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

Genetic toxicity: in vivo

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: Japanese MHW Guidelines on Toxicity Studies

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

EC Guideline B.12

Deviations:

no

Test guideline 4

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

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)

Test material form:

solid: particulate/powder

Test animals

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

Administration / exposure

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.

Doses / concentrations

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.

Examinations

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.

Results and discussion

Test results

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.