Registration Dossier

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:
1998-08-17 to 1998-09-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
see below

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann Gartenstr. 27; D-33178 Borchen
- Age at study initiation: approximately 7 weeks
- Weight at study initiation: M=34.6g; F=27.g
- Assigned to test groups randomly: yes, randomization schemes 98.0665 and 98.0666
- Fasting period before study:- Housing: in fully air-conditioned rooms in makrolon cages type 3 (five animals per cage) on soft wood granulate
- Diet (e.g. ad libitum): rat/mice diet ssniff RIM-H (V 1534), ad libitum ssniff GmbH, Postbox 2039, 59480 Soest
- Water (e.g. ad libitum): tap water in plastic bottles, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 3 degrees C
- Humidity (%): 50± 20%
- Light/dark cycles: 12 hours daily

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
deionized water
Details on exposure:
The test substance was administered once orally by gavage to the test animals at doses of 0 or 2000 mg per kg body weight. The vehicle, deionized water, was administered in the same way to the negative control groups. The study included a concurrent positive control using Endoxan, which was administered once orally by gavage at a dose of 50 mg per kg body weight. Following dosing, the animals were examined regularly for mortality and clinical signs of toxicity.
Duration of treatment / exposure:
According to the test procedure the animals were killed 24 or 48 hours after administration
Frequency of treatment:
once
Post exposure period:
24 or 48h
Doses / concentrations
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
Male: 0 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 0 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 48 hours
Female: 0 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 0 mg/kg; No. of animals: 5; Sacrifice times: 48 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 48 hours
Control animals:
yes, concurrent vehicle
Positive control(s):
The study included a concurrent positive control using Endoxan, which was administered once orally by gavage at a dose of 50 mg per kg body weight.

Examinations

Tissues and cell types examined:
bone marrow cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a preliminary dose range finding study, oral administration of 2000 mg Reactive Red FC73270 per kg body weight did not cause any toxic effects in male and
female mice over an observation period of 7 days. This dose level was therefore the regularly limit dose, selected as the highest dose for the main study.

DETAILS OF SLIDE PREPARATION: Animals were killed by carbon dioxide asphyxiation 24 or 48 hours after dosing. For each animal, about 3 ml fetal bovine serum was poured into a centrifuge tube. Both femora were removed and the banes freed of muscle tissue. The proximal ends of the femora were opened and the bone marrow flushed into the centrifuge tube. A suspension was formed. The mixture was then centrifuged for 5 minutes at approx. 1200 rpm, after which almost all the supernatant was discarded. One drop of the thoroughly mixed sediment was smeared onto a cleaned slide, identified by project code and animal number and air-dried for about 12 hours.

Staining was performed as follows:
-5 minutes in methanol
-5 minutes in May-Grünwald' s solution
-brief rinsing twice in distilled water
-10 minutes staining in 1 part Giemsa solution to 6 parts buffer solution,
-pH 7.2 (Weise)
-rinsing in distilled water
-drying
-coating with Entellan

METHOD OF ANALYSIS:
2000 polychromatic erythrocytes were counted for each animal. The number of cells with micronuclei was recorded, not the number of individual micronuclei. In addition, the ratio of polychromatic erythrocytes to 200 total erythrocytes was determined.
Evaluation criteria:
A substance is considered as positive if there is a significant dose-related increase in the number of micronucleated polychromatic erythrocytes for at least one of the time points compared with the concurrent negative control group. A test substance producing no significant dose-related increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.
Statistics:
A one-sided Wilcoxon-Test was evaluated to check the validity of the study

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
All animals survived after treatment, no signs of toxicity were observed besides observingred-colored feces. Upon dissection there were no macroscopic findings.

Any other information on results incl. tables

Sex Dose
[mg/kg b.w.]
killing
time
Number
of
animals
Poly counted Poly/Ery

Mean
Poly/Ery
SD
Mean
Poly with MN
Mean
Poly with MN [%]
Mean
Poly with MN SD
Mean
Mut. I.
                     
pooled 0 - Control 24 h 10 2000 0,50 0,07 2,00 0,1 0,04 1,0
pooled 2000 24 h 10 2000 0,49 0,05 1,70 0,1 0,05 0,9
pooled 50 - Endoxan 24 h 10 2000 0,49 0,10 59.70* 3,0 0,64 29,9
                     
Mut. I. = Mutagenic Index
Control = Vehicle (deionized water)
* = significantly different from control (p < 0.05)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Reactive Red FC73270 did not lead to a substantial increase of micronucleated polychromatic erythrocytes and is not mutagenic in the micronucleus test under the conditions.
Executive summary:

In a NMRI mouse bone marrow micronucleus assay, 5 animals/sex/dose were treated via oral gavage with Reactive Red FC73270 at doses of 0 or 2000 mg/kg bw.  Bone marrow cells were harvested at 24 or 48 hours post-treatment.  The vehicle was deionized water.

There were no signs of toxicity during the study. Reactive Red FC73270 was tested at an adequate dose based on previous studies. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow.

This study is classified as acceptable and satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.