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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was not mutagenic in the three reported assays (Ames test, in vitro micronucleus assay, in vitro HPRT assay).

Link to relevant study records

Referenceopen allclose all

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
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
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
Target gene:
the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus
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)


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
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.
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
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.


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.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 March 2017 - 19 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human primary culture
Details on mammalian cell type (if applicable):
CELLS USED
- Type and source of cells:
lymphocytes

For lymphocytes:
- Sex, age and number of blood donors:
Two healthy, non-smoking adult humans (between 18 -35 years of age), sex not mentioned
- Whether whole blood or separated lymphocytes were used:
whole blood
- Whether blood from different donors were pooled or not:
yes, two different donors pooled (in equal volumes from each donor)
- Mitogen used for lymphocytes:
phytohaemagglutinin (PHA)

MEDIA USED
- Type and composition of media and incubation temperature:
HML Media: RPMI 1640, supplemented with 10% fetal calf serum, 0.2 IU/mL sodium heparin, 20 IU/mL penicillin / 20 μg/mL streptomycin and 2.0 mM L-glutamine, cultures were incubated at 34 to 39°C
Cytokinesis block (if used):
cytokinesis was blocked using the inhibitor Cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9
rat liver fraction (S9)

- method of preparation of S9 mix
male Sprague-Dawley derived rats dosed with phenobarbital and 5,6-benzoflavone (from Envigo Shardlow)

- concentration or volume of S9 mix and S9 in the final culture medium
S9 homogenate was present in appropriate cultures at a final concentration of 2% v/v.

Test concentrations with justification for top dose:
The highest concentration tested (10 μg/mL) was selected based on solubility.

Preliminary toxicity test: 0.078, 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10 µg/mL
Main tests: -S9 mix (3 hours) 2, 4, 6, 8 and 10 µg/mL; +S9 mix (3 hours) 2, 4, 6, 8 and 10 µg/mL and -S9 mix (20 hours) 2, 4, 6, 8 and 10 µg/mL.

Three test substance concentrations were assessed for determination of induction of micronuclei: 6, 8 and 10 μg/mL (with S9 mix) and 2, 6 and 10 μg/mL (without S9 mix).
Vehicle / solvent:
- Vehicle / solvent used:
ethanol (final volume of ethanol added to the cultures was 1% v/v)

- Justification for choice of solvent/vehicle:
Ethanol was the vehicle that afforded the highest solubility, in which a suspension was formed at 1 mg/mL.

- Justification for percentage of solvent in the final culture medium:
The test substance was formulated as a suspension at 1 mg/mL in ethanol and dosed at 1% v/v to give a maximum initial final concentration of 10 μg/mL.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
colchicine
Remarks:
without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
mitomycin C without S9 mix
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments
1 Preliminary test (3-hour treatment in the absence of S9 mix, 3-hour treatment in the presence of S9 mix, 20-hour treatment in the absence of S9 mix)
1 Main test (3-hour treatment in the absence of S9 mix, 3-hour treatment in the presence of S9 mix, 20-hour treatment in the absence of S9 mix)

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; preincubation
Lymphocyte cultures were incubated for approximately 48 hours following stimulation with PHA, before addition of the test substance.

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period:
Lymphocyte cultures were incubated for approximately 48 hours following stimulation with PHA.
- Exposure duration/duration of treatment:
3-hour treatment (with and without S9 mix): Test substance preparations were added to cultures at 1% v/v. Cultures were incubated at 34 to 39°C for 3 hours. The cells were centrifuged and the medium was replaced with fresh medium. Cytochalasin B, at a final concentration of 6 μg/mL, was then added to all cultures. The cultures were incubated for a further 17 hours until the scheduled harvest time.
20-hour treatment (without S9 mix): 20-hour continuous treatment (1.5 to 2 normal cell cycles) at 34 to 39°C was used in the absence of S9 mix. Test
substance preparations were added to cultures at 1% v/v in the presence of Cytochalasin B (6 μg/mL).

- Harvest time after the end of treatment (sampling/recovery times):
The cells were harvested by centrifugation at 500 g for 5 minutes. The supernatant was removed and the cell pellet re-suspended and treated with a 4 mL hypotonic solution (0.075M KCl) at 34 to 39°C, cultures were then incubated for 3 minutes at 34 to 39°C to cause swelling. Cultures were agitated, 4 mL of ice-cold fixative (3:1 v/v methanol: acetic acid) was added slowly onto the culture surface and the cultures were slowly inverted to mix.

FOR MICRONUCLEUS:
- Cytokinesis blocked method was used for micronucleus assay: Cytochalasin B,
at a final concentration of 6 μg/mL.

- Methods of slide preparation and staining technique used including the stain used (for cytogenetic assays):
The cultures were centrifuged at 500 g for 5 minutes and the supernatant removed. A homogeneous cell suspension was prepared and aliquoted onto the slides. The air-dried slides were stained using an acridine orange solution at 0.0125 mg/mL in purified water.

- Number of replicate cultures and total number of cells scored:
two replicates per test substance concentration, 1000 binucleate cells per culture were scored

- Criteria for scoring micronucleated cells (selection of analysable cells and micronucleus identification):
Cells were included in the analysis provided the cytoplasm remained essentially intact and any micronuclei present were separate in the cytoplasm or only just touching the main nucleus (not connected to the nucleus by a nucleoplasmic bridge). Micronuclei should lie in the same focal plane as the cell, and should possess a generally rounded shape with a clearly defined outline. The main nuclei of the binucleate cells scored for micronuclei should be of approximately equal size. The diameter of the micronucleus should be between 1/16 and 1/3 that of the main nucleus. The color of the micronuclei should be the same or lighter than the main nucleus. There should be no micronucleus-like debris in the surrounding area.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method:
Cytokinesis-block proliferative index (CBPI) values significantly less than the concurrent vehicle control values are indicative of cytotoxicity.

- Any supplementary information relevant to cytotoxicity:
Cytotoxicity = 100-100{(CBPIT-1)/(CBPIC-1)}
Where CBPI = (No. mononucleate cells +2 x No. binucleate cells + 3 x No. multinucleate cells) / Total number of cells
T = test substance treatment culture
C = solvent control culture
Thus, a CBPI of 1 (all cells are mononucleate) is equivalent to 100% cytotoxicity.
Evaluation criteria:
Providing that all of the acceptance criteria have been met, the test item was considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent vehicle control.
- The increase in the frequency of micronucleated cells is dose-related when evaluated with an appropriate trend test.
- Any of the results are outside the distribution of the historical vehicle control data (above the upper control limit).
If all of these criteria are met, the test item was considered able to induce chromosome breaks and/or gain or loss in the test system.
Providing that all of the acceptance criteria have been met, a negative response will be claimed if, in all of the experimental conditions examined:
- None of the test concentrations exhibits a statistically significant increase in the frequency of micronucleated cells compared with the concurrent vehicle control.
- There is no concentration-related increase when evaluated with an appropriate trend test.
- All results are inside the distribution of the historical vehicle control data (below the upper control limit).
If all of these criteria are met, the test item was considered unable to induce chromosome breaks and/or gain or loss in the test system.
Statistics:
The analysis assumed that the replicate was the experimental unit. An arcsine transformation was used to transform the data. Test substance treated groups were compared to the vehicle control using Williams’ tests (Williams 1971, 1972). Positive controls were compared to control using t-tests. Trend tests have also been carried out using linear contrasts by group number. These were repeated, removing the top dose group, until there were only 3 groups.
Statistical significance was declared at the 5% level for all tests.
Key result
Species / strain:
lymphocytes: human
Remarks:
primary culture 20-hour treatment
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: The highest concentration tested (10 μg/mL) was selected based on solubility
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: human
Remarks:
primary culture 3-hour treatment
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: The highest concentration tested (10 μg/mL) was selected based on solubility.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
lymphocytes: human
Remarks:
primary culture 3-hour treatment
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: The highest concentration tested (10 μg/mL) was selected based on solubility.
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:
No fluctuations in pH of the medium were observed at 10 µg/mL of more
than 1.0 unit compared with the vehicle control.

- Data on osmolality:
No fluctuations in osmolality of the medium of more than 50 mOsm/kg were observed compared with the vehicle control.


RANGE-FINDING/SCREENING STUDIES (Preliminary test):
see section "Any other information on results incl. tables" tables 1 - 3.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
see section "Any other information on results incl. tables" tables 4 - 9.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible
no dose-response
- Statistical analysis; p-value
see section "Any other information on results incl. tables" tables 1 - 9.

Micronucleus test in mammalian cells:
- Results from cytotoxicity measurements:
o In the case of the cytokinesis-block method: CBPI; distribution of mono-, bi- and polynucleated cells:
see section "Any other information on results incl. tables" tables 1 - 9.


- Genotoxicity results
o Number of cells with micronuclei separately for each treated and control culture and defining whether from binucleated or mononucleated cells, where appropriate


HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and number of data)
- Positive historical control data:
see section "Any other information on results incl. tables" Historical control data
- Negative (solvent/vehicle) historical control data:
see section "Any other information on results incl. tables" Historical control data

Table 1


Preliminary test: 3-hour treatment in the absence of S9 mix






























































































Treatment
Concentration
(µg/mL)



Mononucleate
cells



Binucleate
cells



Polynucleate
cells



CBPIb



Cytotoxicity (%)



Untreated controls



96
101



367
373



45
37



1.89



-



Vehicle controlsa



101


126



376
357



33
23



1.83



0.0



Mancozeb TK
0.078



127



348



34



1.82



1.7



Mancozeb TK
0.156



140



358



15



1.76



9.1



Mancozeb TK
0.313



109



374



21



1.83



0.7



Mancozeb TK


0.625



106



385



27



1.85



-1.9



Mancozeb TK


1.25



112



381



26



1.83



-0.3



Mancozeb TK


2.5



124



395



36



1.84



-1.2



Mancozeb TK


5



106



391



31



1.86



-3.2



Mancozeb TK
10



88



386



34



1.89



-7.5



a: Vehicle control = Ethanol (1% v/v)


b: Mean value for untreated and vehicle controls


CBPI: Cytokinesis block proliferative index


 


Table 2


Preliminary test: 3-hour treatment in the presence of S9 mix






























































































Treatment
Concentration
(µg/mL)



Mononucleate
cells



Binucleate
cells



Polynucleate
cells



CBPIb



Cytotoxicity (%)



Untreated controls



112
110



381


382



21
18



1.82



-



Vehicle controlsa



120
128



378
368



12
6



1.77



0.0



Mancozeb TK
0.078



129



383



22



1.80



-3.5



Mancozeb TK
0.156



111



381



14



1.81



-4.6



Mancozeb TK
0.313



137



351



14



1.75



2.3



Mancozeb TK


0.625



122



372



20



1.80



-3.7



Mancozeb TK


1.25



123



379



15



1.79



-2.4



Mancozeb TK


2.5



129



358



19



1.78



-1.3



Mancozeb TK


5



112



364



27



1.83



-7.6



Mancozeb TK
10



130



358



14



1.77



0.5



a: Vehicle control = Ethanol (1% v/v)


b: Mean value for untreated and vehicle controls


CBPI: Cytokinesis block proliferative index


 


Table 3


Preliminary test: 20-hour treatment in the absence of S9 mix






























































































Treatment
Concentration
(µg/mL)



Mononucleate
cells



Binucleate
cells



Polynucleate
cells



CBPIb



Cytotoxicity
(%)



Untreated controls



111


86



366
367



23
47



1.87



-



Vehicle controlsa



184
165



311


332



5
3



1.66



0.0



Mancozeb TK
0.078



190



312



9



1.65



2.0



Mancozeb TK
0.156



183



323



6



1.65



0.7



Mancozeb TK
0.313



180



360



8



1.69



-4.1



Mancozeb TK


0.625



195



303



6



1.63



5.2



Mancozeb TK


1.25



151



343



12



1.73



-10.1



Mancozeb TK


2.5



196



291



13



1.63



3.8



Mancozeb TK


5



187



325



6



1.65



1.3



Mancozeb TK
10



306



194



0



1.39



41.1



a: Vehicle control = Ethanol (1% v/v)


b: Mean value for untreated and vehicle controls


CBPI: Cytokinesis block proliferative index


 


Table 4


Main test: 3-hour treatment in the absence of S9 mix - cytotoxicity data
























































































































































Treatment/
Concentration
(µg/mL)



Mononucleate
cells



Binucleate
cells



Polynucleate
cells



CBPI



Mean
CBPI



Mean
Cytotoxicity


(%)



 



102



360



48



1.89



1.88



-



Untreated



120



361



41



1.85



controls



95



372



39



1.89



 



106



358



41



1.87



 



117



367



32



1.84



1.81



0.0



Vehicle



148



332



25



1.76



controlsa



117



360



26



1.82



 



125



356



36



1.83



Mancozeb TK


2



111
109



358
416



34
36



1.85
1.87



1.86



-6.0



Mancozeb TK
4



125
117



367
358



32
30



1.82
1.83



1.83



-1.9



Mancozeb TK
6



107
117



375
378



23
29



1.83
1.83



1.83



-2.9



Mancozeb TK
8



108
115



370
363



23
27



1.83
1.83



1.83



-2.3



Mancozeb TK



91



428



22



1.87



1.84



-4.1



10



116



365



22



1.81



MMC



219



297



1



1.58



1.64



21.5



0.3



162



341



6



1.69



COL


0.07



260
290



229
194



11
17



1.50
1.46



1.48



40.9



a: Vehicle control = Ethanol (1% v/v)


CBPI: Cytokinesis block proliferative index
MMC: Mitomycin C
COL: Colchicine


 


 


 


Table 5


Main test: 3-hour treatment in the absence of S9 mix – micronucleus data


    

























































































Treatment/
Concentration
(µg/mL)



CBPI



Mean
CBPI



Mean
Cytotoxicity
(%)



Binucleated cells containing micronuclei



per 1000
cells



Mean



p-valueb



Trend test
p-valuec



Untreated
controls



1.89
1.85
1.89
1.87



1.88



-



5
5
5
4



4.8



 



 



Vehicle
controlsa



1.84
1.76
1.82
1.83



1.81



0.0



7


5
4


2



4.5



 



 



Mancozeb TK
6



1.83
1.83



1.83



-2.9



7
5



6.0



1.000



 



Mancozeb TK
8



1.83
1.83



1.83



-2.3



4
6



5.0



1.000



0.683



Mancozeb TK
10



1.87
1.81



1.84



-4.1



3
4



3.5



0.733



0.484



MMC


0.3



1.58
1.69



1.64



21.5



32
25



28.5



<0.001***



 



COL


0.07



1.50
1.46



1.48



40.9



30


25



27.5



<0.001***



 



a: Vehicle control = Ethanol (1% v/v)


b: p-values are for comparisons to vehicle control using Williams' test for Mancozeb TK and the t-test otherwise


c: Trend test p-values are for the linear contrast including the vehicle control group and lower concentrations of the same test substance


*** p<0.001


CBPI: Cytokinesis block proliferative index
MMC: Mitomycin C
COL: Colchicine


 


Table 6


Main test: 3-hour treatment in the presence of S9 mix - cytotoxicity data





























































































































































Treatment/
Concentration
(µg/mL)



Mononucleate
cells



Binucleate
cells



Polynucleate
cells



CBPI



Mean
CBPI



Mean
Cytotoxicity


(%)



 



120



358



26



1.81



1.84



-



Untreated



110



366



44



1.87



controls



128



355



22



1.79



 



98



371



33



1.87



 



165



370



14



1.72



1.76



0.0



Vehicle



117



372



23



1.82



controlsa



168



317



20



1.71



 



116



370



20



1.81



Mancozeb TK



158



330



14



1.71



1.75



1.5



2



135



383



23



1.79



Mancozeb TK



147



346



16



1.74



1.75



1.7



4



141



340



21



1.76



Mancozeb TK



137



342



22



1.77



1.78



-2.4



6



135



414



19



1.80



Mancozeb TK
8



135
119



352
367



21
21



1.78
1.81



1.79



-3.5



Mancozeb TK



136



382



23



1.79



1.78



-2.0



10



140



358



20



1.77



CPA
10



262
269



240
242



0
1



1.48
1.48



1.48



37.6



a: Vehicle control = Ethanol (1% v/v)


CBPI: Cytokinesis block proliferative index
CPA: Cyclophosphamide


 


 


Table 7


Main test: 3-hour treatment in the presence of S9 mix – micronucleus data


    















































































Treatment/
Concentration
(µg/mL)



CBPI



Mean
CBPI



Mean
Cytotoxicity


(%)



Binucleated cells containing micronuclei



per 1000
cells



Mean



p-valueb



Trend test
p-valuec



Untreated
controls



1.81
1.87
1.79
1.87



1.84



-



6
6
7
5



6.0



 



 



Vehicle
controlsa



1.72
1.82
1.71
1.81



1.76



0.0



5
5
11


5



6.5



 



 



Mancozeb TK
6



1.77
1.80



1.78



-2.4



7
6



6.5



1.000



 



Mancozeb TK
8



1.78
1.81



1.79



-3.5



5
7



6.0



1.000



0.870



Mancozeb TK
10



1.79
1.77



1.78



-2.0



6
4



5.0



0.626



0.469



CPA
10



1.48
1.48



1.48



37.6



22
30



26.0



<0.001***



 



a: Vehicle control = Ethanol (1% v/v)


b: p-values are for comparisons to vehicle control using Williams' test for Mancozeb TK and the t-test otherwise


c: Trend test p-values are for the linear contrast including the vehicle control group and lower concentrations of the same test substance


*** p<0.001


CBPI: Cytokinesis block proliferative index
CPA: Cyclophosphamide


 


 


Table 8


Main test: 20-hour treatment in the absence of S9 mix - cytotoxicity data
























































































































































Treatment/
Concentration
(µg/mL)



Mononucleate
cells



Binucleate
cells



Polynucleate
cells



CBPI



Mean
CBPI



Mean
Cytotoxicity


(%)



 



68



385



91



2.04



1.99



-



Untreated
controls



101
98



338
311



73
92



1.95
1.99



 



82



342



77



1.99



 



148



350



40



1.80



1.78



0.0



Vehicle



169



328



25



1.72



controlsa



133



327



45



1.83



 



150



320



37



1.78



Mancozeb TK



143



330



35



1.79



1.78



0.6



2



156



343



32



1.77



Mancozeb TK



148



339



21



1.75



1.73



6.8



4



175



344



18



1.71



Mancozeb TK
6



187
210



321
326



22
13



1.69
1.64



1.66



14.9



Mancozeb TK
8



233
218



273
295



5
5



1.55
1.59



1.57



26.9



Mancozeb TK
10



252
251



252
252



5
2



1.51
1.51



1.51



34.6



MMC



148



344



28



1.77



1.78



0.4



0.1



136



369



24



1.79



COL
0.015



289
267



211


230



3
8



1.43
1.49



1.46



41.2



a: Vehicle control = Ethanol (1% v/v)


CBPI: Cytokinesis block proliferative index
MMC: Mitomycin C
COL: Colchicine


 


 


Table 9


Main test: 20-hour treatment in the absence of S9 mix – micronucleus data


    

























































































Treatment/
Concentration
(µg/mL)



CBPI



Mean
CBPI



Mean
Cytotoxicity


(%)



Binucleated cells containing micronuclei



per 1000
cells



Mean



p-valueb



Trend test
p-valuec



Untreated
controls



2.04
1.95
1.99
1.99



1.99



-



4


5
3
6



4.5



 



 



Vehicle
controlsa



1.80
1.72
1.83
1.78



1.78



0.0



3
7
6
4



5.0



 



 



Mancozeb TK


2



1.79
1.77



1.78



0.6



7
3



5.0



1.000



 



Mancozeb TK
6



1.69
1.64



1.66



14.9



3
5



4.0



1.000



0.536



Mancozeb TK
10



1.51
1.51



1.51



34.6



6
8



7.0



0.311



0.354



MMC
0.1



1.77
1.79



1.78



0.4



18


22



20.0



<0.001***



 



COL
0.015



1.43
1.49



1.46



41.2



17
15



16.0



<0.001***



 



a: Vehicle control = Ethanol (1% v/v)


b: p-values are for comparisons to vehicle control using Williams' test for Mancozeb TK and the t-test otherwise


c: Trend test p-values are for the linear contrast including the vehicle control group and lower concentrations of the same test substance


*** p<0.001


CBPI: Cytokinesis block proliferative index
MMC: Mitomycin C
COL: Colchicine


 


HISTORICAL CONTROL DATA


Presented below are the historical negative and positive control data from the period 30 April 2015 to 19 December 2016


Historical control data -S9 3-hour

































































































 



Vehicle control



Mitomycin C
(0.2 or 0.3 µg/mL)



Colchicine
(0.05, 0.06 or 0.07 µg/mL)



 



Binucleate
Individual
MN/1000 cells



Binucleate
Group MN



Binucleate
Individual
MN/1000 cells



Binucleate
Group MN



Binucleate
Individual
MN/1000 cells



Binucleate
Group MN



Minimum



1.0



2.5



16.0



18.0



15.0



19.0



Maximum



14.0



10.5



67.0



64.5



43.0



38.0



Mean



6.3



6.3



34.9



34.9



26.2



26.2



Standard Deviation



2.66



1.9



11.9



11.6



6.4



5.6



Upper Control Limit



 



11.1



 



 



 



 



Number of tests:



24



 



 



 



 



 



Data collection period



30-Apr-15



19-Dec-16



 



 



 



 


           

 


Historical control data +S9 3-hour



































































 



Vehicle control



Cyclophosphamide
(5 or 10 µg/mL)



 



Binucleate
Individual
MN/1000 cells



Binucleate
Group MN



Binucleate
Individual
MN/1000 cells



Binucleate
Group MN



Minimum



0.0



2.5



12.0



13.0



Maximum



15.0



12.5



38.0



31.5



Mean



6.0



6.0



19.9



19.9



Standard Deviation



2.8



2.1



5.3



4.S



Upper Control Limit



 



12.1



 



 



Number of tests:



25



 



 



 



Data collection period:



30-Apr-15



19-Dec-16



 



 



 


Historical control data -S9 20-hour


































































































 



Vehicle control



Mitomycin C
(0.05 or 0.1 µg/mL)



Colchicine
(0.01, 0.015, 0.02 or 0.03 µg/mL)



 



Binucleate
Individual
MN/1000 cells



Binucleate
Group MN



Binucleate
Individual
MN/1000 cells



Binucleate
Group MN



Binucleate
Individual
MN/1000 cells



Binucleate
Group MN



Minimum



0.0



2.0



15.0



16.0



13.0



14.0



Maximum



11.0



9.5



41.0



37.5



22.0



19.5



Mean



6.3



6.3



23.3



23.3



16.8



16.8



Standard Deviation



2.5



1.9



6.5



6.0



1.9



1.4



Upper Control Limit



 



13.5



 



 



 



 



Number of tests:



23



 



 



 



 



 



Data collection period



30-Apr-15



19-Dec-16



 



 



 



 


            

 


The positive control data presented here are pooled from two (Mitomycin C and Cyclophosphamide), three or four (Colchicine; -S9 3 and 20-hour respectively) dose concentrations. The most suitable dose concentration was selected for micronucleus analysis based on the cytotoxicity profile achieved in each test at the Study Directors discretion.

Conclusions:
It was concluded that mancozeb did not show evidence of causing an increase in the induction of micronuclei in cultured human lymphocytes, in this in vitro test system up to the limit concentration for this assay.
Executive summary:

In a guideline and GLP-compliant study, the potential of mancozeb (86.1%) to cause an increase in the induction of micronuclei in cultured human peripheral blood lymphocytes in vitro was investigated. The study consisted of a preliminary toxicity test and a main micronucleus test.


 


In the preliminary toxicity test, human lymphocytes in whole blood culture were exposed to a range of different concentrations of mancozeb for 3 hours in both the absence and presence of (rat) S9 and for 20 hours in the absence of (rat) S9. The maximum final concentration of mancozeb to which the cells were exposed was 10 µg/mL (limit concentration for this test). This was dosed at 1% v/v in ethanol, in order to test up to the maximum achievable concentration in a suitable vehicle. No precipitation was seen up to highest tested concentration. Following 3-hour treatments in the absence and presence of S9, there were no reductions in cytokinesis-block proliferation index (CBPI) at any concentration tested. Therefore, concentrations of mancozeb selected for micronucleus analysis with and without S9 for the 3-hour treatment were 6, 8 and 10 μg/mL. In the absence of S9 following 20-hour treatment, a reduction in CBPI equivalent to 41% cytotoxicity was observed at the highest concentration of 10 µg/mL. Therefore, concentrations of mancozeb selected for micronucleus analysis without S9 for the 20-hour treatment were 2, 6 and 10 µg/mL.


 


In the main test, no precipitation or cytotoxicity was observed up to the top concentration of 10 μg/mL following the 3-hour treatment. In the 20-hour treatment experiment (without S9) no precipitation was seen but a reduction in CBPI equivalent to 34.6% cytotoxicity was observed at the highest concentration of 10 μg/mL. In both the absence and presence of S9 mix, following 3-hour treatment, and in the absence of S9 mix, following 20-hour treatment, mancozeb did not cause any statistically significant increases in the number of binucleate cells containing micronuclei when compared with the vehicle controls. The positive control compounds (mitomycin C, colchicine and cyclophosphamide) caused statistically significant increases in the number of binucleate cells containing micronuclei under appropriate conditions, demonstrating the efficacy of the S9 mix and the sensitivity of the test system.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 December 1989 - 26 March 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
adopted 21 July 1997
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
several genes which govern the biosynhesis of histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
other: rfa mutation, uvr B mutation and R-factor plasmid
Metabolic activation:
with and without
Metabolic activation system:
S9 mix of 5% v/v included S9 fraction (Aroclor 1254 induced rat liver homogenate)
Test concentrations with justification for top dose:
1.95, 3.90, 7.81, 15.62 and 31.25 µg/plate without S9-mix;
15.62, 31.25, 62.5, 125 and 250 µg/plate with 5% v/v S9-mix; limited by cytotoxicity
Vehicle / solvent:
- Vehicle/solvent used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix, strain TA1537
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 mix, strains TA1535 and TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-Nitro-1, 2-phenylene diamine
Remarks:
without S9 mix, strain TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Cumene hydroperoxide
Remarks:
without S9 mix, strain TA102
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Aminofluorene
Remarks:
with S9 mix, strains TA1537, TA1535, TA98, TA100 and TA102
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Plate incorporation

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 1

TREATMENT:
- Exposure duration/duration of treatment:
48 – 72 hours at 37°C

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: clearance of the background lawn
Rationale for test conditions:
Test concentrations were chosen based on cytotoxicity:
Before commencing the mutagenicity study, mancozeb technical was assessed tor cytotoxicity to bacteria using tester strains TA1537. TA1535, TA98, TA100 and TA102. The experiment was conducted using 5% v/v S9 mix and without S9 mix.
Stock solution (5000 µg/plate) of mancozeb technical was prepared by dissolving 0.5 g of mancozeb
technical in 10 mL of Dimethyl sulfoxide. This was serially diluted using Dimethyl sulfoxide to obtain
the desired concentrations, 2500, 1250, 625, 312.5, 156.25 and 78.12 µg plate.
Seven concentrations, 5000, 2500, 1250, 625, 312.5, 156.25 and 78.12 µg/plate were tested for bacterial cytotoxicity.
A quantity of 2 ml of top agar (previously melted and cooled to 45°C ± 2°C) was added to two sets of
sterile test tubes. Later, 500 µL of 5% v/v S9 mix was added to one of the set and 500 µL of 0.2 M
phosphate buffer was added to the second set. 100 µL of the appropriately diluted test substance or solvent control was added to both tubes. Finally 100 µL of standard bacterial suspension was added to both the tubes and were mixed properly. This top agar was added to Minimal Glucose Agar plate and was allowed to solidify.
The petriplates were incubated at 37°C ± 1°C for 48 - 72 hours and then examined to assess the state of the background bacterial growth. Toxicity was assessed by clearance of the background lawn.
Results:
In the experiments with 5% v/v S9 mix, the results revealed that there was no observable cytotoxic effect to bacteria at the dose levels of 78.12 and 156.25 /µg plate, while 312.5 to 5000 µg/plate showed toxic effect i.e., background lawn inhibition in all the five strains.
In case of without S9 mix, the results revealed that there was no observable cytotoxic effect to bacteria at the dose level of 78.12 µg/plate, while 156.25 to 5000/µg/plate showed toxic effect i.e., background lawn inhibition in all five strains.
Evaluation criteria:
A positive result is defined as a statistically significant, dose-dependent increase in the number of histidine independent revertant with at least one dose level inducing a revertant frequency that is two-fold of the spontaneous solvent control value. If the test substance does not induce a statistically significant, dose-dependent increase in revertant frequency but does induce revertant frequency at one dose level that is two-fold the spontaneous control value, the result is considered equivocal. A negative result is defined as the absence of statistically significant or dose-dependent increase in the number of histidine independent revertant.
Statistics:
Simple linear regression analysis was performed for TA1537, TA1535, TA98. TA1OO and TA102, separately, to see the dose dependent increase in the revertant colonies.
Key result
Species / strain:
S. typhimurium TA 102
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
Key result
Species / strain:
S. typhimurium TA 100
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
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 1535
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:
Cytotoxicity test with all five strains

STUDY RESULTS
- Concurrent vehicle negative and positive control data
: see tables in section "Any other information on results incl. tables"

- Concentration-response relationship
: no dose dependent increase was oserved in all strains with and without S9 mix.

- Statistical analysis; p-value
No statistically significant alteration

Ames test:
- Signs of toxicity
: Evaluation of cytotoxicity before conducting the experiment for mutagenic activity:
In the experiments with 5% v/v S9 mix cytotoxic effect to bacteria was observed at the dose levels between 312.5 and 5000 µg/plate. Toxic effect i.e., background lawn inhibition in all five strains.
In case of without S9 mix cytotoxic effect to bacteria was observed at the dose level between 156.25 and 5000 µg/plate. Toxic effect i.e., background lawn inhibition in all five strains.

- Individual plate counts
: see tables in section "Any other information on results incl. tables"

- Mean number of revertant colonies per plate and standard deviation
: see tables in section "Any other information on results incl. tables"

HISTORICAL CONTROL DATA
- no data

TABLE 1 Mean Plate Count


Strain TA1537


 





































































Concentration of
Mancozeb technical
(µg/plate)



His+ revertant colonies/plate
(Mean ± SD)*



Absence of S9 mix



Presence of S9 mix


(5% v/v)



NC



15.00 ± 3.00



22.00 ± 4.58



1.95



14.33 ± 4.16



NA



3.90



19.67 ± 8.51



NA



7.81



19.00 ± 8.54



NA



15.62



16.00 ± 3.46



20.00 ± 7.55



31.25



14.00 ±2.65



23.33 ± 3.51



62.5



NA



25.67 ±4.04



125



NA



25.00 ± 4.58



250



NA



24.00 ± 2.65



PC-9Aa



4333.33 ± 205.58



NA



PC-2Af



NA



85.00 ± 6.56



* Mean of three replicates, SD: Standard deviation, NC: Negative control, PC: Positive control, 9Aa: 9-Aminoacridine (150 µg/plate), 2Af: 2-Aminofluorene (20 µg/plate), NA: Not Applicable


 


 


 


TABLE 1 (continued)


Strain TA1535


 





































































Concentration of
Mancozeb technical
(µg/plate)



His+ revertant colonies/plate
(Mean ± SD)*



Absence of S9 mix



Presence of S9 mix
(5% v/v)



NC



24.33 ± 4.51



29.33 ± 4.73



1.95



25.67 ± 2.52



NA



3.90



19.33 ± 3.51



NA



7.81



21.00 ± 2.65



NA



15.62



20.33 ± 7.10



20.67 ± 6.03



31.25



26.33 ± 4.16



13.00 ± 2.00



62.5



NA



21.00 ±4.36



125



NA



21.33 ± 4.73



250



NA



21.33 ± 2.52



PC-Sa



2808.00 ± 374.12



NA



PC-2Af



NA



41.33 ± 1.53



* Mean of three replicates, SD: Standard deviation, NC: Negative control, PC: Positive control, Sa: Sodium azide (10 µg/plate), 2Af: 2-Aminofluorene (20 µg/plate), NA: Not Applicable


 


 


 


TABLE 1 (continued)


Strain TA98


 





































































Concentration of
Mancozeb technical
(µg/plate)



His+ revertant colonies/plate
(Mean ± SD)*



Absence of S9 mix



Presence of S9 mix
(5% v/v)



NC



23.00 ± 1.73



23.00 ± 1.73



1.95



23.67 ± 5.03



NA



3.90



23.33 ± 1.16



NA



7.81



22.33 ± 3.22



NA



15.62



20.33 ± 3.06



26.33 ± 1.53



31.25



24.00 ± 2.65



23.00 ± 1.73



62.5



NA



23.00 ±4.58



125



NA



25.33 ± 0.58



250



NA



22.33 ± 2.08



PC-4Npd



2544.00 ± 299.76



NA



PC-2Af



NA



5592.00 ± 505.71



* Mean of three replicates, SD: Standard deviation, NC: Negative control, PC: Positive control, 4Npd: 4-Nitro-1,2-phenylene diamine (20 µg/plate), 2Af: 2‑Aminofluorene (20 µg/plate), NA: Not Applicable


 


 


 


TABLE 1 (continued)


Strain TA100


 





































































Concentration of
Mancozeb technical
(µg/plate)



His+ revertant colonies/plate
(Mean ± SD)*



Absence of S9 mix



Presence of S9 mix
(5% v/v)



NC



166.00 ± 4.00



160.00 ± 3.46



1.95



165.00 ± 8.19



NA



3.90



160.00 ± 7.21



NA



7.81



165.00 ± 9.00



NA



15.62



170.00 ± 4.00



150.00 ± 9.17



31.25



155.00 ± 4.36



155.00 ± 4.00



62.5



NA



155.00 ± 12.77



125



NA



150.00 ± 3.46



250



NA



170.00 ± 2.65



PC-Sa



2595.00 ± 26.06



NA



PC-2Af



NA



2603.67 ± 27.79



* Mean of three replicates, SD: Standard deviation, NC: Negative control, PC: Positive control, Sa: Sodium azide (10 µg/plate), 2Af: 2-Aminofluorene (20 µg/plate), NA: Not Applicable


 


 


 


TABLE 1 (continued)


Strain TA102


 





































































Concentration of
Mancozeb technical
(µg/plate)



His+ revertant colonies/plate
(Mean ± SD)*



Absence of S9 mix



Presence of S9 mix
(5% v/v)



NC



254.67 ± 45.49



259.67 ± 29.67



1.95



272.00 ± 60.00



NA



3.90



253.67 ± 19.50



NA



7.81



233.00 ± 23.00



NA



15.62



228.00 ± 36.00



210.67 ± 16.17



31.25



238.00 ± 50.24



248.00 ± 18.19



62.5



NA



249.00 ± 48.82



125



NA



239.67 ± 35.95



250



NA



216.00 ± 44.54



PC-Chp



1224.00 ± 211.41



NA



PC-2Af



NA



584.00 ± 4.00



* Mean of three replicates, SD: Standard deviation, NC: Negative control, PC: Positive control, Chp: Cumene hydroperoxide (100 µg/plate), 2Af: 2‑Aminofluorene (20 µg/plate), NA: Not Applicable


 


Individual plate count in absence of S9 Mix
























































































































































































































Concentration of
Mancozeb
technical



Number of revertant colonies



(µg/plate)



TA1537



TA1535



PA98



TA100



TA102



 



R1



R2



R3



R1



R2



R3



R1



R2



R3



R1



R2



R3



R1



R2



R3



NC



18



15



12



29



20



24



24



21



24



162



166



170



292



204



268



1.95



11



19



13



28



26



23



23



19



29



158



174



163



272



212



332



3.90



26



23



10



23



16



19



24



22



24



166



152



162



276



240



245



7.81



11



28



18



20



19



24



26



20



21



165



156



174



210



256



233



15.62



14



20



14



28



19



14



23



17



21



170



166



174



228



192



264



31.25



17



12



13



31



23



25



21



25



26



158



150



157



208



296



210



PC-4Npd



NA



NA



NA



NA



NA



NA



2880



2448



2304



NA



NA



NA



NA



NA



NA



PC-Sa



NA



NA



NA



2592



2592



3240



NA



NA



NA



2565



2612



2608



NA



NA



NA



PC-9Aa



4096



4456



4448



NA



NA



NA



NA



NA



NA



NA



NA



NA



NA



NA



NA



PC-Chp



NA



NA



NA



NA



NA



NA



NA



NA



NA



NA



NA



NA



1058



1152



1462



 


NC: Negative control, NA: Not Applicable, PC: Positive control, Sa: Sodium azide (10 µg/plate), 4Npd: 4-Nitro-1, 2-Phenylene diamine (20 µg/plate), 9Aa: 9 Aminoacridine (150 µg/plate), Chp: Cumene hydroperoxide (100 µg/plate), R: Replicate


Individual plate count in absence of S9 Mix


































































































































































Concentration of
Mancozeb
technical



Number of revertant colonies



(µg/plate)



TA1537



TA1535



PA98



TA100



TA102



 



R1



R2



R3



R1



R2



R3



R1



R2



R3



R1



R2



R3



R1



R2



R3



NC



23



26



17



31



33



24



24



21



24



158



158



164



256



232



291



15.62



28



19



13



27



20



15



26



25



28



142



148



160



208



228



196



31.25



27



23



20



11



15



13



24



21



24



155



151



159



258



259



227



62.5



21



28



28



23



24



16



22



28



19



152



144



169



236



208



303



125



24



30



21



16



23



25



25



25



26



152



146



152



228



280



211



250



27



22



23



24



19



21



24



20



23



169



173



168



208



264



176



PC-2Af



84



79



92



41



40



43



5544



6120



5112



2635



2582



2594



584



580



588



 


NC: Negative control, PC: Positive control, 2Af: 2‑Aminofluorene (20 µg/plate), R: Replicate

Conclusions:
The substance was tested for mutagenic activity in the Ames-test with the bacterial strains S. typhimurium TA1535, TA1537, TA98, TA100 and TA102 up to cytotoxic concentrations with and without metabolic activation. The test substance did not show mutagenic properties, since no increase of the number of revertants per plate was observed.
Executive summary:

A reverse mutation assay followed the OECD TG 471 was performed using the Salmonella typhimurium strains TA1537, TA1535, TA98, TA100 and TA102 with and without metabolic activation. This study consisted of one independent test.  Based on the cytotoxicity studies, the test substance was evaluated tor its mutagenic effect in all the five strains, in absence of S9 mix at the dose levels 1.95, 3.90, 7.81, 15.62 and 31.25 µg/plate and in presence of 5% v/v S9 mix at the dose levels 15.62, 31.25, 62.5, 125 and 250 µg/plate. Each concentration of the test substance was replicated thrice. No statistically significant alteration in the numbers of revertant colonies of bacteria was recorded for any of the strains of bacteria used, at any test substance concentration, either with or without metabolic activation. No dose dependent increase was found.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance was negative in the in vivo micronucleus assay.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 November 1996 - 02 May 1997
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: Japanese MHW Guidelines on Toxicity Studies
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
EC Guideline B.12
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPP 84-2
Version / remarks:
US EPA Pesticide Assessment Guidelines Subdivision F Series 84-2
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian erythrocyte micronucleus test
Species:
mouse
Strain:
CD-1
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Olac Limited, Shaw's Farm, Blackthorn, Bicester, Oxfordshire.
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: males: 27 - 35 g; females: 20 - 27 g
- Assigned to test groups randomly: yes
- Fasting period before study: 2-3 h period prior to dosing
- Housing: All mice were housed individually in polypropylene cages with stainless steel tops.
- Diet: ad libitum, except for a brief 2-3 h period prior to dosing and 1-2 h after dosing.
- Water: ad libitum, except for a brief 2-3 h period prior to dosing and 1-2 h after dosing.
- Acclimation period: 7 to 8 days between arrival and exposure

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Toxicity study: 16 - 19°C; Micronucleus test: 18 - 20°C
- Humidity (%): Toxicity study: 26 - 51°C; Micronucleus test: 27 - 49°C
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 07 January 1997 To: 20 February 1997
Route of administration:
oral: gavage
Vehicle:
- Vehicle / solvent used: maize oil
- Concentration of test material in vehicle: 50 to 2000 mg/kg (5 to 200 mg/mL)
- Amount of vehicle (gavage): 10 mL/kg body weight
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Immediately prior to dosing, the test material was dissolved in maize oil to give the required dosing concentrations. The dose volume used for both the control and test material treated animals was a constant 10 mL/kg body weight.
Duration of treatment / exposure:
48 h
Frequency of treatment:
twice (at 0 and 24 h)
Post exposure period:
Sampling (scheduled kill) was done at 48 h. Animal health status checks were done at frequent intervals after dosing and prior to the scheduled kill.
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
10 males / 10 females
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral, gavage
- Doses / concentrations: The positive control Cyclophosphamide was prepared freshly as a 5 mg/mL solution in distilled water. It was administered to the positive control animals in dose volumes of 10 mL/kg to give the required target dose of 50 mg/kg bw.
Tissues and cell types examined:
bone marrow cells of femur
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
Based on toxicity investigations, the test substance was judged to be non-toxic at the maximum recommended dose of 2000 mg/kg bw/day. Therefore, in the micronucleus test a single group of male and female CD-1 mice were accordingly dosed orally at 0 h and 24 h with the test substance at the pre-determined concentration of 2000 mg/kg bw/day.

TREATMENT AND SAMPLING TIMES:
In the micronucleus test, groups of CD-1 mice (male and females) were dosed (orally) at 0 h and 24 h with test substance, vehicle or positive control. The animals were sacrificed and the bone marrow samples were taken 24 h after the last treatment (48 h after the first treatment). Animal health status checks were done at frequent intervals after dosing and prior to the scheduled kill. Mice were killed by cervical dislocation. The femora were quickly dissected out and freed of adherent tissue. A small hole was made in the neck of one femur and the bone marrow flushed, using a syringe fitted with a gauge needle.

DETAILS OF SLIDE PREPARATION:
The bone marrow cells were centrifuged for 5 min. at 1000 r.p.m. to pellet the cells. All but a few drops of supernatant fluid were discarded. The cells were then resuspended on a vortex mixer in this residual amount of supernatant liquid. A drop of the suspension was placed at one end of the slide and a smear made by drawing the top of a Pasteur pipette horizontally along the slide. Two slides were prepared from each tube/animal. The smear was left to air dry, fixed in methanol for ca. 5 min. and then immersed for 15 min in 15% Giemsa stain, prepared in tap water, to give optimum erythrocyte discrimination.
The stained smears were finally rinsed in distilled water for ca. 1 min. and left to air dry overnight. Permanent slide preparations were made by sealing coverslips onto the glass slides using DPX mounting medium.

METHOD OF ANALYSIS:
2 prepared slides were selected for examination and the coded slides assessed blind by the same operator. Slides were scored in an ordered sequential fashion using the random number of each slide as guidance. Two thousand (2000) polychromatic erythrocytes (PCE) per animal were scored for micronuclei and the frequency of micro nucleated cells (MN-PCE) determined.
As a control against inclusion of artefacts, or action of a mutagen on the G2 and/or mitotic phase of the cell cycle, the numbers of micro nucleated normochromatic erythrocytes (MN-NCE) in mature red blood corpuscles were also recorded. In addition, scored micronuclei were assigned on the basis of size into small or large categories, historically defined as micronuclei occupying less or more than 25% of the visible cellular area. This classification provided a non-specific measure of compound induced spindle disfunction, as large micronuclei appear to derive from lagging chromosomes caused by damage to the mitotic apparatus during bone marrow erythropoiesis.
The PCE/NCE ratio, a measure of any induced systemic toxicity, was determined by counting a minimum total of 1000 erythrocytes (PCE +NCE) per marrow preparation. Binocular microscopes were used for the assessment. The scoring was done under a nominal magnification of x 1250 using x 12.5 magnification eye pieces and a x 100 oil immersion objective.
Evaluation criteria:
Please refer to the 'Attached background material'.
Statistics:
Please refer to the Evaluation critieria.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
Dose Range Finding Test
In the dose range finding test, 5 groups of one male and one female animal received oral doses of Mancozeb 85% Technical ranging from 50 to 2000 mg/kg bw/day at 0 and 24 h. The highest dose was defined as the maximum routine in vivo exposure level normally administered. One animal death occurred following exposure to 800 mg Mancozeb 85% Technical./kg bw. Clinical signs of subdued behaviour, laboured breathing, hunched appearance, piloerection and hypothermia were observed following treatment.

Main Toxicity Test
In the main toxicity test, 3 groups of 3 male and 3 female animals were given 2 daily doses of Mancozeb 85% Technical ranging from 1200 to 2000 mg/kg bw/day. No deaths or adverse reactions occurred following dosing.
Since no clinical signs or deaths occurred in the main toxicity test, it was considered that the one animal death at 800 mg/kg bw/day in the dose range finding test was of an incidental nature and unrelated to treatment with the test material. Based on these toxicity investigations, Mancozeb 85% Technical was judged to be non-toxic at the maximum recommended dose of 2000 mg/kg bw/d.

MICRONUCLEUS TEST
No animal deaths or clinical signs were observed following treatment.
Vehicle Control Group
The numbers of micronucleated bone marrow polychromatic erythrocytes (MN-PCE) in mice dosed with the vehicle, 10 mL maize oil/kg bw/d averaged 0.04 %. This MN-PCE frequency conformed to the established in-house control range for vehicle treated mice of the CD-I strain (=0.00-0. 28 % per 10 mice or 0.00-0.24 % per 5 mice).
Positive Control Group
Exposure of mice to the positive control agent, 50 mg cyclophosphamide/kg bw, induced large increases in bone marrow micronuclei. The mean MN-PCE frequency for the mice was 1.78%. An evident increase in the number of MN-NCE was also observed. Bone marrow toxicity accompanied these findings a shown by a supression of the PCE/NCE ratios.
Test Material Group
There was no indication that Mancozeb 85% Technical induced bone marrow micronuclei in the treated mice. The highest MN-PCE frequency recorded for the test material was in the females where an incidence of 0.08%o was observed. There was also nothing to suggest bone marrow toxicity in the exposed mice.

Table 1 Summary of Assessment Data














































































































Treatment



Time of



Sex



No. of



Erythrocytes



 



Dosing



 



Mice



Normochromatic Cells (NCE)



Polychromatic Cells (PCE)



PCE/NCE



 



00



 



Assessed



No. ofMN-NCE



PCE Analysed



No. of MN-PCE



%


MN-PCE



Mean ± S.D.



10 mL



0 + 24



m



5



1



10000



4



0.04



0.88 ±0.10



Maize oil/ kg bw/day



 



f



5



6



10000



3



0.03



1.08 ± 0.17



 



 



m+f



10



7



20000



7



0.04



0.98 ±0.17



2000 mg



0 + 24



m



5



4



10000



7



0.07



1.07 ±0.16



Mancozeb 85% Technical/kg bw/



 



f



5



6



10000



8



0.08



0.93 ±0.13



day



 



m+f



10



10



20000



15



0.08



1.00±0.16



50 mg Cyclophosphamide/kg bw/day



0 + 24



m



5



38 <t>



10000



178 a



1.78



0.57 ±0.11



 


PCE = Polychromatic erythrocytes


MN-PCE = Micronucleated PCE


NCE =Normochromatic erythrocytes


MN-NCE = Micronucleated NCE


a = Positive response in PCE


<t>=Evident response in NCE

Conclusions:
Mancozeb 85% Technical did not induce micronuclei in bone marrow cells when tested to the maximum recommended dose of 2000 mg/kg bw/ day in male and female CD-1 mice using a twice (0 h + 24 h) oral dosing and 48 h sampling regimen.
Executive summary:

The in vivo genotoxic potential of Mancozeb 85% Technical was evaluated in a micronucleus test in bone marrow erythrocytes of young, male and female CD-1 mice following a 0 h and 24 h oral dosing and 48 h sampling regimen at a single dose level.
A toxicity study was undertaken to establish a suitable dose level for the micronucleus test. Based on the findings of the toxicity study, Mancozeb 85% Technical was judged to be non-toxic at the maximum recommended dose of 2000 mg/kg bw/day.
In the micronucleus test, one group of CD-1 mice were therefore dosed at 0 h and 24 h orally with the test material at a concentration of 2000 mg/kg bw/day. Bone marrow samples were taken 48 h after the initial 0 h dose. Two control groups of CD-1 mice were also dosed orally with either the vehicle, 10 mL maize oil/kg bw/day, or the positive control agent, 50 mg cyclophosphamide/kg bw/day. The experimental schedule for the control groups followed that of the test material treated mice.
No micronucleus induction was detected in bone marrow erythrocytes of mice dosed with 2000 mg Mancozeb 85% Technical/kg bw/day.
Animals treated with the vehicle alone showed normal background levels of micronuclei, while animals dosed with cyclophosphamide responded with substantial increases in the numbers of bone marrow micronuclei.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

 


The summary below is taken from the CLH report of Mancozeb (Dec 2017).


Mancozeb has been tested in a series of in vitro and in vivo genotoxicity assays. Mancozeb is not mutagenic in bacterial and host mediated bacterial gene mutation assays, or in gene mutation assays in mammalian cells in culture. An equivocal response was observed in vitro in a mouse lymphoma mutation assay. However, mancozeb tested negative in a mammalian cell unscheduled DNA synthesis study. Chromosomal aberrations were induced in vitro, but only in the presence of DMSO and therefore this result is considered invalid. A recently and well conducted in vitro micronucleus study gave negative results for clastogenicity and aneugenicity (Gilby, 2017). A weakly positive response in sister chromatid exchange tests in vitro may be explained by manganese and/or zinc ion interference with enzymes involved in DNA repair and replication. Tight metabolic regulation of these essential nutrients prevents this effect from occurring in vivo.


Mancozeb was tested for photomutagenicity in a bacterial reverse mutation test in the presence of UV light. Under the conditions of the test, mancozeb was found to be non-mutagenic (Schreib, 2014).


Mancozeb was shown not to increase the number of polychromatic erythrocytes containing micronuclei in mice, or to induce chromosome aberrations or cytogenetic abnormalities in mice or rats.


Based on the available data set, mancozeb is concluded not to be a mutagen in somatic cells. As there was no indication of mutagenicity in somatic cells arising from the data set, investigations into the effects of mancozeb in germ cells was not required.

Justification for classification or non-classification

The conclusion below is taken from the CLH report of Mancozeb (Dec 2017).


Mancozeb was not mutagenic in valid in vivo somatic cell mutagenicity tests and so according to the guidance on the application of the CLP criteria no classification is warranted. The overall body of toxicological data from a number of in vitro and in vivo assays indicates that mancozeb is of no genotoxic concern. Therefore no classification for mutagenicity under the CLP regulation is required.