mancozeb (ISO); manganese ethylenebis(dithiocarbamate) (polymeric) complex with zinc salt  

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07-November-1983 to 06-February-1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

non-standard study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

other: Directive 87/302/EEC, part B, in vitro mammalian gene mutation

Version / remarks:

protocol essentially complies with Directive 87/302/EEC, part B, in vitro mammalian gene mutation

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

protocol essentially complies with OECD guideline 476

Principles of method if other than guideline:

The study was conducted at the request of the US.EPA in support of the re-registration of mancozeb. The protocol was derived from OECD guideline 471 and Directive 92/69/EEC, part B .

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Test material

Test material information

Constituent 1

Reference substance name:

Complexation products of manganese and zinc with ethylenebis(dithiocarbamate)

EC Number:

616-995-5

Cas Number:

8018-01-7

Molecular formula:

(x+y)[C4H6N2S4]2- + xMn2+ + yZn2+, x:y ranges between 1:0.062 to 1:0.12 (mean 1:0.091)

IUPAC Name:

Complexation products of manganese and zinc with ethylenebis(dithiocarbamate)

Method

Target gene:

the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
The BH4 subclone of the CHO cell clone K1 was developed by Hsie et al., 1975.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9
Fischer 344 rat or B6C3F1 Mouse liver S-9

- method of preparation of S9 mix
Aroclor 1254-induced Fischer 344 rat or B6C3F1 Mouse liver S-9

- concentration or volume of S9 mix and S9 in the final culture medium
S-9 1 mg protein /mL final concentration in the culture medium.
If the test compound exhibited no adverse effect both without and with activation at 1 mg/mL S-9, then the Gene Mutation Assay is repeated with additional S-9 concentrations (0.3 and 2.0 mg/mL S-9).

Test concentrations with justification for top dose:

without metabolic activation: 0, 0.5, 2, 3, 6 µg/mL (88 % a.i.)
with metabolic activation: 0, 0.5, 1, 2, 7 µg/mL (88 % a.i.)

Vehicle / solvent:

- Vehicle / solvent used: aqueous solvents (sterile distilled water)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with S9 mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments: 1 (with and without S9 mix)

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 5 x 10^5 cells/plate
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Without metabolic activation (S-9), treatment was for 18 to 20 h at 37°C; with metabolic activation (S-9), treatment was for approximately 5 h at 37°C.

FOR GENE MUTATION:
- Expression time:
8-day mutation expression period
- Selection time:
incubated for approximately 7 days
- selective agent:
6-thioguanine (6TG, 2 mg/mL), final concentration of 1.67 µg/mL (10 µM)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells:
200 cells/plate were seeded for survivors at the time of selection
2 x 10^5 cells/ plate were seeded to determine 6TG resistant mutants

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: cloning/plating efficiency

Evaluation criteria:

An evaluation of either no adverse effect or an adverse effect was made independently with and without metabolic activation by the following criteria:

1. To be an adverse effect, the Mutant Frequency must exhibit a reproducible, significant increase, and that increase must be accompanied by an increase in the average number of colonies per selection plate compared to that of the solvent control.
2. If 2 different concentrations of the test compound produce a significant increase in the Mutant Frequency, then duplicates, to confirm the reproducibility of the result, may be in the same test. If the increase in the Mutant Frequency was at only one test compound concentration, then the result must be reproduced in an independent test before accepting it as evidence for an adverse effect.
3. Negative results (no adverse effect) must include evaluation of the test compound to its limits of solubility or over a range of toxicities from 75% or greater survival to 20% or less survival relative to the solvent control. If a test compound is relatively non-toxic, the maximum treatment concentration will be 1000 µg/mL.

Statistics:

The Mutant Frequency at each concentration of test compound is compared to that of both the simultaneous solvent controls and the historical negative control for this laboratory. The results of tests performed at different times are not pooled but analyzed independently.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

with and without Fischer-344 rat liver S9 mix

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

with and without B6C3F1 mouse liver S9

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
Two range finding studies were conducted:
1) without metabolic activation at 9 test substance concentrations from 0.05 to 1000 µg/mL (88% a.i.). The toxicity results as assessed by plating efficiency ranged from 19% survival at 5.0 µg/mL to 88% survival at 0.1 µg/mL. No surviving cells were found following treatment concentrations > 10 µg/mL.
2) with metabolic activating system containing Fischer-344 rat liver (S-9 at 1 mg protein/mL) at test substance concentrations from 0.1 to 1000 µg/mL (88 % a.i.). Approximately 100% or more cell survival occurred at concentrations less than or equal to 2.0 µg/mL. No surviving cells were found following treatment at 1000 µg/mL. Treatment with 5 or 20 µg/mL resulted in cell survivals of 79 and 41%, respectively.

STUDY RESULTS
Without metabolic activation, Dithane M-45 did not induce mutations when tested from 0.5 to 15 µg/mL (0.44 to 13.2 µg/mL a.i.).These treatment concentrations yielded >101 to <52 cell survival relative to solvent controls. Dithane M-45 did not Induce mutations when tested with a metabolic activating system derived from Aroclcr induced Fischer 344 rat liver (1 mg S-9 protein/mL) at compound concentrations of 0.25 to 45 µg/mL (0.22 to 39.6 µg/mL a.i.). These treatments produced >100 to <5% cell survival relative to the solvent controls. In addition, 1 µg/mL Dithane M-45 did not induce mutations when tested with 0.3 and 2.0 mg S-9 protein/mL. Dithane M-45 also did not induce mutations when tested from 1 to 16 µg/mL (0.88 to 14.1 µg/mL a.i.) with a metabolic activation system from B6C3F1 mouse liver (1 mg S9 protein/mL). These treatments produced cell survivals from >100 to <5%. In addition, Dithane M-45 at either 4 or 12 µg/mL did not induce mutations when tested at 0.3 and 0.2 mg S-9 protein/mL.

- Concurrent vehicle negative and positive control data
Experiment 1, Without Metabolic Activation:
H20 controls (mutants/10^6 survivors): 8.4 (average)
positive control: 486.5 (average)

Experiment 2, Without Metabolic Activation:
H20 controls (mutants/10^6 survivors): 2.2 (average)
positive control: 309.4 (average)

Experiment 3, Without Metabolic Activation:
H20 controls (mutants/10^6 survivors): 1.3 (average)
positive control: 380 (average)

Fischer 334 Rat Activation:
In the third experiment positive control values reached assay acceptance critieria:
H20 controls (mutants/10^6 survivors): 6.1 (average)
positive control: 236.9 (average)

B6C3F1Mouse Activation:
1 mg S-9/mL:
H20 controls (mutants/10^6 survivors): 4.2 (average)
positive control: 120.8 (average)

HISTORICAL CONTROL DATA
Results were within the the range of the historical control data.

Applicant's summary and conclusion

Conclusions:

Mancozeb is not mutagenic at the HGPRT locus of the CHO cells up to cytotoxic concentrations with and without metabolic activation.

Executive summary:

Dithane M-45 (Mancozeb, coordination product of zinc and manganese ethylene bis-dithiocarbamate. 88.0% a.i., TD# 83-224, Lot 0842) was tested for mutagenic activity at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus in Chinese hamster ovary (CHO) cells both with and without metabolic activation by Aroclor 1254-induced Fischer 344 rat or B6C3F1 Mouse liver S-9. Without metabolic activation, cells were exposed to the test compound dissolved in water, for 18 to 20 h at 37C in Ham's nutrient medium F-12 supplemented with 5% heat-activated, dialyzed, fetal calf serum. With S-9 metabolic activation, cells were exposed to the test compound for approximately 5 h at 37°C in serum-free Ham's nutrient medium F-12. After treatment, cells were grown for approximately 8 days to permit expression of the mutation, and then cultured in 10 µM 6-thioguanine (6TG) to select for HGPRT locus mutants. After Incubation for approximately 7 days, the mutant colonies were counted and compared to the results of concurrent and historical negative (solvent and untreated) controls.

Without metabolic activation, Dithane H-45 did not induce mutations when tested from 0.5 to 15 µg/mL (0.44 to 13.2 µg/mL a.i.). These treatment concentrations yielded >101 to <5% cell survival relative to solvent controls.

Dithane M-45 did not Induce mutations when tested with a metabolic activating system derived from Aroclor Induced Fischer 344 rat liver (1 mg S-9 protein/mL) at compound concentrations of 0.25 to 45 µg/mL (0.22 to 39.6 µg/mL a.i.). These treatments produced >100 to <5% cell survival relative to the solvent controls. In addition, 1 µg/mL Dithane H-45 did not induce mutations when tested with 0.3 and 2.0 mg S-9 protein/mL. Dithane M-45 also did not induce mutations when tested from 1 to 16 µg/mL (0.88 to 14.1 µg/mL a.i.) with a metabolic activation system from B6C3F1 mouse liver (1 mg S9 protein/mL). These treatments produced cell survivals from >100 to <5%. In addition, Dithane H-45 at either 4 or 12 µg/mL did not induce mutations when tested at 0.3 and 0.2 mg S-9 protein/mL.

Under these test conditions Dithane M-45 does not induce mutations at the HGPRT locus in CHO cells in culture when tested in the absence of metabolic activation, or in the presence of either Fischer 344 rat liver S-9 or B6C3F1 mouse liver S-9.