Ferbam

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1991-12-19 - 1992-09-14

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No positive control

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

No positive control

GLP compliance:

yes

Type of assay:

other: peripheral blood micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Ziram

EC Number:

205-288-3

EC Name:

Ziram

Cas Number:

137-30-4

Molecular formula:

C6H12N2S4Zn

IUPAC Name:

zinc bis(dimethyldithiocarbamate)

Test material form:

solid: particulate/powder

Details on test material:

- State of aggregation: white powder
- Storage condititons: ambient temperature protected from litght

Specific details on test material used for the study:

Expiry and Purity: Analysed at 98.8 % in July 1991 concluding no deterioration, checked at six-month intervals

Test animals

Species:

mouse

Strain:

CD-1

Details on species / strain selection:

CD-1 outbred albino mice of Swiss origin

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, USA
- Age at study initiation: 5-6 weeks
- Weight at study initiation: no data
- Assigned to test groups randomly: Initially the animals were randomised; final group allocation was made (just prior to initiation of treatment) according to body weight to minimise variation in cage mean body weights
- Housing: sexes separated in groups of 2 in solid bottom polypropylene cages with autoclaved sifted sawdust bedding.
- Diet (e.g. ad libitum): ad libitum powdered SDS rat and mouse no. 1 modified maintenance diet.
- Water (e.g. ad libitum): ad libitum tap water
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
Animal room temperature and humidity controls were maintained at 21 ± 3°C and 55 ± 10% respectively; lighting was controlled to give 12 hours continuous light and 12 hours continuous dark per 24 hours.

Administration / exposure

Route of administration:

oral: feed

Vehicle:

no

Details on exposure:

DIETARY FORMULATION
A premix was prepared each week by grinding the test substance directly into SDS rat and mouse No. 1 modified maintenance diet and mixing in a Turbula mixer for a minimum period of 2 minutes.
The required concentrations were then prepared by direct dilution of the pre-mix with further quantities of untreated diet; homogeneity being achieved by further mixing in a double-cone blender for a minimum period of 7 minutes. The concentration of test material in the low dose level was increased by 15% above nominal and the low intermediate by 10% in order to compensate for losses during storage already observed in pre-dosing analytical chemistry.
On completion of the mixing procedure for each group the diets to be fed at each level were divided into aliquots for daily use during the following week. All aliquots were transferred as quickly as possible to storage at approximately 4°C until immediately before feeding.
A constant concentration was administered to treated groups throughout the study.
During the course of the study ZIR/12, samples of the test diet were taken at intervals to confirm accuracy of preparation by chemical analysis.

Duration of treatment / exposure:

89 days

Frequency of treatment:

daily

Post exposure period:

none

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 ppm (nominal)

Remarks:

nominal diet

Doses / concentrations 2

Dose / conc.:

25 ppm (nominal)

Remarks:

nominal diet

Doses / concentrations 3

Dose / conc.:

75 ppm (nominal)

Remarks:

nominal diet

Doses / concentrations 4

Dose / conc.:

225 ppm (nominal)

Remarks:

nominal diet

Doses / concentrations 5

Dose / conc.:

675 ppm (nominal)

Remarks:

nominal diet

No. of animals per sex per dose:

50

Control animals:

yes, plain diet

Positive control(s):

no

Examinations

Tissues and cell types examined:

Peripheral blood

Details of tissue and slide preparation:

PREPARATION OF BLOOD SMEARS
After 89 days dietary exposure, blood smears were prepared from 5 male and 5 female animals in each group (the highest animal numbers in each group were sampled). A drop of blood was taken from a small puncture in a lateral tail vein and a smear prepared on a glass microscope slide in the conventional manner. Four smears were prepared from each animal in case of practical problems with any individual smear.
The prepared smears were fixed in methanol, stained in aqueous 10% (v/v) Gurr's R66 Giemsa (BDH) for 10 minutes, rinsed in distilled water then differentiated in pH 6.8 buffered distilled water for 10 minutes. The slides were allowed to air dry before mounting with glass coverslips using DPX. The mountant was allowed to harden at approximately 37°C before the slides were randomised, encoded and examined by light microscopy for the presence of micronuclei in normochromatic and polychromatic erythrocytes (1000 cells of each type were examined from each animal). The ratio of polychromatic to normochromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal.

Evaluation criteria:

Bone marrow cell toxicity (or depression) is normally indicated by a substantial, statistically significant decrease in the proportion of polychromatic erythocytes in the blood i.e. bone marrow toxicity results in a decrease in the ratio of polychromatic to normochromatic erythrocytes.

Statistics:

Non-parametric statistical methods, based on rank, were chosen for analysis of results because;
(a) They are suited to analysis of data consisting of discrete/integer values such as the incidence of micronucleated polychromatic erythrocytes.
(b) The methods make few assumptions about the underlying distribution of data and therefore the values do not require transformation to fit a theoretical distribution (where data can be approximately fitted to a normal distribution, the results of non-parametric analysis and classical analysis of variance are very similar).
(c) 'Outliers' are frequently found in polychromatic erythrocytes to normochromatic erythrocyte ratios for both control and treated animals; non-parametric analysis does not give these values an undue weighting.
For a comparison of multiple groups with a concurrent control group Kruskal-Wallis' version of Wilcoxon's sum of ranks test was used; Jonckheere's and Spearman's tests were used to analyse for dose-related trends.
A positive response is normally indicated by a dose-related statistically significant increase in the incidence of micronucleated erythrocytes for the treatment groups in comparison with the concurrent control group. In borderline cases further slide reading or sampling is possible.

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not examined

Additional information on results:

MICRONUCLEATED POLYCHROMATIC ERYTHROCYTE COUNTS (mnp)
The test item did not cause any statistical significant increases in the number of micronucleated polychromatic erythrocytes [P>0.01 using Kruskal-Wallis' test, Jonckheere's test for trend and Spearman's correlation test].

MICRONUCLEATED NORMOCHROMATIC ERYTHROCYTES (mnn)
The test item did not cause any statistically significant increases in the incidence of micronucleated normochromatic erythrocytes [P>0.01 using Kruskal-Wallis' test, Jonckheere's test for trend and Spearman's correlation test].

RATIO OF POLYCHROMATIC TO NORMOCHROMATIC ERYTHROCYTES (pin)
The test item failed to cause any significant decreases in the ratio of polychromatic to normochromatic erythrocytes [P>0.01 using Kruskal-Wallis' test, Jonckheere's test for trend and Spearman's correlation test].

Any other information on results incl. tables

Treatment level [ppm]	Ratio p/n (mean)	Incidence mnp (mean)	Incidence mnn (mean)
Control	0.108	0.9	1.1
25*	0.143	0.6	1.7
75**	0.124	1.2	2.0
225	0.095	0.8	0.7
675	0.135	0.5	1.2

p/n Ratio of polychromatic to normochromatic erythrocytes

mnp No. of micronucleated cells observed per 1000 polychromatic erythrocytes

mnn No. of micronucleated cells observed per 1000 normochromatic erythrocytes

* Prepared at 29 ppm initially to allow for loss during storage

** Prepared at 83 ppm initially to allow for loss during storage

Applicant's summary and conclusion

Conclusions:

The test item did not show any evidence of chromosome-damaging activity or bone marrow cell toxicity after sub-chronic dietary administration in this in vivo test procedure.

Executive summary:

This report assess the chromosome-damaging activity of the test item following sub-acute dietary administration to mice during an ongoing oncogenicity study (HRC study number ZIR/12) according to EPA Guideline OPP 84 -2 and in accordance with GLP.

Young adult CD-I outbred albino mice were maintained on diet containing the test material at levels of 25, 75, 225 and 675 ppm using the methods described for HRC Study Schedule No. ZIR/12. A group of control mice were maintained on untreated diet.

After 89 days exposure, peripheral blood smears were prepared from 5 male and 5 female animals in each group. The smears were stained using Giemsa then examined by light microscopy for the presence of micronuclei in normochromatic and polychromatic erythrocytes (1000 cells of each type were examined from each animal). The ratio of polychromatic to normochromatic erythrocytes was assessed by examination of at least 1000 erythrocytes from each animal.

Mice treated with the test item did not show any significant increase in the frequency of micronucleated normochromatic or polychromatic erythrocytes.

Mice treated with the test item did not show any significant decrease in the ratio of polychromatic to normochromatic erythrocytes (a reduction in this ratio is indicative of bone marrow toxicity).

It is concluded that the test material has not shown any evidence of causing chromosome damage in this in vivo test system.