treated with a concentration of captan between 0.0625 and 1 µg/ml in the absence of exogenous metabolic activation and between 1.25 and 100 µg/ml in the presence of S-9 mix. The cell growth was reduced to between 112 and 2% and to between 117 and 9% (compared to the relative solvent control) in the respective tests. In order to induce cytotoxicity the cells in the main study were treated with a concentration range of captan based on this data.
Two independent tests in the absence and three in the presence of exogenous metabolic activation (Arochlor induced rat liver S-9) were carried out. Captan, dissolved in DMSO was tested without metabolic activation at concentrations of 0.025 to 0.4 μg/mL and with metabolic activation at concentrations of 3.125 to 100 μg/L.
Positive controls were carried out in the absence of metabolic activation with ethyl methane sulphonate (EMS) dissolved in DMSO at a final concentration of 500 μg/mL and in the presence of metabolic activation with 20-methylcholanthrene at a final concentration of 7 μg/mL.
Cells were incubated with test substance for 3 hours, washed and then incubated for an additional 48 hours.
Growth in suspension was monitored at 24 and 48 hours after treatment.
Cells were then cloned in soft agar and after 12 days incubation colonies with a diameter of more than 400 μm were counted.
Four of the most appropriate concentrations were chosen for cloning based on toxicity observed at 24 and 48 hours.

Statistics:

The general method of statistical analysis was analysis of variance of the mutant frequncies. This was carried out after the data had been transformed logarithmically sine Irr and Snee (1982) have shown that the random variation in results from the L5178Y cell TK locus mutation asssay can be adequately described by lognormal ditribution.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

In the absence of metabolic activation, captan caused highly significant dose-related increases in mean mutant frequency at concentrations ≥ 0.075 μg/L.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Treatment with captan induced dose dependant cytotoxicity in the absence of metabolic activation as evidenced by decreases in mean cell survival compared to the solvent control.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

The positive controls produced highly significant increases in mutant frequency.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

No two-fold increases in mean mutant frequency were achieved at any treatment concentration.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Treatment with captan induced dose dependant cytotoxicity in the presence of metabolic activation as evidenced by decreases in mean cell survival compared to the solvent control.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

The positive controls produced highly significant increases in mutant frequency.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

not determined

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

n the preliminary toxicity test L5178Y cells were treated with a concentration of captan between 0.0625 and 1 µg/ml in the absence of exogenous metabolic activation and between 1.25 and 100 µg/ml in the presence of S-9 mix.

Positive controls validity:

valid

Additional information on results:

Treatment with captan induced dose dependant cytotoxicity in both the presence and absence of metabolic activation as evidenced by decreases in mean cell survival compared to the solvent control (Table 7.6.1-1).
A summary of the viability of cells in soft agar is given in Table 7.6.1-2. In the absence of metabolic activation, captan caused highly significant dose-related increases in mean mutant frequency at concentrations ≥ 0.075 μg/L (Table 7.6.1-3). In the presence of metabolic activation, captan failed to give rise to statistically significant increases in mean mutation frequency in two of three tests. Significant differences in mean mutation frequency were observed in a third test at concentrations of 25 and 30 μg/mL. At 30 μg/mL a doubling in mutant frequency occurred in one culture. However, no two-fold increases in mean mutant frequency were achieved at any treatment concentration. The positive controls produced highly significant increases in mutant frequency

Table 7.6.1-1 In vitro gene mutation: summary of growth in suspension of L5178Y cells

 

Concentration (μg/mL)	% Control^1
	Without activation		With activation
	test 1	test 2	test 1	test 2	test 3
0	100	100	100	100	100
0.025	117^2	104	-	-	-
0.05	100	97	-	-	-
0.075	-	74	-	-	-
0.1	83	48	-	-	-
0.2	37	8	-	-	-
0.3	9	4^2	-	-	-
0.4	5^2	4^2	-	-	-
3.125	-	-	138^2	-	-
6.25	-	-	114^2	87^2	-
12.5	-	-	111	67^2	-
15	-	-	-	-	58
20	-	-	-	60	30
25	-	-	53	54	33
30	-	-	-	-	26
35	-	-	-	-	14
40	-	-	-	-	13^2
45	-	-	-	-	16^2
37.5	-	-	-	33	-
50	-	-	10	-	-
100	-	-	10	-	-
EMS (500 µg/ml)	60	37	-	-	-
20-methyl-cholanthrene (7 μg/mL)	-	-	100	81	45

 ¹Mean of eight replicates in controls and two replicates in treatments.

²Cultures disregarded prior to cloning in agar in favour of cultures of more acceptable levels of toxicity.

Table 7.6.1 -2: In vitro gene mutation: summary of viability of L5178Y cells in soft agar

 

Concentration (μg/mL)	% Mean survival^1
	Without activation		With activation
	test 1	test 2	test 1	test 2	test 3
0	100	100	100	100	100^2
0.05	97.5	81	-	-	-
0.075	-	63	-	-	-
0.1	83	45	-	-	-
0.2	32	5	-	-	-
0.3	6	-	-	-	-
12.5	-	5	107	-	-
15	-	-	-	-	46
20	-	-	-	70	16
25	-	-	48	58	19
30	-	-	-	-	14
35	-	-	-	-	3^3
37.5	-	-	-	37	-
50	-	-	<1	3	-
100	-	-	<1	-	-
EMS (500 µg/ml)	45	23	-	-	-
20-methyl-cholanthrene (7 μg/mL)	-	-	101	88	22

¹Mean of eight replicates in controls and two replicates in treatments, except where specified.

²Four replicates.

³Only one mutant frequency.

Table 7.6.1-3: In vitro gene mutation: summary of mutation of L5178Y cells in soft agar

 

Concentration (μg/mL)	Mean mutant frequency/10^6viable cells^1
	Without activation		With activation
	test 1	test 2	test 1	test 2	test 3
0	57	48	57	45	60^2
0.05	60	57	-	-	-
0.075	-	79***	-	-	-
0.1	100***	80***	-	-	-
0.2	119***	239***	-	-	-
0.3	244***	-	-	-	-
12.5	-	-	47	-	-
15	-	-	-	-	78
20	-	-	-	60	84
25	-	-	44	46	108**
30	-	-	-	-	116**
35	-	-	-	-	56^3
37.5	-	-	-	42	-
50	-	-	630^4	45	-
100	-	-	0	-	-
EMS (500 µg/ml)	418***	388***	-	-	-
20-methyl-cholanthrene (7 μg/mL)	-	-	195***	220***	260**

¹Mean of eight replicates in controls and two replicates in treatments, except where specified.

²Four replicates.

³Only one mutant frequency, hence disregarded from the replicate

⁴Percentage survival less than 1%, therefore values omitted from statistical analysis. Significant difference compared to the control: ** p = 0.01; *** p = 0.001.

Conclusions:

In the absence of exogenous metabolic activation, captan demonstrated mutagenic activity in vitro under the conditions of the test system. In the presence of exogenous metabolic activation, no reproducible evidence of mutagenic activity was observed.

Executive summary:

Captan was tested for potential mutagenicity in the mouse lymphoma L5178Y cell mutation test (Clive & Spector 1975 = OECD 490 “Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene”). In this in vitro system it is possible to detect and quantitate forward mutation from a wild type cell, which is heterozygous at the thymidine kinase locus (TK +/-), to the homozygous thymidine kinase deficient form (TK -/-). The agent used to select for the mutant phenotype I the thymidine analogue triflourothymidine (TFT): TK -/- mutants, unlike the TK +/- form, are resistant to the otherwise lethal effects of TFT. The experimental methods employed are based on those published by Clive & Spector (1975) & Anacher et al. (179; 1980a; 1980b).

Captan was tested in two independent tests in the absence of exogenous metabolic activation and three independent tests in the presence of S-9 mix using the mouse lymphoma thymidine kinase locus assay.

In the preliminary toxicity test L5178Y cells were treated with a concentration of captan between 0.0625 and 1 µg/ml in the absence of exogenous metabolic activation and between 1.25 and 100 µg/ml in the presence of S-9 mix. The cell growth was reduced between 112 and 2% and to between 117 and 9% (compared to the relative solvent control) in the respective tests. In order to induce cytotoxicity the cells in the main study were treated with a concentration range of captan based on this data.

In the absence of exogenous metabolic activation captan was tested for mutagenic activity using concentrations between 0.05 and 0.3 µg/ml in the first test and between 0.05 and 0.2 µg/ml in the second test. Treatment with captan induced dose-dependent cytotoxicity as assessed by decreases in mean cell survival (compared to solvent control) to between 101 and 6% and to between 81 and 5% in the first and second tests respectively. In both tests treatment with captan stimulated dose-related highly significant increases in mean mutant frequency. In the first test increases in mean mutant frequency of two and four times the solvent control were achieved after treatment with 0.2 and 0.3 µg captan/ml with mean cell survivals of 32 and 6% respectively. In the second test, although the increases in mean mutant frequencies were statistically significant and dose-related, a doubling in mean mutant frequency was achieved only when the mean cell growth was reduced to 5%. In both tests in the absence of exogenous metabolic activation the positive control EMS stimulated a highly significant increase in mean mutant frequency.

In the first test the presence of S-9 mix captan was tested for mutagenic activity using concentrations between 12.5 and 100 µg/ml which induced dose-dependent decreases in mean cell survival to between 107 and less than 1% relative to the solvent control. The mean mutant frequency data at treatment concentrations of 50 and 100 µg captan/ml were disregarded from the statistical analysis due to excessive toxicity. Captan failed to induce statistically significant increases in mean mutant frequency at treatment concentrations of 12.5 and 25 µg/ml with mean cell survivals of 107 and 48% respectively, relative to the DMSO control.

Captan was tested for mutagenic activity using concentrations between 20 and 50 µg/ml in the second test in the presence of S-9 mix. Treatment with captan induced dose-dependent cytotoxicity with decreases in mean cell survival to between 70 and 3% compared to the solvent control. No statistically significant increases in mean mutant frequency were observed in the test.

In view of the results obtained it was decided to perform a third test in the presence of S-9 mix to achieve additional data at the more toxic range. In the third test captan was tested for mutagenic activity using concentrations between 15 and 35 µg/ml. Treatment with capatn between 15 and 30 µg/ml induced decreases in mean cell survival to between 46 and 14% relative to the solvent control. Dose-related statistically significant increases in mean mutant frequnc< were induced after treatment with captan. However, no two fold increases in mean mutant frequency were achieved at any treatment concentration, but at 30 µg captan/ml a doubling in mutant frequency occurred in one culture only. The result was only available from one replicate after treatment with 35 µg captan/ml (due to contamination) which was disregarded from statistical analysis.

In all three tests carried out in the presence of S-9 mix the positive control 20-methylanthrene induced increases in mean mutant frequency of approximately four times the solvent control.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Short description of key information:
1) Genetic toxicity in vivo: mouse micronucleus test study according to OECD 474 (mammalian erythrocyte micronucleus test). There was no increase in the frequency of micronucleated polychromatic cells in captan-treated groups, and no alteration in the ratio of polychromatic to nonnochromatic cells was apparent.

No adverse effects oberserved.

2) Supporting study: Genetic toxicity in vivo: cytogenetic and dominant lethal non-guideline studies on captan.

No negative results were observed.

Endpoint Conclusion: No adverse effects observed (negative)

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 April - 2 May 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Route of administration:

oral: gavage

Vehicle:

0.5% carboxymethyl cellulose (CMC) and 0.5% acetic acid

Duration of treatment / exposure:

preliminary test: 72 hours; main study: 24 hours, 48 hours and 72 hours

Frequency of treatment:

once

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Main Study: one dose; actual ingested

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Remarks:

Main Study: one dose; actual ingested

Dose / conc.:

40 mg/kg bw/day (actual dose received)

Remarks:

Main Study: one dose; actual ingested

Dose / conc.:

5 000 mg/kg bw/day (actual dose received)

Remarks:

Preleminary Toxicity Test: one dose; actual ingested

Dose / conc.:

2 500 mg/kg bw/day (actual dose received)

Remarks:

Preleminary Toxicity Test: one dose; actual ingested

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Remarks:

Preleminary Toxicity Test: one dose; actual ingested

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

Preleminary Toxicity Test: one dose; actual ingested

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

Preleminary Toxicity Test: one dose; actual ingested

No. of animals per sex per dose:

preliminary test: 2 male/2 female per dose (100, 250, 1000, 2500 and 5000 mg/kg)
main study: 5 male/5 female per dose (40, 200 mg/kg): 15 male/15 female per dose (1000 mg/kg); Chlorambucil 5 male/5 female (30 mg/kg)

Control animals:

yes, concurrent vehicle

Positive control(s):

Chlorambucil

Tissues and cell types examined:

femurs, erythrocytes

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

No mutagenic activity under the conditions of this test was found.

Executive summary:

The potential of captan to induce chromosomal damage was investigated by the micronucleus test. The procedures used complied with the recommendations of the OECD Guideline for Testing of Chemicals No. 474.

Charles River CD-I mice were orally administered captan suspended in 0.5% carboxymethyl cellulose (CMC) at dosages of 40, 200 and 1000 mg/kg at a constant volume-dosage of 10 ml/kg on Day 1. Dosages were selected on the basis of a preliminary toxicity test.

There was no increase in the frequency of micronucleated polychromatic cells in captan-treated groups, and no alteration in the ratio of polychromatic to nonnochromatic cells was apparent.

Chlorambucil, a known mutagen, used as a positive control, caused a significant increase in the frequency of micronucleated cells.

The test material, captan, was devoid of mutagenic activity under the conditions of this study.

The result is in accordance with several other in vivo tests on captan (e.g. Tezuka, H.).

Reference 2

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Remarks:

Type of genotoxicity: chromosome aberration

Adequacy of study:

supporting study

Qualifier:

no guideline available

Principles of method if other than guideline:

publication: Mutation Research, 57 (1978) 201-207, © Elsevier/North-Holland Biomedical Press

Type of assay:

chromosome aberration assay

Species:

rat

Strain:

Wistar

Sex:

male

Route of administration:

oral: gavage

Duration of treatment / exposure:

21 h

Frequency of treatment:

single dose or 5 consecutive doses

Dose / conc.:

800 mg/kg bw/day

Remarks:

five consecutive doses

Dose / conc.:

400 mg/kg bw/day

Remarks:

five consecutive doses

Dose / conc.:

200 mg/kg bw/day

Remarks:

five consecutive doses

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

single dose

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

single dose

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

single dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Mitomycin C

Sex:

male

Genotoxicity:

negative

Positive controls validity:

valid

Conclusions:

Chromosomal aberrations were not induced at any dose of captan tested, although mitomycin C resulted in increase of cells with chromatid-type aberrations.

Executive summary:

Possible mutagenic activity of captan was investigated by in vitro and in vivo cytogenetic studies and by the dominant lethal study in mice. In vitro cytogenetic study with cultured human diploid cells revealed a significant increase. In the frequency of cells showing stickiness and a severe mitotic inhibition at concentrations of 3.0 and 4.0 µg of captan per ml, although no chromosomal aberrations were observed. In in vivo cytogenetic study, no chromosomal aberrations were induced in the bone marrow cells of rats treated orally with captan at a single dose of 500, 1000 or 2000 mg/kg or at five consecutive doses of 200, 400 or 800 mg/kg/day.

 

When discussing the biological effects of captan, its reactivity with protein thiols must be considered. Chromosomal stickiness, observed in this in vitro cytogenetic study using nonestablished diploid cells, may be due to interactions between cap tan and cellular proteins.

Legator et al. have reported induction of chromatid-type aberrations in established heteroploid cells in culture. Out present study, however, could not find chromosomal aberrations in the human diploid cells, although they were not established cell line cells. Disagreement between the results of these two studies might be due to difference of cell lines which may have the composition difference of proteins. The problem should be cleared by further studies.

In mammalian test systems, it may be difficult to detect the mutagenicity of captan because of its reactions with thiols. In the host-mediated assay, results obtained are conflicting. Kennedy et al. observed no increase in number of revertants after oral administrations of captan for 14 days to rats. The positive results were shown by Buselmaier et al. in a modified host-mediated assay in mice after s.c. injections of captan. However, Ficsor et al., who followed the methods of Buselmaier at al., have reported no mutation induction after s.c. or p.o. doses in mice or a p.o. dose in rats. They found no mutagenicity in the blood or urine obtained from captan-treated mice and rats. Mutagenic inactivation of captan following incubation with human and rat blood was shown by them. Microbial studies in our laboratory also revealed disappearance of captan mutagenicity after mixing S-9 mixture or cysteine with captan suspension.

The present in vivo cytogenetic study, which had been awaited by the investigators concerned with captan mutagenicity, revealed no chromosomal aberration induction in the bone marrow cells of-rats after p.o. treatments of a single dose or 5 consecutive doses of captan. Negative results in the dominant lethal studies have been reported in several papers, in which mice were given a single i.p. injection of captan. Collins has reported that multiple i.p. or p.o. doses of technical grade captan could induce dominant lethal mutation in rats and mice. Dominant lethal study reported here was attempted to confirm the Collins' findings by using a complete purity and high daily doses of captan. However, no signs of mutation induction were detected by our study. Negative results were also reported by Epstein et al. in their dominant lethal study conducted with multiple p.o. doses of captan.

Several results in in vitro experiments for mutagenicity inactivation and many negative results in in vitro and in vivo mutagenicity experiments, including the present paper, may lead to a speculation that most of dosed captan reacts with extracellular and intracellular proteins and loses its mutagenic activity before reaching cellular DNA. Some discrepancies in the results of captan mutagenicity experiments may be explained by the qualitative and quantitative difference of protein thiols in various tissues and cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Several studies were performed in literature to evaluate genotoxic potential of captan. For the relevant tonnage band several in vitro studies and two additional in vivo studies were used for assessment.

Form literature it is well known that captan may induce genotoxic effects in vitro. The in vitro studies used for the relevant tonnage band also indicated genotoxic effects. Sulfur containing amino acids and peptides showed a diminishing effect on genotoxic activity in comparison to amino acids without sulfur groups. Assuming presence of analogous sulfur containing substances in living organisms, negative in vivo studies are expected. Analogue results were found in an in vitro genotoxic study on mouse lymphoma cells. 

The additional two in vivo studies (Jacoby, O. (1985) and Tezuka, H. (1978)) showed negative results which is in accordance to literature (EFSA report (2009) 296) that captan is not mutagenic in vivo.

Endpoint Conclusion: No adverse effect observed (negative)

 

Justification for classification or non-classification

It is well known from literature that captan may induce genotoxic effects in in vitro studies.

Additional in vivo studies showed negative results which is in accordance to literature (EFSA report (2009) 296) that captan is not mutagenic in vivo.

In consequence captan is not classified as a mutagenic substance.

 

For the relevant tonnage band no studies on carcinogenicity were necessary. Nevertheless, captan is classified in annex VI of regulation 1272/2008 to be carcinogenic cat. 2 with H351 'suspected of causing cancer'.