Reaction products of diazotized 4-amino-6-[(4-aminophenyl)diazenyl]-3-[(4-aminophenyl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid, coupled with benzene-1,3-diamine, sodium salts

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(adopted July 21, 1997)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Reaction products of diazotized 4-amino-6-[(4-aminophenyl)diazenyl]-3-[(4-aminophenyl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid, coupled with benzene-1,3-diamine, sodium salts

EC Number:

812-037-7

Cas Number:

1793011-72-9

Molecular formula:

Unknown for all components

IUPAC Name:

Reaction products of diazotized 4-amino-6-[(4-aminophenyl)diazenyl]-3-[(4-aminophenyl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid, coupled with benzene-1,3-diamine, sodium salts

Test material form:

solid

Test animals

Species:

mouse

Strain:

NMRI

Remarks:

BR

Details on species / strain selection:

These mice are recommended by international guidelines.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Sulzfeld, Germany
- Age at study initiation: 6 to 8 weeks
- Weight at study initiation: Detailed as within 20% of the sex mean
- Assigned to test groups randomly: Yes
- Fasting period before study: No
- Housing: Group housed (5 per cage) in labelled polycarbonate cages containing sterilised saw dust as bedding material.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet.
- Water (e.g. ad libitum): Free access to tap water
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5 to 22.6°C
- Humidity (%): 42 to 85%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle.

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: Physioclogical saline
- Justification for choice of solvent/vehicle: Standard vehicle for this dose route
- Concentration of test material in vehicle: 63, 125 and 250 mg/kg bw (6.3, 12.5 and 25 mg/mL).
- Amount of vehicle (if gavage or dermal): 10 mL/kg bodyweight (dose volume)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The substance was suspended in physiological saline and treated with ultra sound to obtain a homogeneous suspension. All prepared doses were used with 1.5 hours of preparation.

Duration of treatment / exposure:

Animals were dosed on one occassion

Frequency of treatment:

One single dose

Post exposure period:

Bone marrow samples were taken at 24 hours in the controls and at all dose groups with additional samples taken at 48 hours in remaining animals at the high dose, and in the positive control group.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

63 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

125 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

At least 5 male mice were used per sampling time in each dose group.

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide was used as the positive contol.
- Justification for choice of positive control(s): standard positive control substance for studies of this type
- Route of administration: Intraperitoneal
- Doses / concentrations:40 mg/kg bw (4 mg/mL).

Examinations

Tissues and cell types examined:

Bone marrow samples were taken and used to prepare bone marrow smears.

Details of tissue and slide preparation:

Isolation of bone marrow:
The animals were sacrificed by cervical dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with fetal calf serum. The cell suspension was collected and centrifuged for 5 minutes.

Preparation of bone marrow smears:
The supernatant was removed and a drop of serum left on the pellet. The cells in the sediment were carefully mixed with serum by aspiration. A drop of the cell suspension was placed on the end of a clean slide previously immersed in a 1:1 mixture of 96% ethanol/ether and cleaned. The drop was spread and the following preparations were air dried, fixed for 5 minutes in 100% methanol and air dried overnight. Two slides were prepared per animal.

Staining of the bone marrow smears:
The slides were automatically stained using the 'wright-stain-procedure' in an Emes HEMA-tek slide stainer. This staining is based on Giemsa. The dry slides were dipped in xylene before they were embedded in pertex and mounted with a coverslip.

Analysis of the bone marrow:
All slides were randomly coded before examination. Initially the slides were screened at a magnification of 100 x for regions of suitable technical quality. Slides were scored at a magnification of 1000 x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ration of polychromatic to normochromatic erythrocytes was determined by counting and differentiation the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes with averages and standard deviations calculated.

Evaluation criteria:

A test substance is considered positive if it induces a biologically as well as a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) and the number of micronucleated polychromatic erythrocytes in the animals was above the historical control range.

A test substance is considered negative if none of the tested concentrations or sampling times showed a statistically significant increase in the incidence of micronucleated polychormatic erythrocytes and the number of micronucleated polychromatic erythrocytes in the animals was within the historical control data range.

Statistics:

Wilcoxon rank sum test, one sided, p < 0.05.

Results and discussion

Test results

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 250, 500, 1000 and 2000 mg/kg
- Solubility: Formulation suspensions were prepared
- Clinical signs of toxicity in test animals: All animals (1 male and 1 female) died by day 2 post exposure at 2000 and 1000 mg/kg bw. At 500 mg/kg two females died (3 females and 3 males). At 250 mg/kg bw all animals survived (3 males and 3 females). Clinical signs at all doses included ataxia, lethargy, hunched posture, rough coat, quick breathing, closed eyes, and a dark colour of hairless body parts.

- Evidence of cytotoxicity in tissue analyzed: Not analyzed as range finder used to determine appropriate doses.
- Rationale for exposure: Based on available information.
- Harvest times: Bone marrow not harvested

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay): Groups treated with the substance with a 24 hour sampling time showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle controls. The animals treated with 250 mg/kg at the 48 hours sampling time showed a slight decrease in the ratio compared with the vehicle controls, demonstrating toxic effects on erythropoiesis.
- Appropriateness of dose levels and route: Clinical signs were observed in the majority of animals treated at 250 mg/kg bw including ataxia, lethargy, hunched posture, rough coat, closed eyes and a dark colour of the hairless bodyparts. One animal at this does also died by day 2 of exposure.
- Statistical evaluation: Wilcoxon ranked sum test was used.

Any other information on results incl. tables

Mean number of micro nucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes and ratio of polychromatic/normochromatic erythrocytes.

Group	Treatment	Dose (mg/kg bw)	Sampling time (hours)	No. of micronucleated polychromatic erythrocytes per 2000 (mean and SD)	Ratio polychromatic/normochromatic erythrocytes (mean and SD)
A	Solvent control	0	24	0.8 ± 1.3	1.08 ± 0.05
B	Test	250	24	1.0 ± 1.2	0.93 ± 0.11
C	Test	250	48	0.4 ± 0.9	0.72 ± 0.20
D	Test	125	24	0.2 ± 0.4	0.96 ± 0.08
E	Test	75	24	0.4 ± 0.9	0.88 ± 0.04
F	Positive control	40	48	23.6 ± 9	0.39 ± 0.12

Applicant's summary and conclusion

Conclusions:

Based on the results obtained the test substance is not considered to be clastogenic or aneugenic under the conditions of the study in an vivo test system. Evidence of bone marrow exposure was noted based on a reduction in polychromatic and normochromatic erythrocyte ratios, and clinical signs and mortality observed at the highest exposure concentration.