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Toxicological information

Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Acceptable, well documented publication which meets basic scientific principles.

Data source

Reference
Reference Type:
publication
Title:
Cytogenetic Investigations of Mutagenic Action of Caffeine in Premeiotic Spermiogenesis in Mice.
Author:
Adler I-D
Year:
1966
Bibliographic source:
Humangenetik 3: 82-83.

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
no data
GLP compliance:
no
Remarks:
pre-GLP study
Type of assay:
chromosome aberration assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Caffeine
No further data

Test animals

Species:
mouse
Strain:
C3H
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
- Age at study initiation: 10 weeks
No further data

ENVIRONMENTAL CONDITIONS: no data

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: physiol. saline
- Amount of vehicle : 1 ml/animal
Details on exposure:
Experiment 1:
Fourteen mice received a single i.p. injection of the test substance at 200 mg/kg bw in 0.9% saline. Control mice received the vehicle (1 ml).

Experiment 2:
Fourteen mice received daily i.p. injections of the test substance at 250 mg/kg bw in 0.9% saline for 21 consecutive days. Control mice received the vehicle (1 ml).
Duration of treatment / exposure:
- single dose (experiment 1)
- 21 days (experiment 2)
Frequency of treatment:
- single dose (experiment 1)
- daily (experiment 2)
Post exposure period:
- 1 and 7 days (experiment 1)
- 16 hours (experiment 2)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
200 mg/kg bw
Basis:
other: single dose (experiment 1)
Remarks:
Doses / Concentrations:
250 mg/kg bw/d
Basis:
other: repeated dose (experiment 2)
No. of animals per sex per dose:
14 males
Control animals:
yes, concurrent vehicle
Positive control(s):
no

Examinations

Tissues and cell types examined:
testicular tissue
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: maximum tolerated dose

TREATMENT AND SAMPLING TIMES:
- experiment 1: single dose (200 mg/kg bw), preparation of testes on day 1 and 7 post dose
- experiment 2: daily injections (250 mg/kg bw/d for 21 days), preparation of the testes 16 h after the last dose

DETAILS OF TISSUE PREPARATION:
Testicular tissue samples were prepared according to the method of Evans et al. [An air-drying method for meiotic preparations from mammalian testes. Cytogenetics 3: 289-294 (1964)] with a modification. The modification essentially consisted in additional use of Hyaluronidase (150 IU/10 ml 1% Na-acetate solution) in order to loosen the testicular tissue and to obtain a greater number of isolated cells. Airdried preparations of the cell suspensions were stained with 2% OE-solution and evaluated by phase contrast microscopy.
In the first series (experiment 1) testes were studied on the first and seventh day after single injection. According to Marshall's time table of spermiogenesis of mice, Pachytene and Zygotene were covered.
In the 2nd experiment (21-day treatment), mice were sacrificed 16 hours after the last injection and the testes prepared.

METHOD OF ANALYSIS:
contrast microscopy

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
not examined

Any other information on results incl. tables

Experiment 1:

No breaks or translocations could be observed. The rate of univalent chromosome pairs were the same in test and control animals.

Table 1: Comparison of number of observed univalent chromosome pairs in animals treated within different stages of meiosis and in controls.

Stage

No. of bivalents

Univalent chromosome pairs

Univalents in % of total

treated

control

treated

control

treated

control

Diplotene

800

800

5

4

0.625

0.5

Cytogene

800

800

5

5

0.625

0,625

Experiment 2:

In spite of the high number of evaluated chromosome bivalents no breaks or translocations could be observed. The rates of univalent chromosome pairs were the same in testicular preparations of test and control animals.

Table 2: Comparison of number of observed univalent chromosome pairs in animals treated during entire period of premeiotic spermatogenesis and in controls.

Treatment

Observed

bivalents

Univalent chromo-

some pairs

Observed

cells

Univalents

in  % of cells

Caffeine

6000

28

0.46

300

9.3

Controls

6000

26

0.43

300

8.6

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
Executive summary:

Fourteen male mice were treated with maximal tolerated doses of caffeine. Animals were sacrificed and preparations of testicular tissue examined in intervals after treatment. The meiotic chromosomes at Metaphase I (Diakinesis) were studied by phase contrast microscopy for breaks, translocations and univalent chromosomes. No unusual chromosome changes could be found.