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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study; GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
p-(dimethoxymethyl)anisole
EC Number:
218-577-4
EC Name:
p-(dimethoxymethyl)anisole
Cas Number:
2186-92-7
Molecular formula:
C10H14O3
IUPAC Name:
1-(dimethoxymethyl)-4-methoxybenzene
Details on test material:
- Name of test material (as cited in study report): Anisaldehyddimethylacetal
- Purity: 94 %
- Substance No.: 90/645
- Physical state: Colorless to yellowish liquid
- Storage: +4°C to +6°C

Test animals

Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River GmbH, WIGA, D-W8741 Sulzfeld, FRG
- Weight at study initiation: mean ca. 28 g
- Assigned to test groups randomly: yes
- Housing: individually in Makrolon cages, type M I
- Diet (e.g. ad libitum): Standardized pelleted feed Kliba Haltungsdiät, Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland; ad libitum
- Water (e.g. ad libitum): drinking water; ad libitum
- Acclimation period: about one week in groups of 5 in Makrolon cages, type M III


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
- Concentration of test material in vehicle: 3, 6, 12 g/100 ml
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
All test substance formulations were prepared immediately before administration.
Duration of treatment / exposure:
16, 24, 48 h
Frequency of treatment:
single administration
Post exposure period:
not applicable
Doses / concentrations
Remarks:
Doses / Concentrations:
300, 600 and 1200 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide:
- Route of administration: intraperitoneal injection
- Doses / concentrations: 20 mg/kg bw

Vincristine:
- Route of administration: intraperitoneal injection
- Doses / concentrations: 0.15 mg/kg bw

Examinations

Tissues and cell types examined:
polychromatic erythrocytes from femur bone marrow
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
In a pretest for the determination of the acute intraperitoneal toxicity ,deaths were observed down to a dose of 1500 mg/kg body weight. 1250 mg/kg body weight were survived by all animals but led to clinical signs of toxicity such as irregular respiration, abdominal and lateral position, staggering and piloerection about 15 - 60 minutes after test substance administration; the general state of the animals was poor.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
1200 mg/kg bw group: Sacrifice 16h, 24h, 48h after administration.
600 mg/kg bw group: Sacrifice 24h after administration.
300 mg/kg bw group: Sacrifice 24h after administration.
The animals of the negative control were sacrificed 24 hours after administration of the carrier. The control animals were treated at different times so that it was always possible to prepare control animals simultaneously for all the different sacrifice intervals of the test animals

DETAILS OF SLIDE PREPARATION:
The slides were stained in eosin and methylene blue solution for 5 minutes, rinsed in aqua dest. and then placed in fresh aqua dest. for 2 or 3 minutes. They were finally stained in Giemsa solution for 12 minutes. After being rinsed twice in aqua dest. and clarified in xylene, the preparations were embedded in Corbit-Balsam.

METHOD OF ANALYSIS:
In general, 1000 polychromatic erythrocytes from each of the male and female animals of every test group are evaluated and investigated for micronuclei. The normochromatic erythrocytes (= normocytes), which occur, are also scored.
Evaluation criteria:
The increase in the number of micronuclei in polychromatic erythrocytes of treated animals as compared with the solvent control group provides an index of a chromosome-breaking (clastogenic) effect or of a spindle activity of the substance tested.
The number of micronuclei in normochromatic erythrocytes at the early sacrifice intervals represents the situation before test substance administration and may serve as a control value. A substance-induced increase in the number of micronuclei in normocytes may be found with an increase in the duration of the sacrifice intervals.
The ratio of polychromatic to normochromatic erythrocytes indicates an influence of the test substance specifically on the bone marrow .
The size of micronuclei may give an indication on the possible mode of action of the test substance i.e. a clastogenic or a spindle poison effect.
Statistics:
A statistical evaluation was not necessary to perform. The number of polychromatic micronucleated erythrocytes after test substance treatment was nearly the range of the actual control value and within the historical values.

Results and discussion

Test results
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: The single intraperitoneal administration of the carrier in a volume of 10 ml/kg body weight was tolerated by all animals without any signs or symptoms.
Doses of 1200 mg/kg body weight led to irregular respiration, piloerection, abdominal position and staggering about 30 - 60 minutes after treatment and the general state of the animals was poor. The days after treatment only piloerection was observed in a few animals.
The administration of 600 mg/kg body weight led to irregular respiration, piloerection, staggering and squatting posture after about 30 - 60 minutes. The day after treatment only piloerection was found in few cases.
30 - 60 minutes after treatment of the animals with 300 mg/kg body weight only irregular respiration and piloerection was detected which were not observed any longer after two hours.
Neither the single administration of the positive control substance cyclophosphamide in a dose of 20 mg/kg body weight nor that of vincristine in a dose of 0.15 mg/kg body weight caused any evident signs of toxicity.

Any other information on results incl. tables

Results:

Test group Interval: 16 h
Polychromatic erythrocytes Normocytes / total amount polychromatic erythrocytes Cells with micronuclei (%o)
Dose (mg/kg bw) polychromatic normochromatic
vehicle control  
1200 10000 7204 1.1 1
600  
300  
20, cyclophosphamide   
0.15, vincristine        
Test group Interval: 24 h
Polychromatic erythrocytes Normocytes / total amount polychromatic erythrocytes Cells with micronuclei ( %o)
Dose (mg/kg bw) polychromatic normochromatic
vehicle control 10000 6501 0.7 1.7
1200 10000 6705 1.4 1.2
600 10000 5615 1.4 1.1
300 10000 5484 1.6 2.4
20, cyclophosphamide  5000 2152 8.8 0.9
0.15, vincristine 5000 4839 90.6 2.3
Test group Interval: 48 h
Polychromatic erythrocytes Normocytes / total amount polychromatic erythrocytes Cells with micronuclei ( %o)
Dose (mg/kg bw) polychromatic normochromatic
vehicle control  
1200 10000 7326 1.3 1.1
600  
300  
20, cyclophosphamide   
0.15, vincristine        


With 0.88 %, the positive control substance cyclophosphamide for clastogenicity led to the expected increase in the number of polychromatic erythrocytes containing exclusively small micronuclei at a dose level of 20 mg/kg body weight.
With 9.06 % the positive control vincristine for spindle poison effects also led to a clearly enhanced number of polychromatic erythrocytes containing micronuclei with the expected amount of large micronuclei, i.e. 1.34 %.
The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals.
Thus, the test substance Anisaldehyddimethylacetal did not lead to any increase in the rate of micronuclei.

The number of normochromatic or polychromatic erythrocytes containing small micronuclei (d < D/4) did not deviate from the solvent control value at any of the sacrifice intervals. Nor were large micronuclei (d > D/4) observed either in the negative control group or in the three dose groups of Anisaldehyddimethylacetal.

No inhibition of erythropoiesis induced by the treatment of mice with Anisaldehyddimethylacetal was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.


Applicant's summary and conclusion