5-(diisopropylamino)-2-[[4-(dimethylamino)phenyl]azo]-3-methyl-1,3,4-thiadiazolium methyl sulphate

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From February 20 to March 31 and from October 9 to December 14, 1992

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(1983)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(1984)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

5-(diisopropylamino)-2-[[4-(dimethylamino)phenyl]azo]-3-methyl-1,3,4-thiadiazolium methyl sulphate

EC Number:

281-589-3

EC Name:

5-(diisopropylamino)-2-[[4-(dimethylamino)phenyl]azo]-3-methyl-1,3,4-thiadiazolium methyl sulphate

Cas Number:

83969-12-4

Molecular formula:

C17H27N6S.CH3O4S

IUPAC Name:

4-{2-[(2E)-5-[bis(propan-2-yl)amino]-3-methyl-2,3-dihydro-1,3,4-thiadiazol-2-ylidene]hydrazin-1-ylidene}-N,N-dimethylcyclohexa-2,5-dien-1-iminium methyl sulfate

Test material form:

liquid

Test animals

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

SPF Han

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: F. Winkelmann, Borchen
- Age at study initiation: 8-12 weeks of age
- Weight at study initiation: 28-44 g (first trial), 28-43 g (second trial)
- Assigned to test groups randomly: yes
- Housing: Makrolon type I cages; females 3 per gorpu, males singly
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least one week

ENVIRONMENTAL CONDITIONS
- Temperature: 22.5-23 °C
- Humidity: 44-47 % (first trial), 38-45 % (second trial)
- Air changes: 10 times per hour
- Photoperiod: 12 hours light

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

Physiological saline solution.

Details on exposure:

Administered volume: 10 ml/kg body weight

Duration of treatment / exposure:

Negative control: 0 mg/kg, 24 h
Test substance: 8.5 mg/kg, 16 h
Test substance: 8.5 mg/kg, 24 h
Test substance: 8.5 mg/kg, 48 h
Test substance: 8.5 mg/kg, replacement group
Positive control, cyclophosphamide: 20 mg/kg, 24 h

Frequency of treatment:

Single exposure.

Doses / concentrations

Dose / conc.:

8.5 mg/kg bw/day (actual dose received)

Remarks:

based on results of a pilot study

No. of animals per sex per dose:

5/sex/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

yes, cyclophosphamide dissolved in deionized water.

Examinations

Tissues and cell types examined:

Bone marrow cells.

Details of tissue and slide preparation:

Schmid's method was q,etto produce the smears.
At least one intact femur was prepared from each sacrificed animal (not pretreated with a spindle inhibitor).

A suitable tube was filled with sufficient fetal calf serum.
A small amount of serum was drawn from the tube into 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 a4 bone marrow was centrifuged in a suitable centrifuge at approximately 1000 rpm for five minutes.
The supernatant was removed with a suitable 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 td-be stained, they needed to be dried with heat for a short period.

Staining of smears
Smears were stained automatically with an Ames Hema-Tek Slide Stainer from the Miles Company. Slides were then "destained" with methanol, rinsed with deionized water, and left to dry.

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. 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. Slides were not evaluated until the covering agent had dried.

Evaluation
Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. They can be distinguished from artifacts by varying the focus.
Normally, 1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern.
It is expedient to establish the ratio of polychromatic to normochroztic erythrocytes for two reasons:
1.Individual animals with pathological bone-marrow depressions may be identified and excluded from the evaluation.
2. An alteration of this ratio may show that the test compound actually reaches the target.

Evaluation criteria:

The number of normochromatic erythrocytes per 1000 polychromatic ones was noted. 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 relevant, treatment related alteration of the ratio polychromatic to normochromatic erythrocytes can only be concluded if it is clearly lower for a majority ot the animals in the treated group than in the negative control.
In addition to the number of normochromatic erythrocytes per 1000 polychromatic ones, the number of normochromatic erythrocytes showing micronuclei was also established.
This information is useful in two ways. Firstly, it permits the detection of individuals already subject to damage before the start of the test. Secondly, combined with the number of micronucleated pychromatic erythrocytes, it permits a representation of the time-effect curve for positive substances.
An increase in the number of micronucleated normochromatic erythrocytes, without a preceding increase in micronucleated polychromatic erythrocytes, is irrelevant to the assessment of a clastogenic effect, since normochromatic erythrocytes originate from polychromatic ones. Before an effect can be observed in normochromatic erythrocytes, there must be a much greater increase in micronucleated polychromatic erythrocytes, duo to the "dilution effect" of the "old" cells, i.e. normochromatic erythrocytes already present at the start of the test, and this effect would have been observed previously.

Statistics:

Test groups with the highest mean (provided this superceded the negative control mean) and the positive control were checked by Wilcoxon's non-parametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochronatic erythrocytes. A variation was considered statistically significant if its error probability was below 5% and the treatment group figure was higher than that of the negative control.
The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chi-test. A variation was considered statistically significant if the error probability was below 5% and the treatment group figure was higher than that of the negative control.
In addition, standard deviations (ls ranges) were calculated for all the means.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- No. of animals: 5/sex
- Dose range: 5, 7.5, 8.5, 9, 9.5 and 10 mg/kg
- Clinical signs of toxicity in test animals: symptoms were recorded for, up to 48 hours, starting at 5 mg/kg: apathy, roughened4 far, staggering gait, sternal recumbency, spasm, shivering and difficulty in breathing. In addition, 2 of 5 animals died in the 9.5 mg/kg group and 4 of 5 animals dd in the 10 mg/kg group. Based on these results, 8.5 mg/kg was chosen as MTD for this test. Due to the use of another batch in the second trial a toxicity test was performed in advance, in which 3 males and 2 females received 8.5 mg/kg. The following symptoms were recorded for up to 4 days: apathy, reduced motility, roughened fur, staggering gait, spasm and difficulty in breaing. In addition, one male died.

Toleration by the Animals
After single intraperitoneal administration of 8.5 mg/kg, treated animals of both trials, showed the following compound-related symptoms until sacrifice: apathy, roughened fur, staggering gait, spasm shivering and difficulty in breathing. In addition, reduced motility was observed during the first trial. The feedlng behavior of the animals was normal. Two of 40 treated animals died during the test period of the first trial, due to the acute toxicity of 8.5 mg/kg the substance. However, there were no substance-induced mortalities in the second trial. No symptoms were recorded for the control groups. No animals died in these groups.

Microscopic evaluation
Evaluation of trial 1
Concerning the assessment of the clastogenic potential of the substance, there were no relevant variations in results between males and females. Therefore, they were evaluated jointly.
The ratio of polychromatic to normochromatic erythrocytes was altered by the treatment, being 1000: 918 (ls=331) in the negative control, 1000: 1893 (ls=586) in the 16 hours group, 1060: 1864 (ls=838) in the 24 hours group and 1000:147 (ls=2194) in the 48 hours group. Relevant variations were noted.

Any other information on results incl. tables

MAIN STUDY

Toleration by the Animals

After single intraperitoneal administration of 8.5 mg/kg, treated animals of both trials, showed the following compound-related symptoms until sacrifice: apathy, roughened fur, staggering gait, spasm shivering and difficulty in breathing. In addition, reduced motility was observed during the first trial. The feedlng behavior of the animals was normal. Two of 40 treated animals died during the test period of the first trial, due to the acute toxicity of 8.5 mg/kg the substance. However, there were no substance-induced mortalities in the second trial. No symptoms were recorded for the control groups. No animals died in these groups.

Microscopic evaluation: trial 1

Concerning the assessment of the clastogenic potential of the substance, there were no relevant variations in results between males and females. Therefore, they were evaluated jointly. The ratio of polychromatic to normochromatic erythrocytes was altered by the treatment, being 1000: 918 (ls=331) in the negative control, 1000: 1893 (ls=586) in the 16 hours group, 1060: 1864 (ls=838) in the 24 hours group and 1000:147 (ls=2194) in the 48 hours group. Relevant variations were noted.

No biologically important or statistically significant variations existed between the negative control and the groups treated intraperitoneally with 8.5 mg/kg dose, with respect to the incidence of micronucleated polychromatic erythrocytes. The incidence of these micronucleated cells was 2.0/1000 (ls=2.3) in the negative control, and 3.3/1000 (ls=2.9), 2.5/1000 (ls=2.0) and 3.5/1000 (I=2.8) in the treated groups.

Similarly, there could be no biologically significant variation between the negative control and treated groups in the number of micronucleated normochromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones. As expected, relevant variations were not observed.

The positive control, cyclophosphamide, caused a clear increase in the number of polychromatic erythrocytes with micronuclei. The incidence of micronucleated cells was 18.6/1000 (ls=7.8), which represents a biologically relevant increase in comparison to the negative control. There could not have been a biologically relevant effect on the number of micronucleated normochromatic erythrocytes in the positive control since, in conjunction with the cell-cycle duration, normochromatic erythrocytes originated from polychromatic ones.

No further effect of cyclophosphamide was found concerning the ratio of polychromatic to normochromatic erythrocytes, since this ratio did not vary to a biologically relevant degree (1000: 1070 (1s=385), as against 1000: 918 in the negative control). This clearly demonstrates that an alteration of the ratio of polychromatic to normochromatic erythrocytes is not necessary for the induction of micronuclei.

The means of micronucleated polychromatic erythrocytes of the 16 and 48 hours groups treated with 8.5 mg/kg test substance exceeded the range of historical negative control means. Despite the negative results of this trial but due to the assay assessment criteria, the results of the test must be rated as equivocal and a second test had to be performed.

Microscopic evaluation: trial 2

Concerning the assessment of the clastogenic potential of test substance, there were no relevant variations in results between males and females. Therefore, they were evaluated jointly. The ratio of polychromatic to normochromatic erythrocytes was altered by the treatment with test substance, being 1000: 556 (ls=110) in the negative control, 1000: 1742 (ls=522) in the 16 hours group, 1000: 1096 (ls=428) in the 24 hours group and 1000: 1155 (ls=489) in the 48 hours group. Relevant variations were thus noted.

No biologically important or statistically significant variations existed between the negative control and the groups treated intraperitoneally with dose of 8.5 mg/kg, with respect to the incidence of micronucleated polychromatic erythrocytes. The negative result of the first trial was reproduced by the second trial and no mean exceeded the range of historical negative controls. The incidence of micronucleated cells was 1.6/1000 (ls=1.6) in the negative control, and 1.3/1000 (ls=0.8), 1.9/1000 (ls=2.0) and 2.9/10b0 (ls=3.0) in treated groups.

Similarly, there could be no biologically significant variation between the negative control and test groups in the number of micronucleated normochromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones. As expected, relevant variations were not observed. The positive control, cyclophosphamide, caused a clear increase in the number of polychromatic erythrocytes with micronuclei. The incidence of micronucleated ce1ls was 24.3/1000 (ls=10.8), which represents a biologically relevant increase in comparison to the negative contro1. There could not have been a biologically relevant effect on the number of micronucleated normochromatic erythrocytes in the positive control since, in conjunction with the cell-cycle duration, normochromatic erythrocytes originated from polychromatic ones.

No further effect of cyc1ophosphide was found concerning the ratio of polychromatic to normochromatic erythrocytes, since this ratio did not vary a biologically relevant degree (1000: 578 (ls=192N as against 1000: 556 in the negative control). This clearly demonstrates that an alteration of the ratio of polychromatic to normochromatic erythrocytes is not necessary for the induction of micronuclei.

Assessment

Normally, cells with micronuclei (Howell-Jolly bodies) occur in polychromatic erythrocytes with an incidence of up to approximately 3.0/1000 (5, 6, and own experience). The increase in micronucleated polychromatic erythrocytes, due, for example, to chromosome breaks or spindle disorders, is the criterion for clastogenic effects in this test model.

The combined results of both trials gave no relevant indications of clastogenic effects after a single intraperitoneal treatment with 8.5 mg/kg. The known mutagen and clastogen, cyclophosphamide, had in both trials a clear clastogenic effect at an intraperitoneal dose of 20 mg/kg body weight. The number of micronucleated polychromatic erythroytes increased to a biologically relevant degree.

The number of micronucleabed normochromatic erythrocytes did not increase relevantly in any of the groups.

It is of further interest to establish the number of normochromatic cells, to learn whether the ratio of polychromatic to normochromatic erythrocytes was altered by treatment. In both trials this ratio did vary to a biologically relevant degree in treated groups in comparison to the negative control. Cyclophosphamide did not change this ratio.

Applicant's summary and conclusion

Conclusions:

There was no indication of a clastogenic effect of an intraperitoneal dose of 8.5 mg/kg test substance in the micronucleus test on the mouse, i.e. in a somatic test system in vivo.

Executive summary:

Method

The micronucleus test was carried out on male and female mice to assess a possible clastogenic effect of test substance on chromosomes of bone-marrow erythroblasts. Cyclophosphamide, i.e. a known clastogen and cytostatic agent, served as positive control.

Two independent trials with identical setup were performed. Treated animals of both trials received a single intraperitoneal administration of either test substance or cyclophosphamide. The femoral marrow of groups treated with test substance was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours.

Test substance and positive control, cyclophosphamide, were administered at doses of 8.5 and 20 mg/ bw, respectively.

Results

After administration both trials, test substance caused symptoms of toxicity, in terms of apathy, roughned fur, staggering gait, spasm, reduced motility,....

Two of forty animals of the first trial died before the end of the test due to acute intraperitoneal toxicity of the 8.5 mg/kg dose, while all animals of the second trial survived until the end of this test.

In both trials there was an altered ratio between polychromatic and normochromatic erythrocytes. Combined results of both trials gave no indications of relevant clastogenic effect of test substance after a single intraperitoneal treatment with 8.5 mg/kg.

Positive control had in both trials a clear clastogenic effect, as is shown by the biologically relevant increase in polychromatic erythrocytes with micronuclei; however, the ratio of polychromatic to normochromatic erythrocytes was not altered.