Coffee, bean, roasted, ext.

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

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. Vol 23, No.8, pp. 747-752.

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

- Principle of test: human lymphocyte mutagenicity test
- Short description of test conditions: heparinized whole blood is added to the culture tissue and then incubated with the test item at 37ºC. Three hours prior to harvest, the spindle inhibitor was added. At the time of harvest, the cells culture is gently centrifuged, supernatant removed and cells are re-suspended in the hypotonic saline and further cells are fixed and stained.
- Parameters analysed / observed: gaps, breaks and other chromatid or chromosome aberrations

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

not applicable - High molecular weight constituents (Mwt > 719 g mol-1) such as polysaccharides, proteins and melanoidin polymers

Molecular formula:

not applicable - High molecular weight constituents (Mwt > 719 g mol-1) such as polysaccharides, proteins and melanoidin polymers

IUPAC Name:

not applicable - High molecular weight constituents (Mwt > 719 g mol-1) such as polysaccharides, proteins and melanoidin polymers

Constituent 2

Reference substance name:

not applicable - Inorganic constituents (including K, Mg and Ca) of Coffee, bean, roasted, ext.

Molecular formula:

not applicable - Inorganic constituents (including K, Mg and Ca) of Coffee, bean, roasted, ext.

IUPAC Name:

not applicable - Inorganic constituents (including K, Mg and Ca) of Coffee, bean, roasted, ext.

Constituent 3

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

Constituent 4

Reference substance name:

1-methylpyridinio-3-carboxylate

EC Number:

208-620-5

EC Name:

1-methylpyridinio-3-carboxylate

Cas Number:

535-83-1

Molecular formula:

C7H7NO2

IUPAC Name:

1-methylpyridinium-3-carboxylate

Constituent 5

Reference substance name:

5-O-(3,4-dihydroxycinnamoyl)-L-quinic acid

EC Number:

206-325-6

EC Name:

5-O-(3,4-dihydroxycinnamoyl)-L-quinic acid

Cas Number:

327-97-9

Molecular formula:

C16H18O9

IUPAC Name:

3-{[3-(3,4-dihydroxyphenyl)acryloyl]oxy}-1,4,5-trihydroxycyclohexanecarboxylic acid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
1) Home brew coffee samples (Melita filter coffee, 50 g/litre) were prepared from the same blend of coffee beans as that used in the production of the instant coffee. Both home brew and instant coffee were produced from caffeine-containing and decaffeinated beans.
2) Caffeine was obtained from Fluka AG, Buchs.
- Expiration date of the lot/batch: not available
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Coffee solutions were stored overnight at -20°C.
- Stability under test conditions: not available
- Solubility and stability of the test substance in the solvent/vehicle: not available
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
1) The home brew coffee was lyophilized. The home brew and instant coffee powders (caffeine-containing and decaffeinated) were dissolved in distilled water at room temperature and heated in a water-bath at 60°C. These coffee stock solutions were then filtered through 0.8-µm Millipore filters and autoclaved at 121°C for 20 min.
2) Caffeine was diluted in warm distilled water and made up to the required concentrations.
- Preliminary purification step (if any): not available
- Final dilution of a dissolved solid, stock liquid or gel: not available
- Final preparation of a solid: not applicable

OTHER SPECIFICS: none

Method

Species / strain

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: human blood
- Suitability of cells: not available
- Cell cycle length, doubling time or proliferation index: not available
- Sex, age and number of blood donors if applicable: not available
- Whether whole blood or separated lymphocytes were used if applicable: the whole blood
- Number of passages if applicable: not available
- Methods for maintenance in cell culture if applicable: not available
- Modal number of chromosomes: not available
- Normal (negative control) cell cycle time: not available

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Chromosome 1A medium, supplemented with 20% heat-activated fetal bovine serum
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not available
- Periodically checked for karyotype stability: not available
- Periodically 'cleansed' against high spontaneous background: not available

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

0, 2.5, 2.9, 3.4, 3.9, 4.3 mg/mL culture (instant and home brew coffee)
0, 10, 25, 50, 75, 100 microL/mL culture (caffeine)

The test substances were administered at doses up to maximally tolerated levels, 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.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: none

Controls

Untreated negative controls:

yes

Remarks:

sterile distilled water

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: endoxan and bleomycin

Details on test system and experimental conditions:

METHOD OF APPLICATION: pre-incubated test solutions with S9 mix were added to the 48-hour cultures
- Cell density at seeding (if applicable): not available

DURATION
- Preincubation period: 2 hours at 37ºC
- Exposure duration: 8 hours at 37ºC for instant, home brew coffee and caffeine
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): 2 days at 4ºC

SELECTION AGENT (mutation assays): not applicable

SPINDLE INHIBITOR (cytogenetic assays): Vincristine at 0.5 µg/ml culture for 3-4 hours prior harvesting

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: not available

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The cells were harvested, washed with balanced Hanks solution and swollen in a hypotonic solution (1 ml Hanks plus 4 ml double-distilled water) for 10 min at 37°C. The cells were then fixed in glacial acetic acid-absolute methanol (1:3, v/v). They were left in this fixative for 2 hours at 4ºC. After fixation, chromosome preparations were made, stained with Giemsa, cleared in xylol and made permanent with Merckoglass spray.

NUMBER OF CELLS EVALUATED: not available

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): Fifty metaphases from each of two slides were scored for each concentration of the test substance.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: not applicable

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

OTHER EXAMINATIONS:
- Determination of polyploidy: not applicable
- Determination of endoreplication: not applicable
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): none

- OTHER: none

Evaluation criteria:

Since gaps and breaks might be induced by different mechanisms, gaps and breaks were recorded separately. The remaining chromatid and chromosome aberrations were classified collectively as 'other' aberrations because these types of aberration did not occur frequently in these experiments.
Fifty metaphases from each of two slides were scored for each concentration of the test substance.

Statistics:

The objective was to test for a positive linear trend with increasing concentration. As a basis for statistical analysis, the proportion of cells with one or more aberrations was judged to be the most appropriate parameter. Accordingly, the tabulated values represent the percentage of cells with one or more aberrations of the same type. Since two-sided exact probabilities in 2 x 2 tables revealed homogeneity of results obtained with two slides for each concentration, the following procedures have been 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.
These three approaches were chosen because computer simulations have clearly indicated that no single one of them was always the most powerful. Therefore a positive linear trend was accepted as significant if
(a) procedure 1 gave a P value of less than 0.1, or
(b) both procedures 2 and 3 indicated a P value of less than 0.05 (even if procedure 1 indicated no significant effects).
Furthermore, for each test substance, the slope (linear regression coefficient) with and without S9 mix was compared by Student's t test. The method of Stead, Hasselblad, Creason & Claxton (1981) was used to plot curves for the total number of aberrations. This method was considered adequate for illustration since multi-aberrant metaphases were infrequent.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

lymphocytes: human blood

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

lymphocytes: human blood

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

lymphocytes: human blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: home brew and instant coffee

Any other information on results incl. tables

Instant coffee

Both caffeine-containing and decaffeinated instant coffees, when tested without S9 mix, significantly increased the number of gaps and breaks and total aberrations In the presence of S9 mix both instant coffees (with and without caffeine) induced a much smaller number of gaps and breaks and total aberrations than in its absence. Furthermore, comparison of regression slopes showed that the induction of total aberrations by either of the instant coffees was significantly lower in the presence of S9 mix than in its absence.

Complete deactivation did not occur in all cases in the presence of S9 mix. Caffeine-containing instant coffee still had a significant but weak effect on gaps and total aberrations, whereas with the decaffeinated coffee only chromosomal breaks were significantly elevated.

Home brew coffee

Both caffeine-containing and decaffeinated home brew coffee significantly increased the number of gaps, breaks and total aberrations (except for gaps with the caffeine-containing sample) when tested without S9 mix. Fewer aberrations were observed with S9 mix than without it but the effects were significant (compared with negative controls), except for gaps induced by decaffeinated home brew.

Statistical comparisons of regression slopes revealed that total aberrations for decaffeinated home brew coffee were significantly lower in the presence of S9 mix than in its absence.

Caffeine

Pure caffeine tested with or without S9 mix 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.

See attached additional information on results.

Applicant's summary and conclusion

Conclusions:

In an in vitro human lymphocytes mutagenicity test instant coffee and home brewed coffee, increased the number of total aberrations, this effect was weak in the presence of metabolic activation. Caffeine did not increase the number of total aberrations.

Executive summary:

Incubation of instant and home brew coffees (caffeinated and decaffeinated) with cultured human lymphocytes in the presence and absence of S9 mix increased the number of total aberrations. However, this increase was smaller in the presence of S9 mix. Pure caffeine tested with or without S9 mix at doses equivalent to levels in caffeine-containing coffee did not give statistically significant increases of any type of aberration when compared with controls. The effect observed in the lymphocyte test was very weak in comparison with that observed for active mutagens.

In other in vitro test systems, coffee mutagenic activity seems to be metabolically deactivated in the presence of S9 mix. This could explain the negative results obtained in mutagenicity assays in vivo.