Caffeine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

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:

Instant and brewed coffees in the in vitro human lymphocyte mutagenicity test.

Author:

Aeschbacher HU et al.

Year:

1985

Bibliographic source:

Fd Chem Toxic 23 (8): 747-752.

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

The test procedure was carried out in principle according to Evans HJ et al (1980). Cytogenetic damage as an endpoint in short-term assay systems for detecting environmental carcinogens. In Long-term and Short-term Screening Assays for Carcinogens: A Critical Appraisal. Edited by Montesano r et al. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Suppl. 2, p. 227. International Agency for Research on Cancer, Lyon.

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

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, pure
No further data.

Method

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: chromosome 1A medium (GIBCO) supplemented with 20% heat-inactivated foetal bovine serum

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S-9

Test concentrations with justification for top dose:

5, 10, 25, 50, 75, 100 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile distilled water

Controls

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: Endoxan, 40 µg/culture, in 0.1 ml physiol. saline

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation
The preincubation method was chosen to avoid effects of the rat liver S-9 on the lymphocytes

DURATION
- Preincubation period: 2 h
- Exposure duration: 8 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

SPINDLE INHIBITOR (cytogenetic assays): Vincristine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 50 metaphases per slide (i.e. 100 metaphases per dose)

DETERMINATION OF CYTOTOXICITY
- Method: The test substance was administered at doses up to maximally tolerated level, i.e. just below cytotoxic levels. A marked reduction in the number of metaphases and a greater proportion of unscorable metaphases served as the criteria for cytotoxicity.

Statistics:

The following procedures were used to check the linear trend:
(1) The one degree of freedom chi-square test for linear trend (Cochran, 1954), in the context of a 2 x k contingency table, where k is the number of dose levels;
(2) linear regression using the angular transformation of the 2k proportions;
(3) linear regression using the angular transformation of the k proportions pooled over the two slides at each dose level.
Significance levels: P<0.1, P<0.05
The slope (linear regression coefficient) with and without S-9 mix was compared by Student's t test.

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

The test substance tested with or without S-9 at doses equivalent to levels in caffeine-containing coffee did not give statistically significant increases of any type of aberration, compared with negative controls, although they were slightly increased in most cases.

Table: Frequency of cells with chromosomal aberrations among cultured human lymphocytes incubated for 8 h with caffeine pre-incubated with or without S-9 mix

Caffeine concentration [µg/ml culture]	No. of cells with aberrations/100 cells
	- S-9		+ S-9
	gaps	breaks	gaps	breaks
Negative control	5	4	5	8
5 10 25 50 75 100	6 6 11 5 5 5	4 5 7 5 5 8	6 4 8 5 6 6	3 5 7 7 3 8
Positive control	-	-	5	17

Negative (vehicle) control: sterile distilled water

Positive control: Endoxan, 40 µg/culture

The results are the numbers of cells with one or more aberrations the same type, per 100 cells examined on two slides (50 cells on each). No other aberrations were seen in the cells incubated with caffeine.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Executive summary:

Pure caffeine tested with or without S-9 at doses equivalent to levels in caffeine-containing coffee did not give statistically significant increases of any type of aberration in human lymphocytes when compared with controls.