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Toxicological information

Genetic toxicity: in vivo

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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
Study period:
16. August - 30. September 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report date:
1988

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
reaction mass of: tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-) tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-) tert-alkyl(C12-C14)ammonium bis[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-chromate(1-) tert-alkyl(C12-C14)ammonium [[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-) tert-alkyl(C12-C14)ammonium [[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-) tert-alkyl(C12-C14)ammonium ((1-(4-nitro-2-oxidophenylazo)-2-naphtholato)(1-(3-nitro-2-oxido-5-(1,1-dimethylpropyl)phenylazo)-2-naphtholato))chromate(1-)
EC Number:
938-781-3
Cas Number:
117527-94-3
Molecular formula:
C21H21N304.C16H11N304.Cr unspecified.C12-C14 chain unspec.
IUPAC Name:
reaction mass of: tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-) tert-alkyl(C12-C14)ammonium bis[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]-chromate(1-) tert-alkyl(C12-C14)ammonium bis[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-chromate(1-) tert-alkyl(C12-C14)ammonium [[1-[(2-hydroxy-4-nitrophenyl)azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-) tert-alkyl(C12-C14)ammonium [[1-[[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]azo]-2-naphthalenolato(2-)]-[1-[(2-hydroxy-5-nitrophenyl)azo]-2-naphthalenolato(2-)]]-chromate(1-) tert-alkyl(C12-C14)ammonium ((1-(4-nitro-2-oxidophenylazo)-2-naphtholato)(1-(3-nitro-2-oxido-5-(1,1-dimethylpropyl)phenylazo)-2-naphtholato))chromate(1-)
Details on test material:
- Lot/batch No.: 217342.89

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, Sulzfeld, FRG
- Age at study initiation: approx. 8 weeks of age
- Weight at study initiation: males: 25.1 - 33.8 g and females: 18.9 - 28 g
- Fasting period before study: Feed was withheld overnight before dsing until about 4 hours after administration of the test substance.
- Housing: groups of 5 in polycarbonate cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2 °C
- Humidity (%): 60-80 %
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: 09. August - 30. September 1988

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: Corn oil
- Concentration of test material in vehicle: 5000 mg/kg bw
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw
Duration of treatment / exposure:
single stomach intubation
Frequency of treatment:
once
Post exposure period:
Test item group and vehicle group animals dosed at 5000 mg/kg bw were sacrificed 24, 48 and 72 hrs following dosing.
Positive control animals were sacrificed 48 hrs following dosing.
Doses / concentrations
Remarks:
Doses / Concentrations:
5000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide dissolved in 0.9% NaCl solution was used as a positive control and dosed once orally at a level of 50 mg/kg bw.

Examinations

Tissues and cell types examined:
Bone marrow smears from femur.

Details of tissue and slide preparation:
Preparation of the bone marrow smears
The cell suspension was 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 resuspended in 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 animal number). The drop was spread by moving a clean slide with round-whetted sides at an angle of 45" 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.

Staining of the bone marrow smears
The slides were stained for 3 min in undiluted May-Grünwald solution followed by 2 min in May-GrOnwald solution diluted 1:1 with Sörensen buffer pH 6.8. Thereafter slides were rinsed in this buffer and stained for 25 min in 5% (v/v) Giemsa solution in Sörensen 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.

Analysis of the bone marrow smears for micronuclei
All slides were randomly coded before examination. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells are well spread, undamaged and well stained. Slides were scored at a magnification of 1000x. 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 counted in polychromatic erythrocytes only.
Evaluation criteria:
Acceptability of assay and criteria for response
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 at P<0.05) increase in the frequencies of micronuclei.
b) The incidence of micronuclei in the control animals should reasonably fall within the laboratory historical control data range.
A test substance is considered positive in the micronucleus test if:
a) It induces a statistically (P<0.05) as well as biologically significant increase in the frequencies 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 (at P<0.05) increase in the incidence of micronuclei either in the combined data for both sexes or in the data for male or female groups alone. The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.
Statistics:
The Wilcoxon rank-sum test was used to assess significant differences between the numbers of micronuclei in the treatment and control groups, in which P<0.05 was used as the lowest level of significance.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
In a preliminary study 12 animals (3 males and 3 females per group) were dosed orally with 5000 and 2000 mg/kg bw. No signs of reaction to treatment were observed. Based on the results of this pilot study 5000 mg/kg bw was selected as an appropriate dose for the Micronucleus test.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
No increase in the frequency of micronuclei was observed. The incidenceof micronuclei in the test substance and control animals were found to be in the range of the historical data (0.75 +/- 0.97; mean +/- deviation, N = 700). The positive control substance induced in both sexes a statistically sgnificant (P < 0.05) increase in the number of micronuclei.

- Ratio of PCE/NCE (for Micronucleus assay):
The ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all test substance dose groups.
The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a toxic effect of this compound on the erythropoiesis.

- Statistical evaluation:
No statistically significant differences in the frequency of erythrocytes containing micronuclei either between the solvent control and the dose group (5000 mg/kg bw) or between the various sacrifice intervals (24, 48 and 72 hours) was observed.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative