Disodium [6-[(2-anilino-1-naphthyl)azo]-2,4-dinitrophenolato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]naphthalene-1-sulphonato(3-)]chromate(2-)

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:

From October 26th to November 24th, 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1994

Report date:

1994

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Similar Substance 01

IUPAC Name:

Similar Substance 01

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann Borchen Germany.
- Age at study initiation: 8 to 12 weeks, young adult male and virgin female mice.
- Weight at study initiation: 28 to 43 g.
- Assigned to test groups randomly: yes.
- Housing: the females were kept in groups of a maximum of three mice in Makrolon type I cages. Males were kept singly in type I cages.
- Diet: Altromin 1324 Standard Diet, ad libitum.
- Water: tap water and feed were available ad libitum.
- Acclimation period: at least one week.
- Health check: the breed's state of health was regularly spot-checked for the major specific pathogens.

ENVIRONMENTAL CONDITIONS
- Temperature: 22.5 - 23 °C
- Humidity: 44 - 52 %
- Air changes: ten times per hour.
- Photoperiod: 12 hrs dark / 12 hrs light, electrical lighting daily of about 500 lux.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle used: physiol. saline.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS
The test item was dissolved in physiological saline solution, using a magnetic stirrer for 30 minutes, stirred with a magnetic mixer during administration and injected intraperitoneally.
Administration volume was 10 ml/kg bw.

Duration of treatment / exposure:

Administration at 16, 24, or 48 hours

Frequency of treatment:

Single dose

Doses / concentrations

Dose / conc.:

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

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

- Positive control: cyclophosphamide.
- Justification for choice of positive control: routinely used positve control.
- Vehicle: physiological saline.
- Route of administration: intraperitoneal.
- Doses / concentrations: 20 mg/kg bw.

Examinations

Tissues and cell types examined:

Bone marrow (femur)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
The selection of test item dose was based on a pilot test, in which groups of five animals, including both males and females, were intraperitoneally administered 100 mg/kg, 150 mg/kg, 250 mg/kg and 500 mg/kg.
Based on these results, 150 mg/kg test item was chosen for the main test.

DETAILS OF 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-Ieg 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. The femur was then completely immersed in the calf serum and pressed against the wall of the tube, to prevent its slipping off.

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 HemaTek Slide Stainer from the Miles Company. The slides were then "destained" with methanol, rinsed with deionized water and left to dry.

METHOD OF ANALYSIS
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.

Evaluation criteria:

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.
A 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.
In addition, a test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group. In this case, a second test had to be performed at the most sensitive interval.

An assay was considered acceptable if the figures of negative and positive controls were within the expected range, in accordance with the laboratory's experience and/or the available literature data.

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:

The results with test item gave no relevant indications of clastogenic effects after a single intraperitoneal treatment with 150 mg/kg.

Toleration by the animals
After single intraperitoneal administration of 150 mg/kg test item treated animals showed the following compound-related symptoms until sacrifice: apathy back discoloration of hairless parts of skin, staggering gait, spasm, difficulty in breathing and eyelids stuck together.
Their feeding behavior was normal. One of 40 treated animals died during the test period, due to the acute toxicity of 150 mg/kg test item.
No symptoms were recorded for the control groups. No animals died in these groups.

Microscopic Evaluation
Concerning the assessment of the clastogenic potential of test item, there were no relevant variations in results between males and females. Therefore, they were evaluated jointly.
As animal 12 of the 48 hours group exhibited a pathological change of the ratio polychromatic to normochromatic erythrocytes, it was not included in the evaluation.
The ratio of polychromatic to normochromatic erythrocytes was altered by the treatment with test item, being 1000: 1056 (1s=386) in th gative control, 1000: 2370 (1s=1258) in the 16 hogts group, 1000: 1537 (1s=593) in the 24 hours group and 1000: 1733 (1s=1012) in the 48 hours group. Relevant vaiations were thus noted.
No biologically important or statistically significant variations existed between the negative control and the groups treated intraperitoneally with 150 mg/kg, with respect to the incidence of micronucleated polychromatic erythrocytes. The incidence of these micronucleated cells was 1.3 / 1000 (1s=0.7) in the negative control, and 1.6 / 1000 (1s=1.6), 2.0 / 1000 (1s=1.7) and 1.6 / 1000 (1s=0.9) in the test item groups.
Similarly, there could be no biologically significant variation between the negative control and Ttest item groups in the number of micronucleated normechromatic erythrocytes, since normochromatic erythrocytes originated from polychromatic ones. As expected, relevant variations were not observed.

POSITIVE CONTROL
The positive control, cyclophosphamigg, caused a clear increase in the number of polychromatic erythrocytes with micronuclei. The incidence of micronucleated cells was 14.9 / 1000 (1s=9.8), which represe ts a biologically relevant increase in comparison to the negative control.
There could not have been a biologically relevant effect on the number of micronuclhated normochromatic erythrocytes in the positive control since, in conjunction with the cell-cycle duration, normochromatic erythrocytes originated from polychromat ones.
No further effect of cyclophosphamide was found concerning the ratio of polychromatic to normochromatic erythrocytes, since the ratio did not vary to a biologically relevant degree [1000: 674 (1s=232), as against 1000: 1056 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.

RESULTS OF RANGE-FINDING STUDY
The following symptoms were recorded for up to 48 hours, starting at 100 mg/kg: apathy, lack discoloration of hairless parts of skin, staggerig gait, sternal recumbency, spasm, difficulty in breathing and eyelids stuck together. In addition, 1 of 5 animals died in the 150 mg/kg group and 5 of 5 animals died in the 250 and 500 mg/kg groups.

Any other information on results incl. tables

Results of micronucleus test with test item after acute peritoneal treatment with 150 mg/kg bw

Group	Number of evaluated PCE	NCE/1000 PCE	Micronucelated cells per 1000 NCE	Micronucleated Cells per 1000 PCE
negative control	10000	1056 ± 386	1.2 ± 1.2	1.3 ± 0.7
test 16 hours	10000	2370 ± 1258	1.2 ± 1.0	1.6 ± 1.6
test 24 hours	10000	1537 ± 593	1.1 ± 0.8	2.0 ± 1.7
test 48 hours	10000	1733 ± 1012	0.5 ± 0.7	1.6 ± 0.9
positive control	10000	674 ± 232	0.4 ± 0.8	14.9 ± 9.8

Applicant's summary and conclusion

Conclusions:

The test item did not cause the formation of micronuclei in mice in vivo.

Executive summary:

Test substance was assayed in the micronucleus test in male and female mice for a possible clastogenic effect on the chromosomes of bone-marrow erythroblasts. The known clastogen and cytostatic agent, cyclophosphamide, served as positive control. Treated animals received a single intraperitoneal administration of either test item or cyclophosphamide. The femoral marrow of groups treated with the substance was prepared 16, 24 and 48 hours after administration. All negative and positive control animals were sacrificed after 24 hours. Doses of test item and positive control, cyclophosphamide, were 150 and 20 mg/kg body weight, respectively.

Animals treated with test item showed symptoms of toxicity after administration. One of forty animals died before the end of the test due to the acute intraperitoneal toxicity of 150 mg/kg. There was an altered ratio between polychromatic and normochromatic erythrocytes.

No indications of a clastogenic effect of test item were found after a single intraperitoneal treatment with 150 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.

Conclusion

Test item did not cause the formation of micronuclei in mice in vivo.