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EC number: 272-823-5 | CAS number: 68916-18-7
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Link to relevant study records
- 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
- 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
- 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 - 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
- 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. - 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
- 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
- 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
- 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.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Version / remarks:
- Original protocol of B.N. Ames
- Deviations:
- yes
- Remarks:
- To the Ames protocol: Plates were incubated for 3 days at 37 degrees C.
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: “home brew” powder coffee and Nescafé standard (instant coffee)
- 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: not available
- Stability under test conditions: not available
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water
- 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: “home brew” coffee was prepared on a Melitta filter exactly as for human consumption (50 g/L water), the solution was then freeze-dried
- Preliminary purification step (if any): not available
- Final dilution of a dissolved solid, stock liquid or gel: the commercially available Nescafé instant coffee and freeze-dried sample of “home brew” coffee were diluted in hot tap water and autoclaved at 120ºC
- Final preparation of a solid: not applicable
OTHER SPECIFICS: none - Target gene:
- His
- Species / strain / cell type:
- S. typhimurium TA 98
- Species / strain / cell type:
- S. typhimurium TA 100
- Species / strain / cell type:
- S. typhimurium TA 1535
- Species / strain / cell type:
- S. typhimurium TA 1537
- Species / strain / cell type:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- 5, 15, 25, 35, 50 mg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: tap water
- Justification for choice of solvent/vehicle: solubility in water - Untreated negative controls:
- yes
- Remarks:
- buffer
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): not applicable
DURATION
- Preincubation period: not specified
- Exposure duration: 3 days
- Expression time (cells in growth medium): not specified
- Selection time (if incubation with a selection agent): not specified
- Fixation time (start of exposure up to fixation or harvest of cells): not specified
SELECTION AGENT (mutation assays): histidine-free medium
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable
NUMBER OF REPLICATIONS: 4
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable
NUMBER OF CELLS EVALUATED: not applicable
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not applicable
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: not applicable
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: not applicable
- Any supplementary information relevant to cytotoxicity: not applicable
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): not applicable
- OTHER: none
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- above 15 mg/plate
- Untreated negative controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Remarks:
- Observed at 35 mg/plate
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- above 15 mg/plate
- Untreated negative controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- above 15 mg/plate
- Untreated negative controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- above 15 mg/plate
- Untreated negative controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- above 15 mg/plate
- Untreated negative controls validity:
- not specified
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- not specified
- Remarks on result:
- other: Home brew + Nescafé
- Conclusions:
- In an in vitro gene mutation test in bacteria performed according to the original Ames protocol two coffee types, freeze-dried home brew and Nescafé standard solutions were concluded to have negative mutagenic potential in the presence of metabolic activation.
- Executive summary:
Two coffee types, freeze-dried home brew and Nescafé standard solutions, were tested in an in vitro gene mutation test in bacteria performed according to the original Ames protocol.
Both coffees increased the number of Salmonella TA 100 revertants to about 2 to 2.7-fold above the spontaneous mutation level in absence of S9. This effect was observed at 35 mg per plate. For all the other strains, TA 98, TA 1535, TA 1537 and TA 1538, no consistent increase of revertants was observed when coffees were applied up to the cytotoxic concentrations (above 15 mg coffee per plate).
When both coffees were tested at concentrations up to 50 mg per plate in the presence of S9, the mutagenic induction as well as the cytotoxic effect on the complete agar plates was completely abolished. This was observed for all Salmonella strains.
Based on the results, it can be concluded that both coffee types had a weak mutagenic effect on one of the five Salmonella strains without metabolic activation, however, this effect was abolished by addition of S9.
- Endpoint:
- in vitro gene mutation study in bacteria
- 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
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- not specified
- GLP compliance:
- not specified
- Remarks:
- Study was performed in a research laboratory, probably not GLP compliant.
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Americano coffee collected from five international coffee chains in Taiwan (without additives)
- 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: not available
- Stability under test conditions: not available
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water
- 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: not applicable
- Preliminary purification step (if any): not available
- Final dilution of a dissolved solid, stock liquid or gel: not applicable
- Final preparation of a solid: not applicable - Target gene:
- His
- Species / strain / cell type:
- S. typhimurium TA 98
- Species / strain / cell type:
- S. typhimurium TA 100
- Species / strain / cell type:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- Original, dilution 2x, 4x, 8x,16x
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: none
- Untreated negative controls:
- yes
- Remarks:
- sterile water
- Positive controls:
- yes
- Positive control substance:
- not specified
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in agar (plate incorporation)
- Cell density at seeding (if applicable): not applicable
DURATION
- Preincubation period: not specified
- Exposure duration: 24 hours
- Expression time (cells in growth medium): not specified
- Selection time (if incubation with a selection agent): not specified
- Fixation time (start of exposure up to fixation or harvest of cells): not specified
SELECTION AGENT (mutation assays): histidine-free medium
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable
NUMBER OF REPLICATIONS: not specified
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable
NUMBER OF CELLS EVALUATED: not applicable
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not applicable
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: not applicable
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: not applicable
- Any supplementary information relevant to cytotoxicity: not applicable
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): not applicable
- OTHER: none - Evaluation criteria:
- To determine the cellular toxicity of coffee samples for tester bacteria, the level of bacteria on the sample plates was compared with that of the negative control by evaluating the dose-response effects of toxicity or by using
Student’s t tests (Excel, Microsoft, Redmond, WA). Differences were considered significant at P < 0.05.
To determine the mutagenic activity of coffee, the two-fold criterion and the significant effect of the dose-response were used. A coffee sample was considered mutagenic when
(i) the number of revertant colonies was at least two-fold higher than the number of control colonies or
(ii) a significant dose-response was observed.
When suspicions about the mutagenicity of a sample arose, Student’s t test was performed to verify the significance (P < 0.05) of the difference in the number of revertant colonies in comparison with the negative control. - Statistics:
- Student’s t tests
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- In an in vitro gene mutation test in bacteria performed according to the Ames protocol using three Salmonella typhimurium strains (TA 98, TA 100 and TA1535) five Americano coffees from the selected coffee chains were concluded to have negative mutagenic potential in the presence and absence of metabolic activation.
- Executive summary:
Five Americano coffees from the selected coffee chains were tested in an in vitro gene mutation test in bacteria performed according to the Ames protocol.
Initially, five volumes of Americano coffee samples (i.e., 100, 200, 300, 400, and 500µL) were tested against the growth of Salmonella typhimurium tester strains TA98, TA100, and TA1535 to assess cytotoxicity.
Furthermore, the non-cytotoxic doses were further subjected to the Ames test. Several dilutions (original, 2x, 4x, 8x and 16x) were mixed with diluted bacteria and soft agar, plated on nutrient agar plates and incubated for 24 h.
The levels of bacterial revertants in Americano coffee samples from all coffee chains were lower than the two-fold criterion of the control sets, and no significant dose-response effect was observed with or without rat liver enzyme activation. These data indicate that Americano coffees from the selected coffee chains do not possess direct mutagenic activity with or without enzyme activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: test done according to Ames et al., (1975)
- Deviations:
- yes
- Remarks:
- different cofactor solution to the one provided by Ames et al.,
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: urine of the human volunteers that ingested instant coffee or water
- 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: during 4-day experiment all the urine was collected and stored in plastic flasks at 4ºC. After each 24 hours, all the flasks were collected, the volumes divided into 2 equal parts, one part was frozen unchanged (-20ºC) and the second part was incubated with beta-Glucuronidase. The same procedure was done for the urine collected after 2-hour exposure.
- 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: For the first experiment, half of the total volumes (4 x 0.5 volume of the 24 hour collection) were thawed at 4ºC, pooled and subjected to XAD-2 column chromatography. The same procedure was carried out on the urine incubated with beta-glucuronidase for 24 hours at 37ºC in a shaking water-bath. The columns were rinsed with 100 ml water before and with 50 ml water after the urine passage. The columns were then eluted with 100 ml acetone, which was later evaporated to dryness at about 45ºC in a rotary evaporator.
- Preliminary purification step (if any): not available
- Final dilution of a dissolved solid, stock liquid or gel: The residue obtained through the XAD-2 column chromatography was diluted in DMSO, 3 ml for 4-day exposure and 0.4 ml for 2-hour exposure.
- Final preparation of a solid: not applicable
OTHER SPECIFICS: none - Target gene:
- His
- Species / strain / cell type:
- S. typhimurium TA 98
- Species / strain / cell type:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- 0, 25, 50, 100 and 200 microL/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Untreated negative controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- 2-acetylaminofluorene
- other: 2-nitro-1,4-phenylenediamine
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium: in agar (plate incorporation)
- Cell density at seeding (if applicable): not available
DURATION
- Preincubation period: none
- Exposure duration: 3 days at 37ºC
- 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): not applicable
SELECTION AGENT (mutation assays): histidine-free medium
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable
NUMBER OF REPLICATIONS: 4 petri plates were used per concentration, except in rare instances where only 3 petri plates per concentration could be used due to the restricted DMSO volume available
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable
NUMBER OF CELLS EVALUATED: not applicable
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not applicable
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: not applicable
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: not applicable
- Any supplementary information relevant to cytotoxicity: not applicable
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): not applicable
- OTHER: none - Statistics:
- Paired t test
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- not valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at the highest concentration tested
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at the highest concentration tested
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other:
- Remarks:
- w/o beta-Glucuronidase
- Conclusions:
- The non-polar urine fractions obtained from coffee and non-coffee drinkers did not increase the number of revertants in the Ames assay in the presence and absence of metabolic activation.
- Executive summary:
Urine of human coffee drinkers who had ingested 48 g (or an equivalent of about 24 cups) of instant coffee was collected during 4 days, the same was done for subjects who only drank water. The urine volume was divided into halves, one of which was treated with beta-Glucuronidase, the other half remaining untreated. The two halves of the urine were fractionated by XAD-2 column chromatography and subjected to the Ames tester strains TA98 and TA 100 with and without metabolic activation.
In a second assay subjects ingested 1L of water or coffee within 2 hours, and the urine was collected in the following 7 hours and as in the first experiment it was fractionated by XAD-2 column chromatography and subjected to the Ames tester strains TA98 and TA 100 with and without metabolic activation.
Treatment with or without beta-Glucuronidase gave no mutagenic effect in either strain with urine of non-coffee or coffee drinkers. The absence of a mutagenic effect of these urine fractions, especially in Salmonella TA100, confirmed the detoxifying effect of the mammalian metabolizing system.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: Original Ames test (Ames et al., 1975) with modifications (Yahagi et al., 1975)
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: instant coffee
- 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: not available
- Stability under test conditions: not available
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water
- 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: Instant coffee was dissolved in cold 0.1 M phosphate buffer, pH 7.4
- 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 - Target gene:
- His
- Species / strain / cell type:
- S. typhimurium TA 100
- Species / strain / cell type:
- S. typhimurium TA 102
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix
- Test concentrations with justification for top dose:
- 0, 10, 20, 30, 40 and 50 mg/plate
- Untreated negative controls:
- yes
- Remarks:
- 0.1 M phosphate buffer
- Positive controls:
- other: in some experiments methylglyoxal (25%) was added to the coffee stock solutions or tested alone for its mutagenicity
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium; in agar (plate incorporation)
- Cell density at seeding (if applicable): not available
DURATION
- Preincubation period: none
- Exposure duration: 48 hours at 37ºC
- 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): not applicable
SELECTION AGENT (mutation assays): histidine-free medium
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable
NUMBER OF REPLICATIONS: 3
METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable
NUMBER OF CELLS EVALUATED: not applicable
NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): not applicable
CRITERIA FOR MICRONUCLEUS IDENTIFICATION: not applicable
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: not applicable
- Any supplementary information relevant to cytotoxicity: not applicable
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): not applicable
- OTHER: none - Evaluation criteria:
- For each dose the mean of the total number of revertants and the standard deviation of 3 replicate plates was calculated. The standard deviation normally was approximately 10% of the total number of revertants.
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 40 and 50 mg/plate
- Untreated negative controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Untreated negative controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 102
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- at 50 mg/plate
- Untreated negative controls validity:
- valid
- Conclusions:
- In an in vitro gene mutation test in bacteria performed according to the Ames protocol, instant coffee was concluded to have negative mutagenic potential in the presence of metabolic activation.
- Executive summary:
The instant coffee was tested in an in vitro gene mutation test in bacteria performed according to the Ames protocol.
Coffee increased the number of Salmonella TA 100 and 102 revertants in absence of S9 mix. The cytotoxic effects were observed above 30 mg/plate in TA 100 strain.
When the coffee was tested in the presence of S9 mix, the mutagenic induction was completely abolished in TA 100, the only strain tested.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: read across from surrogate substance
- Justification for type of information:
- Please see Read across justification document in IUCLID section 13.
- Reason / purpose for cross-reference:
- read-across source
- Conclusions:
- In an in vitro mouse lymphoma L5178Y Tk+/- assay instant coffee did not increase the number of mutant colonies, moreover, the addition of instant coffee decreased the mutagenic effect of N-methyl-N-nitro-N-nitrosoguanidine on the L5178Y Tk+/- cells. Based on the results from the analogue substance, it is concluded that similar effects can be expected for Coffee, bean, roasted, ext.
- Executive summary:
The main aim of this study was to test the hypothesis that standard (caffeinated) instant coffee can inhibit in mammalian cells the in vitro mutagenicity of N-methyl-N-nitro-N-nitrosoguanidine (MNNG), a directly acting genotoxin. Cultures of mouse lymphoma L5178Y Tk+/- were treated with MNNG at 25, 50 and 75 ng/ml, instant coffee alone at 125 µg/ml and MNNG at 25, 50 and 75 ng/ml together with 125 µg/ml of instant coffee. Exposure to instant coffee did not increase the mutant frequency at the Tk locus of mouse lymphoma L5178Y cells. MNNG application increased number of mutants in a dose-response manner, however, instant coffee and MNNG co-treatment decreased the number of mutant colonies. These results demonstrate that addition of instant coffee inhibited the in vitro mutagenicity of MNNG, suggesting the significant protective effects of caffeinated instant coffee.
Based on the results from the analogue substance, it is concluded that similar effects can be expected for Coffee, bean, roasted, ext.
Referenceopen allclose all
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.
See attached additional information on results
The levels of bacterial revertants in samples from coffee chains were lower than the two-fold criterion of the control sets, and no significant dose-response effect was observed with or without rat liver enzyme activation. See attached additional information on results.
4-day experiment
With Salmonella strains TA98 and TA 100 (±S9 mix) the number of revertants observed with the non-polar urine fraction of non-coffee drinkers and coffee drinkers was in the range of the spontaneous revertants. The highest concentration of beta-Glucuronidase-treated urine fractions caused a bactericidal effect. This effect was more pronounced in TA 100 than in TA98 and occurred more frequently in urine fractions of coffee drinkers than of non-coffee drinkers.
2-hour exposure
When the same subjects drank water, or one week later coffee, their urine extracts did not increase the number of revertants with or without S9 mix in Salmonella TA 98 and TA 100. The strong inhibitory effect of beta-Glucuronidase-treated urine on revertant colonies observed in the 4-day experiment was less pronounced in this experiment. This was attributed to the lower urine volume extracted.
Exposure to instant coffee did not increase the mutant frequency in at the Tk locus of mouse lymphoma L5178Y cells. MNNG induced mutation in a dose response manner and its co-treatment with caffeinated instant coffee at three different concentrations resulted in reduction of mutagenicity (P <0.05).
Genetic toxicity in vivo
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- 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
- Qualifier:
- according to guideline
- Guideline:
- other: original method
- Version / remarks:
- The micronucleus test. Schmid W. Mutation Research 31 (1975) 9-15
- Principles of method if other than guideline:
- - Principle of test: micronucleus test
- Short description of test conditions: test substance was applied sub-acutely to mice, and the effect was read in direct smears from bone marrow
- Parameters analysed / observed: bone-marrow metaphase chromosomes - GLP compliance:
- no
- Type of assay:
- mammalian erythrocyte micronucleus test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Instant coffee was obtained in sealed 100 g jars
- 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: not available
- Stability under test conditions: not available
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: coffee was always freshly prepared just before use, with boiling tap water and cooled to room temperature.
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not available
- Final preparation of a solid: not applicable
OTHER SPECIFICS: none - Species:
- mouse
- Strain:
- Swiss
- Remarks:
- OF-1
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Iffa Credo, France
- Age at study initiation: 10 weeks
- Weight at study initiation: not available
- Assigned to test groups randomly: yes
- Fasting period before study: not available
- Housing: individually in cages after random distribution
- Diet: ad libitum, Nafag diet 875
- Water: ad libitum
- Acclimation period: at least 1 week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±1
- Humidity (%): 55±5
- Air changes (per hr): not available
- Photoperiod (hrs dark / hrs light): not available
IN-LIFE DATES: From: To: not available - Route of administration:
- oral: gavage
- Vehicle:
- Tap water was used as a test substance vehicle and negative control, administered at 1 mL/40 g bw/day
- Details on exposure:
- Instant coffee was administered to the animals by gavage in volumes up to 1 mL. Volumes were adjusted for body weight. Coffee doses were applied up to the highest tolerated dose level, which were determined in the preliminary assay. Instant coffee was administered for five consecutive days at 24-hour intervals, the last dose was given 6 hours before killing. The negative-group animals received water at the same time as the test animals were treated. The positive control animals were always treated 30 minutes and 6 hours before killing by intraperitoneal injection.
- Duration of treatment / exposure:
- 5 days
- Frequency of treatment:
- 24-hour intervals
- Post exposure period:
- 6 hours
- Dose / conc.:
- 500 mg/kg bw/day
- Remarks:
- instant coffee
- Dose / conc.:
- 1 000 mg/kg bw/day
- Remarks:
- instant coffee
- Dose / conc.:
- 2 000 mg/kg bw/day
- Remarks:
- instant coffee
- Dose / conc.:
- 3 000 mg/kg bw/day
- Remarks:
- instant coffee
- No. of animals per sex per dose:
- 9-10
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Triethylenethiophosphoramide
- Justification for choice of positive control(s): not available
- Route of administration: intraperitoneal
- Doses / concentrations: 1.5 mg/kg bw, injected at 30 and 6 hours prior to killing - Tissues and cell types examined:
- marrow cells from femurs
- Details of tissue and slide preparation:
- After killing, the femurs were removed and the marrow expelled with 2 ml of fetal bovine serum. After one or two flushings, the marrow cells obtained were dispersed in the serum as a fine suspension, which was centrifuged at 200 g for 10 minutes. The suparnatant was removed and the cells were resuspended in a small drop of serum and the slides were prepared. After drying for 24 hours, the slides were fixed for 5 minutes in absolute methanol and stained in 10% Giemsa for 45 minutes. They were then thoroughly rinsed with distilled water, air dried, cleared in xylitol and covered with Merckoglas spray.
- Evaluation criteria:
- 1000 polychromatic were scored for micronuclei
- Statistics:
- Counts were checked for their mathematical distribution. Analysis of variance (ANOVA) based on the Poisson distribution was used to compare the difference in micronuclei frequency between animals receiving water only and those receiving instant coffee.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- not specified
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Instant coffee induced no significant effect in the micronucleus test when mice received up to 3 g coffee/kg body weight/day during five consecutive days. There was no difference in micronuclei frequency between the mice receiving water only and those receiving instant coffee.
- Conclusions:
- Administration of oral doses of instant coffee given to Swiss OF-1 mice up to 3000 mg/kg/ body weight per day for five executive days did not induce increases in micronuclei above spontaneous levels.
- Executive summary:
Instant coffee was administered to Swiss OF-1 mice by gavage up to 3000 mg/kg/ body weight per day for 5 consecutive days. The negative-group animals received water at the same time as the test animals were treated and positive control animals received triethylenethiophosphoramide at 1.5 mg/kg bw 30 minutes and 6 hours before killing by intraperitoneal injection.
After killing, the femurs were removed, bone marrow expelled, washed, stained and 1000 polychromatic were scored for the presence of micronuclei.
Instant coffee did not have any significant effect in the micronucleus test when mice received up to 3000 mg coffee/kg body weight/day during five consecutive days. There was no difference in micronuclei frequency between the mice receiving water only and those receiving instant coffee.
Under conditions of this study, instant coffee was considered non-genotoxic in mice.
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline followed
- Principles of method if other than guideline:
- - Principle of test: detects the ability of a chemical to enhance the exchange of DNA between two sister chromatids of a duplicating chromosome.
- Short description of test conditions: test substance was applied acutely to hamsters, and the effect was read in direct smears from bone marrow
- Parameters analysed / observed: bone-marrow metaphase chromosomes - GLP compliance:
- no
- Type of assay:
- sister chromatid exchange assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Instant coffee was obtained in sealed 100 g jars
- 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: not available
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: coffee was always freshly prepared just before use, with boiling tap water and cooled to room temperature
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not available
- Final preparation of a solid: not applicable
OTHER SPECIFICS: none - Species:
- hamster
- Strain:
- other: Chinese
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Kleintier-Farm, Fullinsdorf
- Age at study initiation: 10-12 weeks
- Weight at study initiation: not available
- Assigned to test groups randomly: yes
- Fasting period before study: not available
- Housing: individually in cages after random distribution
- Diet: ad libitum, Nafag diet 924
- Water: ad libitum
- Acclimation period: at least 1 week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23±1
- Humidity (%): 55±5
- Air changes (per hr): not available
- Photoperiod (hrs dark / hrs light): not available
IN-LIFE DATES: From: To: not available - Route of administration:
- oral: gavage
- Vehicle:
- Tap water was used as a test substance vehicle and negative control, administered as a a single dose of 0.1 mL/10 g bw by gavage
- Details on exposure:
- Instant coffee as well as the negative and positive controls were administered once, 2 hours post bromodeoxyuridine (BrdU) implantation. The BrdU tablet (50mg) was implanted subcutaneously in the neck area just behind the ears of animals under anaesthesia (Ketalar). At 24 hours after implantation, 0.02 mg vincristine/animal were injected intraperitoneally and 3-3.5 hours later the animals were killed
- Duration of treatment / exposure:
- 22 hours
- Frequency of treatment:
- single dose
- Post exposure period:
- 3-3.5 hours
- Dose / conc.:
- 1 000 mg/kg bw/day
- Dose / conc.:
- 1 500 mg/kg bw/day
- Dose / conc.:
- 2 000 mg/kg bw/day
- Dose / conc.:
- 2 500 mg/kg bw/day
- No. of animals per sex per dose:
- 7-10/trial
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Endoxan
- Justification for choice of positive control(s): not available
- Route of administration: intraperitoneal
- Doses / concentrations: 10 mg/kg bw, injected once - Tissues and cell types examined:
- marrow from femurs
- Details of tissue and slide preparation:
- After killing, the femurs were removed and the marrow was expelled with 2 mL Hanks solution. The bone marrow cells, finely suspended in Hanks solution, were centrifuged and the Hanks solution was removed. Warm 0.075 M KCl (5 ml) was added and the cells were resuspended and incubated for 10 minutes at 37°C. After this hypotonic treatment, the cells were fixed in the usual manner with fixative consisting of 1 part glacial acetic acid and 3 parts absolute methanol. Then a hazy suspension was made. Clean grease-free slides were embedded in crushed ice. The cell suspension was dropped on to a slide immediately upon its removal from the ice and the slide was carefully passed through a flame so as to spread the chromosomes.
The slides were stained for 15 min in 2 µg Hoechst fluorescent dye/ml phosphate buffered saline (PBS). After rinsing they were arranged in a tray, covered with PBS, and set under a DESAG UV lamp at 366 nm for 45 min. They were then incubated for 1 hour in 2 x SSC at 60°C. After rinsing they were stained for 10 minutes in 7% Giemsa. The slides were dried, cleared in xylol and made permanent with Merckoglas spray. - Evaluation criteria:
- For each hamster a well-stained slide was selected on which 30 metaphases were evaluated and all exchanges in each metaphase were registered
- Statistics:
- Counts were checked for their mathematical distribution; results were analyzed statistically using a nested analysis of variance (ANOVA) from 3 sources: between treatment groups, between animals within a treatment group and between metaphases within an animal. As the animals placed in each group were a random sample, the variation between groups was tested for significance against the second source of variance. Accordingly, the variation between the metaphases within animals was not directly involved in these analyses.
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- not specified
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- In a first trial, where doses up to 2500 mg/kg body weight were given to hamsters, no significant variation in SCEs was observed between treatments (P=0.05). Comparison of the three coffee treatments with the negative control, however, showed a significant difference at the 5% level (P = 0.042) in a one-sided t test. The trend of the dose-response relationship over the three coffee treatments was negative, so the assay was repeated (Trial 2). In this second assay no significant difference was found between any of the coffee treatments and the negative control when ANOVA was used as for the first trial. Thus, coffee was considered to have no effect in the SCE test, since even traces of reactive compounds would have caused a dose-dependent reproducible effect under the conditions used.
- Conclusions:
- Administration of a single dose of instant coffee given to Chinese hamsters up to 2500 mg/kg/ body weight per day did not increase the frequency of sister chromatid exchange in comparison to the animals receiving water.
- Executive summary:
A single dose of instant coffee was administered to Chinese hamsters by gavage up to 2500 mg/kg body weight per day. The negative-group animals received water at the same time as the test animals were treated and positive control animals received endoxanat 10 mg/kg bwby intraperitoneal injection.
After killing, the femurs were removed, bone marrow expelled, washed, stained and 30 metaphases were evaluated per each animal.
The instant coffee did not increase the frequency of sister chromatid exchange in treated animals in comparison to the animals receiving water.
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- no guideline available
- Principles of method if other than guideline:
- - Principle of test: the test was carried out to evaluate the possible dose-dependent effects of orally administered standard instant coffee on the genotoxicity of the potent carcinogens in the mouse bone marrow micronucleus test
- Short description of test conditions: test substance and carcinogens were applied acutely to mice, and the effect read in direct smears from bone marrow
- Parameters analysed / observed: bone-marrow metaphase chromosomes - GLP compliance:
- no
- Type of assay:
- mammalian erythrocyte micronucleus test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Standard instant coffee (obtained from a large coffee producer)
- 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: not available
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in water
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not applicable
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: dissolved in water at room temperature
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: not available
- Final preparation of a solid: not applicable
OTHER SPECIFICS: none - Species:
- mouse
- Strain:
- Swiss
- Remarks:
- Albino
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: not available
- Age at study initiation: 9-11 weeks
- Weight at study initiation: 28-32 g
- Assigned to test groups randomly: not available
- Fasting period before study: not available
- Housing: university animal house
- Diet: ad libitum, standard mouse diet (Hindustan Lever Limited)
- Water: ad libitum
- Acclimation period: not available
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25±2
- Humidity (%): not available
- Air changes (per hr): not available
- Photoperiod (hrs dark / hrs light): not available
IN-LIFE DATES: From: To: not available - Route of administration:
- oral: gavage
- Vehicle:
- Tap water was used as a test substance vehicle and negative control (10 mL/kg body weight)
- Details on exposure:
- Instant coffee was administered orally (10 mL/kg body weight) twice, 2 and 20 hours before animals were exposed to the carcinogens including 7,12-dimethylbenz[a]anthracene (DMBA), aflatoxin B~ (AFB1),benzo[a]pyrene (BP), urethane (UR) and cyclophosphamide (CP).
At the time of administration (either at 2 or 20 hours), the first 3 groups were given 125, 250 and 500 mg coffee/kg body weight respectively, while the control group received the same volume (10 mL/kg body weight) of tap water. In addition, another experiment was carried out to monitor the time course of micronucleus induction in bone marrow cells of mice, which received either water (control) or coffee, 2 and 20 hours before exposure to DMBA. For this purpose, 2 groups of 10 mice each (coffee and control) were killed at 6 hours intervals in order to sample the bone marrow cells.
The chemical carcinogens were injected intraperitoneally (10 ml/kg body weight). Prior to this, BP and DMBA were suspended in peanut oil. AFB1 was dissolved in dimethyl sulfoxide. CP and UR were dissolved in saline. - Duration of treatment / exposure:
- 1st test: Coffee was orally administered 2 and 20 h before injecting the carcinogens. The animals were killed after either 28 h (AFBI, CP and UR) or 48 h (DMBA and BP).
2nd test: Coffee was orally administered 2 and 20 h before injecting DMBA. The time course of micronucleus induction by DMBA with or without the administration of coffee was monitored by killing the animals 48, 54, 60, 66 and 72 h after exposure to the carcinogen. - Post exposure period:
- 1st test: 28 or 48 hours
2nd test: 48, 54, 60, 66 and 72 - Dose / conc.:
- 125 mg/kg bw (total dose)
- Dose / conc.:
- 250 mg/kg bw (total dose)
- Dose / conc.:
- 500 mg/kg bw (total dose)
- No. of animals per sex per dose:
- 10 animals per dose
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- No
- Tissues and cell types examined:
- Bone marrow
- Details of tissue and slide preparation:
- Not available
- Evaluation criteria:
- From each experimental animal 2500 polychromatic erythrocytes were scored from a single slide to detect the presence of micronucleated polychromatic erythrocytes.
- Statistics:
- One-way analysis of variance (ANOVA) was applied for each carcinogen separately and the comparison of each of the 3 doses with the control was based on Dunnett's t statistic. Two-way ANOVA was performed to evaluate the effect of sampling (48, 54, 60, 66 and 72 h) and treatment (water or coffee) on the incidence of DMBA-induced micronucleated polychromatic erythrocytes. In addition the trend test was employed.
- Additional information on results:
- A significant dose-response reduction was observed in the incidence of micronucleated polychromatic erythrocytes (MnPCEs) induced by the carcinogens DMBA, AFB1, BP, and UR, following the administration of 250 and 500 mg coffee/kg body weight. A similar dose response was observed for CP. However, when a lower dose of coffee (125 mg/kg) was administered, the reduction observed in the incidence of MnPCEs was not significantly different from that in the control mice, which received water instead of coffee). Furthermore, the trend test has confirmed that the observed dose effect of coffee for the inhibition of genotoxicity is significantly linear.
In the second experiment, irrespective of the sampling time, a significant reduction was observed in the incidence of DMBA-induced MnPCEs in bone marrow cells of mice, which received coffee instead of water. The trend test showed that the time effect is significantly linear. - Conclusions:
- Administration of oral doses of instant coffee given to Swiss albino mice up to 500 mg/kg/day significantly inhibited the in vivo genotoxicity of several carcinogens.
- Executive summary:
The mouse bone marrow micronucleus test was carried out to evaluate the possible inhibitory effects of 3 doses (125, 250 and 500 mg/kg) of standard instant coffee on the in vivo genotoxicity of 7,12-dimethylbenz[a]anthracene (DMBA), benzo[a]pyrene (BP), aflatoxin B1 (AFB1), urethane (UR) and cyclophosphamide (CP).
Coffee was orally administered to Swiss albino mice twice, 2 and 20 h before the carcinogens were injected intraperitoneally. From the results obtained, it was evident that the administration of 250 and 500 mg coffee/kg body weight could significantly inhibit the in vivo genotoxicity of these carcinogens. A linear dose response was observed for the inhibitory effect of coffee. Furthermore, inhibition of genotoxicity by coffee was observed in bone marrow cells which were sampled at 6-hour intervals (48, 54, 60, 66 and 72 h) from the time of peak induction of micronuclei by DMBA.
Dose of 250 mg instant coffee/kg body weight administered to mice is equivalent to the intake of 8-10 cups of instant coffee per day, by a person weighing ~60 kg.
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See attached additional information on results.
Additional information
Coffee may cause a relatively weak direct-acting activity in some bacterial and in vitro mammalian cell test systems. Mutagenicity is mainly attributed to coffee components such as aliphatic carbonyls, however, coffee’s mutagenicity is readily deactivated by detoxifying mammalian enzyme systems. Additionally, the mutagenic and genotoxic potential of coffee and caffeine was demonstrated at doses several orders of magnitude greater than the estimated lethal dose for caffeine in humans. Despite any positive in vitro results, coffee has subsequently demonstrated a lack of mutagenicity and genotoxicity in vivo. Coffee and caffeine are also able to inhibit the mutagenic effects of numerous chemicals in the in vitro and in vivo systems.
In the last decade, many studies have reported coffee consumption to be associated with a reduced risk of several chronic diseases; the physiological chemistry that underlies these claims often involves a number of bioactive compounds that exhibit antioxidant and anti-peroxidation activities, respectively. Additionally, none of the aliphatic carbonyls such as methylglyoxal, propylglyoxal or ethylglyoxal responsible for mutagenic activities of coffee are reported in the composition of Coffee, bean, roasted, ext.
Justification for classification or non-classification
Based on the available experimental results for suitable analogues of Coffee, bean, roasted, ext. and the intrinsic properties for the main components of Coffee, bean, roasted, ext, it can be concluded that Coffee, bean, roasted, ext. does not represent mutagenic or genotoxic risk for human health and therefore, does not meet the criteria for classification as such in accordance with CLP Regulation (EC) 1272/2008.
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