polymeric zinc propylenebis(dithiocarbamate);propineb (ISO)

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:

1996/10/15 to 1997/05/09

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Principles of method if other than guideline:

The administration was via the i.p. route, which is generally not recommended since it is not an intended route of human exposure. Based on evidence of target tissue exposure (altered NCE:PCE ratio) and clear evidence of systemic toxicity, including mortality at 700 mg/kg bw, it is concluded that dosing route was appropriate. Two thousand (2000) instead of recommended 4000 immature erythrocytes per animal were tested, however since the total number of animals tested/ concentration was 10 (instead of the recommended 5), the overall number of analysed cells was concluded to be sufficient.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

polymeric zinc 1,2-propylenebis(dithiocarbamate)

EC Number:

620-365-5

Cas Number:

9016-72-2

IUPAC Name:

polymeric zinc 1,2-propylenebis(dithiocarbamate)

Test material form:

solid

Remarks:

Powder

Specific details on test material used for the study:

The test material was a formulation containing 70% propineb

Test animals

Species:

mouse

Strain:

other: Hsd/Win

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: approximately 6 to 12 weeks.
- Weight at study initiation: males: 37-44 g, females: 28-34 g
- Housing: The animals were kept singly in type I cages.
- Water: Tap water ad libitum.
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±1.5
- Humidity (%): 40% to 70%
- Air changes (per hr): 10

Husbandry was standardized, with twelve hours of electrical lighting daily (6.00 hours to 18.00 hours, about 500 lux), 22.5-23°C room temperature, and 47-54% (1st trial) and 33-38% (2nd trial) mean relative humidity. EP IT Elb. 2 (engineering department) gives the following settings for the animal room: 22±1.5°C, 40% to 70% humidity and air change about ten times per hour.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Water

Details on exposure:

The treated animals received a single intraperitoneal administration of either propineb or cyclophosphamide. The femoral marrow of groups treated with propineb was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours. The doses of propineb were 500 mg/kg bw in the first trial and 700 mg/kg bw in the second experiment. For the positive control, cyclophosphamide, 20 mg/kg body weight were used for both studies.

Including both males and females, were intraperitoneally administered 250 mg/kg bw, 500 mg/kg bw and 1000 mg/kg bw of the test material.

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Once

Post exposure period:

16, 24, 48 hours

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

250 mg/kg bw/day

Remarks:

First experiment: equivalent to 175 mg/kg bw/d propineb

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day

Remarks:

First experiment: equivalent to 350 mg/kg bw/d propineb

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day

Remarks:

First experiment: equivalent to 700 mg/kg bw/d propineb

Doses / concentrations 4

Dose / conc.:

500 mg/kg bw/day

Remarks:

Second experiment: equivalent to 350 mg/kg bw/d propineb

Doses / concentrations 5

Dose / conc.:

700 mg/kg bw/day

Remarks:

Second experiment: equivalent to 490 mg/kg bw/d propineb

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes

Positive control(s):

20 mg/kg Cyclophosphamide

Examinations

Tissues and cell types examined:

bone marrow

Details of tissue and slide preparation:

At least one intact femur was prepared from each sacrificed animal (not pretreated with a spindle inhibitor). A suitable instrument was used to sever the pelvic bones and lower leg.
The femur was separated from muscular tissue. The lower-leg stump, including the knee and all attached soft parts, was separated in the distal epiphyseal cartilage by a gentle pull at the distal end. The proximal end of the femur was opened at its extreme end with a suitable instrument, e.g. fine scissors, making visible a small opening in the bone-marrow channel. A suitable tube was filled with sufficient fetal calf serum. A small amount of serum was drawn from the tube into a
suitable syringe with a thin cannula. The cannula was pushed into the open end of the marrow
cavity. The femur was then completely immersed in the calf serum and pressed against the wall of the tube, to prevent its slipping off. The contents were then flushed several times and the bone
marrow was passed into the serum as a fine suspension. Finally, the flushing might be repeated from the other end, after it had been opened. The tube containing the serum and bone marrow was centrifuged in a suitable centrifuge at approximately 1000 rpm for five minutes.
The supernatant was removed with a suitable pipette (e.g. Pasteur pipette), leaving only a small remainder. The sediment was mixed to produce a homogeneous suspension. One drop of the viscous suspension was placed on a well cleaned slide and spread with a suitable object, to allow
proper evaluation of the smear. The labeled slides were dried overnight. If fresh smears needed to be stained, they needed to be dried with heat for a short period.
The Staining of Smears:
The smears were stained automatically with an Ames Hema-Tek Slide Stainer from the Miles Company. The slides were then "destained" with methanol, rinsed with deionized water, and left to dry.
The Covering of Smears:
Following this treatment, the smears were transferred to a holder. A cuvette was filled with xylene, into which the holder was immersed for approximately ten minutes. The slides were removed singly (e.g. with tweezers) to be covered. A small amount of covering agent was taken from a bottle with a suitable object (e.g. glass rod) and applied to the coated side of the slide. A cover glass was then placed in position without trapping bubbles. The slides were not evaluated until the covering agent had dried.

Evaluation criteria:

Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the a nucleated erythrocytes. They can be distinguished from artifacts by varying the focus.

If the ratio for a single animal amounts to distinctly more than 3000 normochromatic erythrocytes per 1000 polychromatic ones, or if such a ratio seems likely without other animals in the group showing similar effects, then the case may be regarded as pathological and unrelated to treatment, and the animal may be omitted from the evaluation. A test was considered positive if, at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control. test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory's experience was within the range of negative controls.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

A negative result is reported for a mouse bone marrow micronucleus assay with propineb, using single doses of 500 and 700 mg/kg bw (sampling at 16, 24 and 48 hours).

Any other information on results incl. tables

There is evidence of target tissue exposure (altered NCE:PCE ratio) and clear evidence of systemic toxicity, including mortality at 700 mg/kg bw. Furthermore, target tissue exposure can be assumed due to the dosing method used.

Applicant's summary and conclusion

Conclusions:

No relevant indications of a clastogenic effect of propineb were found after a single intraperitoneal treatment with 500 and 700 mg/kg bw.

Executive summary:

The micronucleus test was employed to investigate propineb in male and female mice for a possible clastogenic effect on the chromosomes of bone-marrow erythroblasts in two independent experiments. The known clastogen and cytostatic agent, cyclophosphamide, served as positive control. The treated animals received a single intraperitoneal administration of either a propineb product (containing 70% propineb) or cyclophosphamide.

The femoral marrow of groups treated with propineb was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours. The doses of propineb were 500 mg/kg bw in the first trial and 700 mg/kg bw in the second experiment. For the positive control, cyclophosphamide, 20 mg/kg body weight were used for both studies. The animals treated with propineb showed symptoms of toxicity after administration. Two of forty animals died before the end of the test due to the acute intraperitoneal toxicity of 700 mg/kg bw propineb. There was an altered ratio between polychromatic and normochromatic erythrocytes. No relevant indications of a clastogenic effect of propineb were found after a single intraperitoneal treatment with 500 and 700 mg/kg.

Cyclophosphamide, the positive control, had a clear clastogenic effect, as is shown by the biologically relevant increase in polychromatic erythrocytes with micronuclei. The ratio of polychromatic to normochromatic erythrocytes was not altered.