Disodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-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 February 18th to May 12th, 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to internationally accepted testing guidelines and performed according to GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)

EC Number:

274-490-1

EC Name:

Disodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)

Cas Number:

70236-60-1

Molecular formula:

C36H21CrN8O11S.2Na

IUPAC Name:

disodium [2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)][3-hydroxy-4-[(2-hydroxy-1-naphthyl)azo]-7-nitronaphthalene-1-sulphonato(3-)]chromate(2-)-

Test animals

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Únětice, Czech Republic, RČH CZ 21760118.
- Weight at study initiation: 215 - 216 g.
- Housing: SPF animal house according to the guideline.
- Acclimation period: at least 5 days.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: aqua pro injection

Duration of treatment / exposure:

21-day application period

Doses / concentrations

Remarks:

Doses / Concentrations:
50, 125, 500 and 1000 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

20 females, 5 per dose-group

Control animals:

yes, concurrent vehicle

Examinations

Tissues and cell types examined:

Bone marrow and peripheral blood.

Details of tissue and slide preparation:

BONE MARROW HARVESTING
Bone marrow cells were from the femora immediately after euthanasia of animals.
After excising and careful cleaning of the bone both femur ends were clipped off Marrow was gently flushed from the bone by 1 ml of bovine serum into the tube. Acquired bone marrow was mixed several times by syringe with thin needle.

Preparation of the bone marrow smears
The bone marrow with serum in tubes was centrifuged (5 min - 1000 rpm). The supernatant was gently removed, one drop of bovine serum was added to the sediment and this cell suspension was mixed on mixer. Clean and degreased slides were marked by the number of study, number of animal, sex and dose level. One drop of cell suspension was placed onto the slide and a smear was prepared using a pusher slide. Two bone marrow smears were prepared per animal.

Staining of the bone marrow smears
After drying (20 minutes, 60 °C) the smears were fixed by ethanol - 5 minutes. Then they were twice rinsed by distilled water and stained by 5 % solution of Giemsa - 15 minutes.

PERIPHERAL BLOOD SMEARS
The blood samples were collected from the orbital plexus by glass micropipette under the light ether narcosis into the PVC test tubes containing anticoagulation systems. Smears preparations were made and then stained. The frequency of micronuclei was scored in mature normochromatic erythrocytes (NCE).
Four slides from each animal were made.

EXAMINATION
Stained bone marrow smears and peripheral blood smears were coded and examined blindly using light microscopy.
At bone marrow smears the incidence of micronucleated cells per 2000 polychromatic erythrocytes (PCE - blue stained immature cells) per animal was scored.

The ratio of polychromatic (immature erythrocytes) among 200 total normochromatic erythrocytes per animal was calculated together with appropriate group mean values and standard deviations to identify toxicity of the test substance.

For peripheral blood smears examination, about 4000 mature normochromatic erythrocytes from each animal were scored. The frequency of micronuclei was scored in mature normochromatic erythrocytes (NCE). The final number of mieronuclei was adjusted for 1000 erythrocytes per animal.

Evaluation criteria:

A comparison was made between the number of micronucleated polychromatic erythrocytes (in case of the bone marrow) and norrnochromatic erythrocytes (in case of the blood) occurring in each of the test group and the number occurring in the corresponding vehicle control group.
Genotoxic activity is indicated by a statistically significant, dose-related increase in the incidence of micronucleated immature erythrocytes for the treatment group compmed with the negative control group.
If these criteria are not demonstrated, then the test material is considered to be non-genotoxic under the conditions of the test.
A positive response for bone marrow toxicity is demonstrated when the dose group mean polychromatic to normochromatic ratio is statistically lower than the concurrent vehicle control group.

Statistics:

The Excel software was used for calculation of mean values and standard deviations for each group of animals.
The statistical analysis was performed by the non-parametric Kruskal-Wallis test for the comparison of the measured effect in all treatment groups with the vehicle control group, as global test. Mann- Whitney test was used for confirmation of results.

Results and discussion

Test results

Sex:

female

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

test substance did not significantly affect the formation of new erythrocytes

Vehicle controls validity:

valid

Negative controls validity:

valid

Additional information on results:

Statistically significant increase in the number of micronucleated polycbromatic erythrocytes in the bone marrow and normochromatic erythrocytes in peripheral blood compared to the control was not recorded at any dose level.
Negative results in the micronucleus test indicate that under the test conditions, the test substance does not produce micronuclei in polychromatic erythrocytes in bone marrow and normochromatic erythrocytes in peripheral blood of rat.
The test substance is considered to be non-genotoxic under the condition of the test.

Any other information on results incl. tables

Proportion of immature erythrocytes among total erythrocytes - "cytotoxicity index"

The number of 200 erythrocytes was evaluated per animal for the proportion of polychromatic (immature) and mature erythrocytes (index cytotoxicity) in bone marrow.

In any group of animals administered by test substance, statistically significant changes of proportion of immature erythrocytes from total number of erythrocytes were not found out.

Comparison of values between treated groups and control group revealed that the test substance did not significantly affect the formation of new erythrocytes.

Cytotoxicity index - group means and standard deviations

Group	Mean	Standard deviation
1000 mg/kg	0.401	0.05
500 mg/kg	0.397	0.06
125 mg/kg	0.422	0.05
50 mg/kg	0.381	0.05
Negative control	0.388	0.05

Number of micronucleated polychramatic erythrocytes

In any group of animals administered by test substance, statistically significant changes of number of micronucleated polychromatic erythrocytes were not found out.

The number of polychromatic erythrocytes with micronuclei was slightly increased in animals of the dose 500 mg/kg, but the result is not statistically significant.

Micronucleated polychromatic erythrocytes - group means and standard deviations

Group	Number of micronucleated PCE per animal (per 2000 PCE)		Percentage expression
	Mean	Standard deviation	Mean	Standard deviation
1000 mg/kg	2.17	0.83	0.109	0.04
500 mg/kg	2.37	1.13	0.119	0.06
125 mg/kg	2.17	0.82	0.109	0.04
50 mg/kg	1.98	0.99	0.099	0.05
Negative control	1.98	0.7	0.099	0.03

Examination of the peripheral blood smears

The frequency of micronuclei was scored in mature normochromatic erythrocytes (NCE) in the circulating blood obtained from the eye socket of the rat.

Red blood samples were collected at the termination of Dose Range Finding Experiment from treated females only; Slides were prepared, fixed and stained as for the bone marrow studies. Four slides from each animal were made. About 4000 mature red blood cells from each animal were scored. The count of micronuclei from treated animals was compared with the count hom negative control group of animals.

Micronucleated erythrocytes - gong means and standard deviations

Group	Number of micronucleated NCE per animal (per 1000 NCE)
	Mean	Standard deviation
1000 mg/kg	0.98	0.39
500 mg/kg	0.84	0.48
125 mg/kg	0.85	0.71
50 mg/kg	0.79	0.32
Negative control	0.79	0.37

NCE - normochromatic (mature) erythrocytes

A dose-related increase in the number of mNCE was observed up to the dose of 1000 mg/kg, though the modulation in the number of micronucleated erythrocytes is not statistically significant.

In any group of animals administered by test substance, statistically significant changes of number of micronucleated erythrocytes were not found out.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test substance is considered to be non-genotoxic under the condition of the test.

Executive summary:

Method

The examination of the bone marrow and peripheral blood was performed with females only. At the termination of the Dose Range Finding Experiment with the test substance performed for Combined Study, the bone marrow harvesting and the blood taking was done.

Extra negative control group of 5 females, treated with vehicle only (aqua pro injection), was added to the DRF experiment. Positive control group of animals normally used in micronucleus test was waived.

Evaluation and interpretation of results was performed according to OECD Test Guideline 474 (1997) Mammalian Erythrocyte Micronucleus Test.

Results

Statistically significant increase in the number of micronucleated polycbromatic erythrocytes in the bone marrow and normochromatic erythrocytes in peripheral blood compared to the control was not recorded at any dose level.

Negative results in the micronucleus test indicate that under the test conditions, the test substance does not produce micronuclei in polychromatic erythrocytes in bone marrow and normochromatic erythrocytes in peripheral blood of rat.

The test substance is considered to be non-genotoxic under the condition of the test.