Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-02-27 to 1989-05-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report date:
1989

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Version / remarks:
1983
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

Constituent 1
Chemical structure
Reference substance name:
A mixture of isomers of: 1,1'-[(3,5(or 2,4 or 4,6 or 2,6)-dihydroxy-o(or m or p)-phenylene)bis(azo-meta-phenyleneazo{1-[3-(dimethylamino)propyl]-1,2-dihydro-6-hydroxy-4-methyl-2-oxopyridine-5,3-diyl})]dipyridinium-dichloride-dihydrochloride; 1-(1-[3-(dimethylamino)propyl]-5-{3-[x-(4-{1-[3-(dimethylamino)propyl]-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-5-pyridinio-3-pyridylazo}phenylazo)-2,4(or 2,6 or 3,5 or 4,6)-dihydroxyphenylazo]phenylazo}-1,2-dihydro-6-hydroxy-4-methyl-2-oxo-3-pyridyl)pyridinium-dichloride-dihydrochloride (where x is variable)
EC Number:
404-540-1
EC Name:
A mixture of isomers of: 1,1'-[(3,5(or 2,4 or 4,6 or 2,6)-dihydroxy-o(or m or p)-phenylene)bis(azo-meta-phenyleneazo{1-[3-(dimethylamino)propyl]-1,2-dihydro-6-hydroxy-4-methyl-2-oxopyridine-5,3-diyl})]dipyridinium-dichloride-dihydrochloride; 1-(1-[3-(dimethylamino)propyl]-5-{3-[x-(4-{1-[3-(dimethylamino)propyl]-1,6-dihydro-2-hydroxy-4-methyl-6-oxo-5-pyridinio-3-pyridylazo}phenylazo)-2,4(or 2,6 or 3,5 or 4,6)-dihydroxyphenylazo]phenylazo}-1,2-dihydro-6-hydroxy-4-methyl-2-oxo-3-pyridyl)pyridinium-dichloride-dihydrochloride (where x is variable)
Cas Number:
159405-95-5
Molecular formula:
C50H56Cl4N14O6
IUPAC Name:
1'-[3-(dimethylamino)propyl]-5'-(2-{3-[2-(4-{2-[3-(2-{1'-[3-(dimethylamino)propyl]-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1lambda5-[1,3'-bipyridin]-1-ylium-5'-yl}diazen-1-yl)phenyl]diazen-1-yl}-2,6-dihydroxyphenyl)diazen-1-yl]phenyl}diazen-1-yl)-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1lambda5-[1,3'-bipyridin]-1-ylium 1'-[3-(dimethylamino)propyl]-5'-(2-{4-[2-(4-{2-[4-(2-{1'-[3-(dimethylamino)propyl]-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1lambda5-[1,3'-bipyridin]-1-ylium-5'-yl}diazen-1-yl)phenyl]diazen-1-yl}-2,6-dihydroxyphenyl)diazen-1-yl]phenyl}diazen-1-yl)-6'-hydroxy-4'-methyl-2'-oxo-1',2'-dihydro-1lambda5-[1,3'-bipyridin]-1-ylium tetrahydrochloride tetrachloride
Test material form:
solid

Test animals

Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, FRG
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: 19.9- 28.8 gr females; 28.1- 35.3 gr males
- Assigned to test groups randomly: randomly allocated to treatment groups as they came to hand from delivery boxes.
- Fasting period before study: no data
- Housing: In groups of 5 per sex in polycarbonate cages.
- Diet (e.g. ad libitum): standard laboratory animal diet. Feed was withheld overnight prior to dosing until approximately 3-4 hours after administration of the test substance.
- Water (e.g. ad libitum): tap water, ad libitum.
- Acclimation period: at least 6 days under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 3 °C
- Humidity: 40 - 70 %
- Air changes (per hr): 7.5
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: water
- Concentration of test material in vehicle: 200 mg/ml
- Amount of vehicle (if gavage or dermal): 10 mg/kg bw
- Purity: Milli-RD water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test item well soluble in water, no special solution preparation procedure necessary.
Duration of treatment / exposure:
24, 48, and 72 h
Frequency of treatment:
single application
Post exposure period:
not applicable
Doses / concentrations
Dose / conc.:
2 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide
- Route of administration: oral (intubation)
- Doses / concentrations: 50 mg/kg body weight dissolved in 0.9 % NaCl

Examinations

Tissues and cell types examined:
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 approximately 2 ml of foetal calf serum. The cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a preliminary study 24 animals (3 males and 3 females per group) were dosed orally with 5000, 4000, 3000 and 2000 mg/kg body weight (groups 1, 2, 3 and 4, respectively). In group 1 all animals died within 2 hours post-dosing. In group 2 five animals died within 3 hours post-dosing (three males, two females). In group 3 five animals died within 3 hours post-dosing (two males, three females). The animals of group 4 did not show any signs of reaction to treatment. Based on the results of this pilot study 2000 mg/kg body weight was selected as an appropriate dose for the micronucleus test.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Sampling of the bone marrow was done 24, 48 and 72 hours after treatment.

DETAILS OF SLIDE PREPARATION:
The bone marrow cell suspension was collected and centrifuged at 1000 rpm (approximately 100 g) for 5 min.The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed by aspiration with the serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 hours immersed in a 1:1 mixture of 96 % ethanol/ether and cleaned with a tissue) and marked (with the RCC NOTOX study identification number and the animal number). The drop was spread by moving a clean slide with round-whetted sides at an angle of 45 °C over the slide with the drop of bone marrow suspension. The preparations were then air-dried and thereafter fixed for 5 min in 100 % methanol and air-dried overnight. Two slides were prepared per animal.
The slides were stained for 3 min in undiluted May-Grunwald solution followed by 2 min in May-Grunwald solution diluted 1:1 with Sorensen buffer pH 6.8. Thereafter slides were rinsed in this buffer and stained for 25 min in 5 % (v/v) Giemsa solution in SOrensen buffer pH 6.8. The preparations were rinsed for 1 min in running tap-water and blotted dry between filter paper. The dry slides were cleared by dipping them in xylol before they were embedded in DePeX and mounted with a coverslip.

METHOD OF ANALYSIS:
All slides were randomly coded before examination. An adhesive label with RCC NOTOX study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of micronuclei was counted in 1000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.
Evaluation criteria:
A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (Wilcoxon Rank Sum Test, two-sided test at P < 0.05) increase in the frequency of micronuclei.
b) The incidence of micronuclei in the control animals should reasonably fall within the laboratory historical control data range.
Statistics:
A test substance is considered positive in the micronucleus test if:
a) It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test; two-sided test at p < 0.05) increase in the frequency of micronuclei (at any dose or at any sampling time) in the combined data for both sexes or in the data for male or female groups separately.

A test substance is considered negative in the micronucleus test if:
a) None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronuclei neither in the combined data for both sexes nor in the data for male or female groups alone.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Remarks:
≤ 2000 mg/kg bw
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range: Dead animals occurred when being dosed orally with 5000, 4000, 3000 mg/kg body weight. At 2000 mg/kg bw no signs of reaction to trreatment, thus 2000 mg/kg body weight was selected as an appropriate dose for the micronucleus test.
- Solubility: test item very well soluble in water
- Clinical signs of toxicity in test animals: no
- Evidence of cytotoxicity in tissue analyzed: no
- Rationale for exposure: it is generally recommended to use the maximum tolerated dose for the micronucleus assay.
- Harvest times: 24, 48, and 72 h
- High dose with and without activation: 2000 mg/kg bw

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: no increase in the frequency of micronuclei
- Ratio of PCE/NCE: no effects
- Appropriateness of dose levels and route: the maximum tolerated dose was used for the micronucleus assay. The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use.
- Statistical evaluation: see "statistics" above

Any other information on results incl. tables

treatment dose (mg/kg bw) sampling time (h) number o micronuclei per 1000 polychromatic erythrocytes (mean ± S.D.) ration polychromatic/normochromatic erythrocytes (mean ± S.D.)
males
vehicle - 24 0.6±0.9 0.82±0.19
vehicle - 48 0.6±0.9 0.96±0.19
vehicle - 72 1.0±1.0 1.14±0.20
test substance 2000 24 0.0±0.0 0.84±0.18
test substance 2000 48 0.5±1.0 0.99±0.08
test substance 2000 72 0.6±0.5 1.03±0.26
positive control 50 48 13.8±6.6 0.45±0.21
females
vehicle - 24 0.6±0.9 1.04±0.14
vehicle - 48 0.4±0.5 1.07±0.30
vehicle - 72 0.4±0.5 1.28±0.20
test substance 2000 24 0.2±0.4 0.93±0.25
test substance 2000 48 1.0±1.0 1.19±0.12
test substance 2000 72 0.6±0.9 1.01±0.15
positive control 50 48 15.8±6.5 0.52±0.35

Applicant's summary and conclusion

Conclusions:
The test substance can be considered as not mutagenic in the mouse micronucleus test, performed according to OECD guideline 474.
Executive summary:

Aim of the study was to assess the mutagenicity of the test substance when administered to mice in a maximum tolerated acute dose, by measuring the increase of the number of micronuclei per 1000 polychromatic erythrocytes in mouse bone marrow. The micronucleus test is a mammalian in vivo test, which detects damage to the chromosome or to the mitotic apparatus induced by the test substance. The study followed the OECD guideline 474.

Three groups (5 animal/sex/group) were dosed at 2000 mg/kg bw. Bone marrow was sampled at 24, 48 and 72 hours after dosing. Corresponding vehicle treated groups served as negative controls. Bone marrow from a positive control group, treated with a single oral dose of cyclophosphamide at 50 mg/kg bw, was harvested at 48 hours after dosing only.

The test substance was found to respond negatively in the micronucleus test, whereas the positive control substance produced a statistically significant increase in the incidence of micronuclei in polychromatic erythrocytes.

It is concluded that the substance can be considered as not mutagenic in this assay.