Calcium 4-chloro-2-(5-hydroxy-3-methyl-1-(3-sulfonatophenyl)pyrazol-4-ylazo)-5-methylbenzenesulfonate

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

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

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:

Versálová žluť HGR (Pigment Yellow 191)

IUPAC Name:

Versálová žluť HGR (Pigment Yellow 191)

Constituent 2

Reference substance name:

Calcium 4-chloro-2-(5-hydroxy-3-methyl-1-(3-sulfonatophenyl)pyrazol-4-ylazo)-5-methylbenzenesulfonate

EC Number:

403-530-4

EC Name:

Calcium 4-chloro-2-(5-hydroxy-3-methyl-1-(3-sulfonatophenyl)pyrazol-4-ylazo)-5-methylbenzenesulfonate

Cas Number:

129423-54-7

Molecular formula:

C17H15ClN4O7S2.Ca

IUPAC Name:

calcium 4-chloro-5-methyl-2-{2-[3-methyl-5-oxo-1-(3-sulfonatophenyl)-4,5-dihydro-1H-pyrazol-4-yl]diazen-1-yl}benzene-1-sulfonate

Details on test material:

Molecular formula: C17H15ClN4O7S2.Ca
Molecular weight: 524.9
Appearance: Yellow solid powder
Storage: the sample was stored in supplied container in dry room at the temperature > 30oC

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals: Albino laboratory rat, strain Wistar, healthy young adult males and females (nulliparous and non-pregnant)
Age at receiving: 6 – 7 weeks (at arrival)
Total number of animals: Pilot experiment: 9 animals (females), Main test: 100 animals (50 males and 50 females)
Random selection: According to SOP No.42
Acclimatisation: at least 5 days
Microclimatic conditions: Room temperature 22 + 3°C, permanently monitored
Relative humidity 30 – 70 %, permanently monitored
Light: 12 hours light/dark cycle: 6am-6pm/6pm-6am
Water: Drinking tap water ad libitum
Diet: Pelleted standard diet for experimental animals ad libitum
Bedding: Sterilized shavings of soft wood
Prophylactic arrangement: Cleaning and disinfection of animal room was regularly performed, as it is described in appropriate SOP No.10.

Administration / exposure

Route of administration:

oral: drinking water

Duration of treatment / exposure:

24 and 48 hours for each dose level and each animal group

2000 mg/kg, exposition – 24 h: 5 males + 5 females
2000 mg/kg, exposition – 48 h: 5 males + 5 females
1000 mg/kg, exposition – 24 h: 5 males + 5 females
1000 mg/kg, exposition – 48 h: 5 males + 5 females
500 mg/kg, exposition – 24 h: 5 males + 5 females
500 mg/kg, exposition – 48 h: 5 males + 5 females

Frequency of treatment:

Continuous exposure.

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
500
Basis:
nominal in water

Doses / concentrations 2

Remarks:

Doses / Concentrations:
1000
Basis:
nominal in water

Doses / concentrations 3

Remarks:

Doses / Concentrations:
2000
Basis:
nominal in water

No. of animals per sex per dose:

5 males, 5 females

Control animals:

yes

Examinations

Tissues and cell types examined:

Bone marrow cells.

Details of tissue and slide preparation:

Bone marrow cells were obtained 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 several times mixed by syringe with thin needle. 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.

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.

Before examination, the slides were coded.
Stained smears were examined by light microscope. 200 erythrocytes were evaluated per
animal for the proportion of immature (polychromatic) and mature (normochromatic)
erythrocytes („cytotoxicity index“) in bone marrow.
At least 2000 polychromatic erythrocytes per animal were scored for the incidence of
micronucleated immature erythrocytes.

Evaluation criteria:

The criteria for determining a positive result are dose-related increase in the number of
micronucleated cells or a clear increase in the number of micronucleated cells in a single dose
group at a single sampling time. Biological relevance of the results is considered first.

Statistics:

The statistical analysis was performed by the ANOVA test - Analysis of Variance (software
QC.Expert 2.5, Trilobyte, Statistical Software, Czech Rep.).

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Clinical observation

No animal died during the main experiment.

No symptoms of toxicity were observed in all animals of all dose levels.

No symptoms of toxicity were observed in the animals of positive control group and

negative control groups.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test substance Versálová žluť HGR, was tested for the assessment of cytogenetic damage using micronucleus test.
Statistically significant increase in the count of micronucleated immature erythrocytes compared to the control was not recorded at any dose level.
Under the given test conditions, the test substance, Versálová žluť HGR, has negative result in micronucleus test.
Negative results in the micronucleus test indicate that under the test conditions the test
substance does not produce micronuclei in immature erythrocytes in bone marrow of rat.