Coffee, bean, roasted, ext.

Endpoint summary

Description of key information

Not irritating to skin (based on the experimental results from a suitable analogue substance).

Not irritating to eyes (OECD TG 437).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vitro / ex vivo

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 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

GLP compliance:

not specified

Remarks:

Study was performed in a research laboratory, probably not GLP compliant.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: coffee silver skin samples were provided by a national coffee roaster (BICAFÉ, Portugal).
- 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: 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: coffee silver skin was milled to particle size of approximately 0.1 mm using a A11 basic analysis mill (IKA Wearke, Staufen, Germany) and stored in silicone tubes at room temperature (25–28ºC) until extract preparation. Samples (1 g) were submitted to solvent extraction by maceration with 20 mL of water, ethanol: water (1:1 v/v) or ethanol for 30 min at 40ºC. The three different extracts obtained were filtered through Whatman No. 1 paper filter and the filtrates collected.
- Preliminary purification step (if any): not applicable
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid: not applicable

OTHER SPECIFICS: none

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

The in vitro reconstructed human skin tissue (EpiSkin) method, proposed to replace animal testing for skin corrosivity and skin irritation is based on determining cell viability, and cytokine release (IL-1a) as an additional endpoint. The reconstructed 0.38 cm2 skin inserts were used and the assay was performed according to the manufacturer instructions and protocol.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Duration of treatment / exposure:

15 minutes

Duration of post-treatment incubation (if applicable):

After exposing to the extracts and controls for 15 min, the epidermis samples were washed with sterile PBS and then incubated in the maintenance medium. After 42 h, the medium was collected and frozen at -20ºC for further determination of IL-1a.

Number of replicates:

3 replicates per coffee sliverskin extract

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

water extract

Value:

117.7

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

water/ethanolic extract

Value:

105.9

Negative controls validity:

valid

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

ethanolic extract

Value:

117.8

Negative controls validity:

valid

Positive controls validity:

valid

The results of skin irritability assay. Cell viability was assessed by MTT while the post-exposure basal media was analyzed for IL-1a release (pg/mL)

Coffee silverskin extract	Viability (%)	IL-1a (pg/mL)
Water	117.7 ± 11.7	8.5 ± 0.1
Water/ethanol (1:1 v/v)	105.9 ± 15.9	76.4 ± 2.0
Ethanol	117.8 ± 9.8	17.6 ± 3.2
Positive control (SLS)	11.6 ± 2.4	552.9 ± 32.1
Negative control (PBS)	100 ± 9.7	28.4 ± 2.6

EpiSkin method distinguishes between irritants and non-irritants. Irritant chemicals are identified by their ability to decrease cell viability below defined threshold levels of 50%. The three coffee silverskin extracts were not considered skin irritants, as the viability in all cases was higher than 50%, with values ranging between 105.9 ± 15.9 and 117.8 ± 9.8.

The low irritating potential was also confirmed by the low release of IL-1a, a highly active and pro-inflammatory cytokine produced by keratinocytes.

Additionally, histological analysis did not reveal any morphological differences between extracts and negative control, in contrast to the positive control, where the adverse effects were observed in all the epidermal layers.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results from the in vitro reconstructed human epidermis test, the coffee silverskin extracts did not meet the classification criteria for skin irritation under the CLP Regulation (EC) No 1272/2008.

Executive summary:

The reconstructed skin inserts (EpiSkin method) were exposed to three types of coffee silverskin extracts (water, water/ethanol, ethanol) and controls for 15 min. The cell viability was determined by MTT assay, while the post-exposure basal media were analyzed for IL-1a release. Three extracts were not considered skin irritants in this test as the cell viability in all the cases was above 50% with the values ranging between 105 ± 15.9 and 117 ± 9.8 %. This potential was also confirmed by the low release of IL-1a, a highly active and pro-inflammatory cytokine produced by keratinocytes. Additionally, no morphological differences were observed between extracts and negative control (PBS), in contrast to the positive control (SLS) where the adverse effects were observed in all the epidermal layers.

Under the conditions of this assay, the coffee silverskin extracts did not meet the classification criteria for skin irritation under the CLP Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

Guideline version from 9 October 2017

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: CF02
- Expiration date of the lot/batch: 31.01.2019
- Purity test date: not available

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: approximately at 4 °C in the dark
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: not applicable
- 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: not available
- 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 available

OTHER SPECIFICS: none

Species:

cattle

Strain:

not specified

Vehicle:

Hank's balanced salt solution

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration (if solution): not applicable; applied undiluted

Duration of treatment / exposure:

10 minutes

Duration of post- treatment incubation (in vitro):

120 minutes

Number of animals or in vitro replicates:

Triplicate

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing Hanks’ Balanced Salt Solution (HBSS) until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1ºC for 60 minutes. At the end of the incubation period each cornea was examined for defects.

QUALITY CHECK OF THE ISOLATED CORNEAS
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer.

NUMBER OF REPLICATES
Three corneas were randomly allocated to the test item, negative and positive control.
NEGATIVE CONTROL USED
Sodium chloride, 0.9% w/v

SOLVENT CONTROL USED (if applicable)
Not applicable

POSITIVE CONTROL USED
Ethanol, >99.8%

APPLICATION DOSE AND EXPOSURE TIME
0.75 mL of the test item or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1ºC for 10 minutes.
TREATMENT METHOD: closed chamber
POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red.

- POST-EXPOSURE INCUBATION:
120 minutes at 32 ± 1ºC

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader] (OD492)
- Others (e.g, pertinent visual observations, histopathology): (please specify):
The condition of the cornea was visually assessed post treatment and post incubation. No histopathology was performed.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.

DECISION CRITERIA:
For an acceptable test the following positive control criterion should be achieved:
Neat ethanol was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during 2016 for this testing facility. Therefore, the In Vitro Irritancy Score should fall within the range of 31.6 to 58.7.
For an acceptable test the following negative control criteria should be achieved:
Sodium chloride 0.9% w/v was used for negative control purposes. The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2016 for bovine corneas treated with the respective negative control. When testing liquids the negative control limit for opacity should be =3.0 and for permeability =0.077.

The test item was classified according to the following prediction model:
IVIS Classification
= 3: No category. Not requiring classification to UN GHS or EU CLP;
> 3: =55 No prediction of eye irritation can be made;
> 55: Category 1. UN GHS or EU CLP Causes serious eye damage.

Irritation parameter:

in vitro irritation score

Value:

1.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

The In Vitro irritancy scores:

Treatment	In Vitro Irritancy Score
Test Item	1.6
Negative Control	0.0
Positive Control	45.4

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results from the Bovine Corneal Opacity and Permeability (BCOP) test, conducted according to OECD test guideline 437 and to GLP, the Coffee, bean, roasted, ext. does not meet the classification criteria for eye irritation or serious eye damage in accordance with CLP Regulation (EC) 1272/2008.

Executive summary:

The Bovine Corneal Opacity and Permeability (BCOP) test, conducted according to OECD test guideline 437 and to GLP, was performed to evaluate the eye hazard potential of Coffee, bean, roasted, ext.

The undiluted test item was applied for 10 minutes then rinsed followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score.

The In Vitro Irritancy Score for Coffee, bean, roasted, ext. was determined at 1.6; therefore, GHS criteria for eye irritation or serious eye damage are not met.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Based on their composition, coffee sliverskin extracts were identified as suitable analogues to assess irritation potential of Coffee, bean, roasted, ext. The reconstructed skin inserts (EpiSkin method) were exposed to three types of coffee silverskin extracts (water, water/ethanol, ethanol) and controls for 15 min. The cell viability was determined by MTT assay, while the post-exposure basal media were analyzed for IL-1a release. Three extracts were not considered skin irritants in this test as the cell viability in all the cases was above 50% with the values ranging between 105 ± 15.9 and 117 ± 9.8 %. This potential was also confirmed by the low release of IL-1a, a highly active and pro-inflammatory cytokine produced by keratinocytes. Additionally, no morphological differences were observed between extracts and negative control (PBS), in contrast to the positive control (SLS) where the adverse effects were observed in all the epidermal layers. Under the conditions of this assay, the coffee silverskin extracts were not considered skin irritants, based on these results it is concluded that Coffee, bean, roasted, ext. does not meet the classification criteria for skin irritation under the CLP Regulation (EC) No 1272/2008.

 

The Bovine Corneal Opacity and Permeability (BCOP) test, conducted according to OECD test guideline 437 and to GLP, was performed to evaluate the eye hazard potential of Coffee, bean, roasted, ext. The undiluted test item was applied for 10 minutes then rinsed followed by an incubation period of 120 minutes. Negative and positive control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score. The In Vitro Irritancy Score for Coffee, bean, roasted, ext. was determined at 1.6; therefore, criteria for eye irritation or serious eye damage set in the CLP Regulation (EC) No 1272/2008 are not met.

Justification for classification or non-classification

Adequate skin and eye irritation studies were performed either on Coffee, bean, roasted, ext. or its suitable analogue substance. Under the conditions of the assays, no skin or eye irritation was observed. Based on these results, Coffee, bean, roasted, ext. does not meet the classification criteria for skin and eye irritation under the CLP Regulation (EC) No 1272/2008.