Caffeine

Genetic toxicity: in vivo

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

Test material information

Constituent 1

Reference substance name:

Caffeine

EC Number:

200-362-1

EC Name:

Caffeine

Cas Number:

58-08-2

Molecular formula:

C8H10N4O2

IUPAC Name:

1,3,7-trimethyl-3,7-dihydro-1H-purine-2,6-dione

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 or test system 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 / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
200 mg/kg bw
Basis:
other: single dose (experiment 1)

Doses / concentrations 2

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.