Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Principles of method if other than guideline:
This study was performed to investigate the potential of zinc ethylphenyl dithiocarbamate to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was formulated in corn oil. his vehicle was used as negative control. The volume administered intraperitoneally was 10 ml/kg bw. 16h, 24 h and 48 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. The occurrence of micronuclei in ten animals (5 males, 5 females) per test group was evaluated. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.
GLP compliance:
yes
Type of assay:
mammalian germ cell cytogenetic assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Zinc bis(N-ethyl-N-phenyldithiocarbamate)
EC Number:
238-677-1
EC Name:
Zinc bis(N-ethyl-N-phenyldithiocarbamate)
Cas Number:
14634-93-6
Molecular formula:
C18H20N2S4Zn
IUPAC Name:
zinc(2+) bis([ethyl(phenyl)carbamothioyl]sulfanide)
Specific details on test material used for the study:
Name: Zinc ethylphenyl dithiocarbamate
CAS-No.: 14634-93-6
Aggregate State at RT: solid
Colour: white
Purity: 99.6 %

Test animals

Species:
mouse
Strain:
NMRI
Details on species / strain selection:
Reasons for the Choice of the Experimental Animal Species:
The mouse is an animal which has been used for many years as suitable experimental animal in cytogenetic investigations. There are many data available from such investigations which may be helpful in the interpretation of results from the micronucleus test. In addition, the mouse is an experimental animal in many physiological, pharmacological and toxicological studies. Data from such experiments also may be useful for the design and the performance of the micronucleus test.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Strain: NMRI
Source: Charles River Wiga GmbH
Number of animals: 108 (54 males/54 fe ales)
Initial age at start of acclimatization: minimum 10 weeks
Initial Body eight at start of treatment: 28.2 - 37.2 g

The animals were kept conventionally. The experiment was conducted under standard laboratory conditions.
Housing: single
Cage Type: Makrolon Type I, with wire mesh top
Bedding: granulated soft wood bedding
Feed: pelleted standard diet, ad libitum
Water: tap water, ad libitum
Environment: temperature 21 ± 3°C, relative humidity 30-70%, artificial light 6.00 a.m. - 6.00 p.m.

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
corn oil
Details on exposure:
Six males and six females were assigned to each test group.
The following dose levels of the test article were investigated:
16 h preparation interval: 10 mg/kg b.w.
24 h preparation interval: 1, 3, and 10 mg/kg b.w..
48 h preparation interval: 10 mg/kg b.w..
In pre-experiments the highest dose administered was estimated to be the maximum tolerated dose.

At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article once. Twelve animals, six males and six females, were treated per dose group. Sampling of the bone marrow from animals treated with the highest dose was done 16, 24 and 48 hours after treatment. Bone marrow samples fro animals treated with the low and medium dose were taken only at preparation interval 24 hours.
Duration of treatment / exposure:
16h, 24 h, or 48 hour.
Frequency of treatment:
Single treatment.
Doses / concentrationsopen allclose all
Dose / conc.:
10 other: mg/kg bw
Remarks:
16 h preparation interval: 10 mg/kg b.w.
24 h preparation interval: 10 mg/kg b.w..
48 h preparation interval: 10 mg/kg b.w..
Dose / conc.:
3 other: mg/kg bw
Remarks:
24 h preparation interval: 3 mg/kg b.w..
Dose / conc.:
1 other: mg/kg bw
Remarks:
24 h preparation interval: 1 b.w..
No. of animals per sex per dose:
6 male and 6 female animals/dose/interval
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide

Examinations

Tissues and cell types examined:
1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 1000 PCE.
Details of tissue and slide preparation:
Study Procedure
Test Groups:
Six males and six females were assigned to each test group.
The following dose levels of the test article were investigated:
16 h preparation interval: 10 mg/kg bw.
24 h preparation interval: 1, 3, and 10 mg/kg bw
48 h preparation interval: 10 mg/kg bw

Treatment:
During the study period the animals received feed and water ad libitum . At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article once. Twelve animals, six males and six females, were treated per dose group. Sampling of the bone marrow from animals treated with the highest dose was done 16, 24 and 48 hours after treatment. Bone marrow samples from animals treated with the low and medium dose were taken only at preparation interval 24 hours.

Preparation of the Animals:
The survived animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald. Cover slips were mounted. At least one slide was made from each bone marrow sample.

Analysis of Cells:
Evaluation of the slides was performed using microscopes with 100 x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated. The
microscopic slides of the remaining animals were scored if an animal died in a test group (same time and dose group, same sex).
Evaluation criteria:
A test article is considered positive if, at any of the intervals, there is a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparisonn to the negative control.
A test article is considered negative if there is no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time interval. A test is also considered negative if there is a significant increase in that rate which, according to the laboratory s experience is within the range of negative controls.
The biometric evaluation can be performed by means of the nonparametric Mann-Whitney test.

Results and discussion

Test results
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
In pre-experiments the highest dose administered was estimated to be the maximum tolerated dose. The animals expressed toxic reactions. Reduction of spontaneous activity and eyelid closure followed by apathy were observed.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Under the experimental conditions the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Any other information on results incl. tables

In comparison to the corresponding negative controls there was no enhancement in the frequency of micronuclei at any preparation interval after ap lication of the test article and with any dose

level used.

30 mg/kg b.w. cyclophosphamide administered intraperitoneally was used as positive control which induced a distinct increase of the micronucleus frequency.

Applicant's summary and conclusion

Conclusions:
Zinc ethylphenyl dithiocarbamate was negative (non-mutagenic) in this micronucleus assay.
Executive summary:

This study was performed to investigate the potential of Zinc ethylphenyl dithiocarbamate to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse. The test article was formulated in corn oil. This vehicle was used as negative control. The volume administered intraperitoneally was 10 ml/kg bw. 16h, 24 h and 48 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis. The occurrence of micronuclei in ten animals (5 males, 5 females) per test group was evaluated. 1000 polychromatic erythrocytes (PCE) per animal were scored for micronuclei.

After treatment with the test article the number of NCEs was not substantially increased as compared to the corresponding negative controls thus indicating that Zinc ethylphenyl dithiocarbamate had no cytotoxic effect.

In comparison to the corresponding negative controls there as no enhancement in the frequency of micronuclei at any preparation interval after ap lication of the test article and with any dose level used.

In conclusion, it can be stated that during the study described and under the experimental conditions reported the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse.

Therefore, Zinc ethylphenyl dithiocarbamate is considered to be negative (non-mutagenic) in this micronucleus assay.