Ibuprofen

Endpoint summary

Description of key information

The test substance was not corrosive or irritative to the skin.

The test substance was not corrosive to the eyes. Eye irritation cannnot be excluded.

Irritating to respiratory tract based on worker anecdotal evidence.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

13 April 2004

Qualifier:

according to guideline

Guideline:

other: EU Method B: Methods for the determination of toxicity and other health effects: In Vitro Skin Corrosion: Human Skin Model Test; Official Journal of the European Union, No. L 142

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

24 April 2002; for determination of a corrosive/irritant property

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test substance: Ibuprofen
- Test substance No.: 02/0156-6
- Batch ID: IB1S1290
- Purity: 99.7%
- pH: ca. 4.5 (undiluted test substance, moistened with water)

Test system:

human skin model

Source species:

human

Cell type:

other: EpiDerm™ human epidermis model

Justification for test system used:

Based on the results of ECVAM (European Center for Validation of Alternative Methods) funded validation studies, it was concluded by the ECVAM Scientific Advisory Committee that the EpiDerm™ human epidermis model is suitable to be used for distinguishing between corrosive and non-corrosive chemicals (ECVAM: ESAC statement on the application of the EpiDerm™ human skin model for skin corrosivity testing of 14-15 Mar 2000) as well as between irritant and non-irritant chemicals (ECVAM: ESAC statement on the scientific validity of in-vitro tests for skin irritation testing of 5 Nov 2008).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The EpiDerm™ model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the humanepidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm Ø) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
- Origin: MatTek Corporation, Ashland MA, USA

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (3 min exposure) and 37°C (1 hour exposure)

REMOVAL OF TEST MATERIAL AND CONTROLS
The tissues were washed with PBS to remove residual test material 3 minutes or 1 hour after start of the application treatment. Rinsed tissues were kept in 24-well plates (holding plates) at room temperature on assay medium until all tissues per application time were dosed and rinsed.

VIABILITY TEST
The assay medium was replaced by MTT solution and tissues were incubated for 3 hours. After incubation, tissues were washed with PBS and the formazan produced by the tissues was extracted with isopropanol over night at room temperature. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 µL of highly de-ionized water was applied first. Thereafter, a bulk volume of 25 µL of test material was applied with a sharp spoon and homogeneously distributed with water

VEHICLE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL

Duration of treatment / exposure:

3 minutes and 1 hour

Number of replicates:

2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

106

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

99

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Interpretation of results:

other: Not corrosive

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Version / remarks:

24 April 2002; for determination of a corrosive/irritant property

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test substance: Ibuprofen
- Test substance No.: 02/0156-6
- Batch ID: IB1S1290
- Purity: 99.7%
- pH: ca. 4.5 (undiluted test substance, moistened with water)

Test system:

human skin model

Source species:

human

Cell type:

other: EpiDerm™ human epidermis model

Justification for test system used:

Based on the results of ECVAM (European Center for Validation of Alternative Methods) funded validation studies, it was concluded by the ECVAM Scientific Advisory Committee that the EpiDerm™ human epidermis model is suitable to be used for distinguishing between corrosive and non-corrosive chemicals (ECVAM: ESAC statement on the application of the EpiDerm™ human skin model for skin corrosivity testing of 14-15 Mar 2000) as well as between irritant and non-irritant chemicals (ECVAM: ESAC statement on the scientific validity of in-vitro tests for skin irritation testing of 5 Nov 2008).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: The EpiDerm™ model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm Ø) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
- Origin: MatTek Corporation, Ashland MA, USA
- Pre-incubation: On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3hours.

TEST SYSTEM
The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period.

VIABILITY TEST
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol for at least 2 hours at room temperature on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

Control samples:

yes, concurrent vehicle

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 µL sterile PBS was applied first. Thereafter, a bulk volume of 25 µL of test material was applied with a sharp spoon and homogeneously distributed together with the fluid

VEHICLE CONTROL
- Amount(s) applied: 30 µL

POSITIVE CONTROL
- Amount(s) applied: 30 µL

Duration of treatment / exposure:

1 hour (25 min overall, 35 min in the incubator)

Duration of post-treatment incubation (if applicable):

42 ± 4 hours

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

irritation test

Value:

104

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Interpretation of results:

other: Not irritating

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

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 Ocular Corrosives and Severe Irritants)

Version / remarks:

7 September 2009

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Version / remarks:

24 April 2002; for determination of eye irritation/corrosion

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

- Name of test substance: Ibuprofen
- Test substance No.: 02/0156-6
- Batch ID: IB1S1290
- Purity: 99.7%
- pH: ca. 4.5 (undiluted test substance, minimally moistened with water); ca. 4 (20% aqueous preparation)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Schlachthof Bensheim, Am Schlachthof 7-9, 64625 Bensheim
- Characteristics of donor animals: age of animals: minimum 12 months, maximum 60 months
- Species: Bovine (eyes are obtained as a by-product of freshly slaughtered cattle)

Details on study design:

SELECTION AND PREPARATION OF CORNEAS & QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in specially designed corneal holders that consists of anterior and posterior chambers. Both chambers were filled to excess with prewarmed Eagles’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour. After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that show macroscopic tissue damage or an opacity value < 566 opacity units were discarded. Corneas with opacity values close to the median value of all corneas were selected as negative (vehicle) control. The remaining corneas were then distributed into treatment and positive control groups. Each corneal holder was uniquely identified with a number written on the chambers with a felt pen.

NUMBER OF REPLICATES
Each treatment group (test substance, vehicle control and positive control) consisted of 3 corneas.

SOLVENT CONTROL USED
Highly deionized water

POSITIVE CONTROL USED
20% (w/v) solution of Imidazole in highly de-ionized water

APPLICATION DOSE AND EXPOSURE TIME
750 μL of the 20% (w/v) test-substance preparation (non-surfactant), vehicle control or positive control was applied using a pipette. The corneas were incubated in a horizontal position at about 32 °C for approximately 4 hours (non-surfactant solids).

TREATMENT METHOD: open chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
The vehicle control and positive control were removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red). Because the test substance could not be removed using a syringe, the epithelium was rinsed with the open chamber method.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: visual observations and opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

ACCEPTANCE CRITERIA
In case one of the below given acceptance criteria is not covered, repetition of the test was considered.
- A study is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean.
- The negative control responses should result in opacity and permeability values that are less than the established upper limits.
- Since the IVIS per treatment group is determined from the mean of three single corneas, the variability between the corneas treated per test substance should be acceptably low. If the prediction is not clearly identified in all corneas, the test will be repeated.

DECISION CRITERIA
IVIS > 55: risk of serious damage to eyes
IVIS ≤ 55: no risk of serious damage to eyes

Irritation parameter:

cornea opacity score

Run / experiment:

1st

Value:

28.7

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Irritation parameter:

cornea opacity score

Run / experiment:

2nd

Value:

32.7

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

1st

Value:

1.357

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Irritation parameter:

fluorescein retention score

Run / experiment:

2nd

Value:

1.05

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

1st

Value:

49

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Irritation parameter:

in vitro irritation score

Run / experiment:

2nd

Value:

48.4

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

Results of negative and positive controls see "any other information on results incl. tables"

First run

	Mean Opacity Value	Mean Permeability Value	In Vitro Irritancy Score
Test substance	28.7	1.357	49.0
Negative Control	2.2	0.020	2.5
Positive Control	81.6	3.738	137.6

Second run

	Mean Opacity Value	Mean Permeability Value	In Vitro Irritancy Score
Test substance	32.7	1.050	48.4
Negative Control	4.1	-0.005	4.0
Positive Control	63.2	3.466	115.2

Interpretation of results:

other: Not corrosive to the eyes

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Additional information

Skin irritation/corrosion

In two GLP-compliant studies, in accordance with OECD guideline 431 and 439, the potential to cause dermal corrosion/irritation was assessed by a single topical application of 25μL bulk volume (about 14 mg) of the test substance to a reconstructed three dimensional human epidermis model (EpiDerm™). For the corrosion test two EpiDerm™ tissue samples were incubated with the test substance for 3 minutes and 1 hour, respectively. The irritation test was performed with three EpiDerm™ tissue samples, which were incubated with the test substance for 1 hour followed by a 42-hours post-incubation period. Tissue destruction was determined by measuring the metabolic activity of the tissue after exposure/post-incubation using a colorimetric test. The reduction of mitochondrial dehydrogenase activity, measured by reduced formazan production after incubation with a tetrazolium salt (MTT) was chosen as suitable endpoint. The formazan production of the test substance treated epidermal tissues is compared to that of negative control tissues. The quotient of the values indicates the relative tissue viability. The EpiDerm™ skin corrosivity/irritation test showed the following results: the test substance is not able to reduce MTT directly. Corrosion test: the mean viability of the test-substance treated tissues determined after an exposure period of 3 minutes was 106%, and it was 99% after an exposure period of 1 hour. Irritation test: the mean viability of the test-substance treated tissues determined after an exposure period of 1 hour with about 42 hours post-incubation was 104%. Based on the observed results and applying the evaluation criteria it was concluded, that the test substance does not show a skin irritation potential in the EpiDerm™ skin corrosion/irritation test under the test conditions chosen.

 

Eye corrosion

In a GLP-compliant study, in accordance with OECD guideline 437, the potential to cause serious damage to the eyes was assessed by a single topical application of 750 μL of a 20% aqueous test- substance preparation to the epithelial surface of isolated bovine corneas. Three corneas were treated with the test- substance preparation for an exposure period of 4 hours (1st test run). In order to clarify the borderline result of the 1st test run (2 corneas produced IVIS values below the threshold for classification and 1 cornea above this value) a 2nd test run was performed with another 3 corneas. Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea. Both measurements were used to calculate an In Vitro Irritancy Score of the test substance relative to the control corneas. The mean corneal opacity value was 28.7, 2.2, and 81.6 in the 1st test run and 32.7, 4.1, and 63.2 in the 2nd test run for the test substance, vehicle control, and positive control, respectively. The mean permeability was 1.357, 0.02, and 3.737 in the 1st test run and 1.05, -0.005, and 3.466 in the 2nd test run for the test substance, vehicle control, and positive control, respectively. The in vitro irritancy score (IVIS) was 49.0, 2.5, and 137.6 in the 1st test run and 48.4, 4.0, and 115.2 in the 2nd test run for the test substance, vehicle control, and positive control, respectively. A substance is considered to cause serious eye damage if the IVIS is >55. The test substance is therefore not considered to cause serious eye damage. However, based on the results of the study it is not possible to determine the eye irritative potential of the test substance.

Justification for classification or non-classification

Based on the available information classifcation for skin irritation/corrosion is not warranted in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.

Based on the available information ibuprofen does not lead to serious eye damage in accordance in accordance with EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008. However, an eye irritation potential cannot be excluded in absence of further data.

Irritating to respiratory tract based on worker anecdotal evidence.