Benzaldehyde, 4-[(3-fluorophenyl)methoxy]-

Endpoint summary

Description of key information

Skin irritation

Key, RhE, OECD 439, GLP, negative

Eye irritation

Key, BCOP, OECD 437, GLP, negative

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

Study period:

02 OCT 2014 - 29 JAN 2015

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:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Unwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D-65189 Wiesbaden (18 November 2014)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: The RHE™-model was obtained by culturing adult human keratinocytes on a polycarbonate filter under conditions which permit their terminal differentiation and the reconstruction of an epidermis with a functional horny layer.

Justification for test system used:

To reduce animal testing, this alternative in vitro method was used. The human skin RHE™-model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: RHE™-model (SkinEthic Laboratories, Lyon, France)
- Tissue batch number(s): 14-RHE-024
- Expiration date: October 27, 2014
- Date of initiation of testing: October 22, 2014

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: Using a multi pipette the tissues were gently rinsed with a minimum volume of 25 mL DPBS to remove any residual test item. Excess DPBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: microplate reader (ELx800, BioTek Instruments GmbH, Bad Friedrichshall, Germany)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD=1.286 (±0.076) (Acceptance criterion: OD > 0.7)
- Barrier function: 5.7 h (Acceptance criterion: 4.0 h <= ET50 <= 10.0 h)
- Morphology: Well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum. 6 viable cell layers present. Absence of significant histological abnormalities.

NUMBER OF REPLICATE TISSUES: 3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 42 minutes exposure is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 42 minutes exposure is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5 %

Duration of treatment / exposure:

42 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean relative viability

Value:

78.43

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
The negative control OD values were 2.445, 2.588 and 2.510 and, thus, in the range of >= 0.8 and <= 3.0.
- Acceptance criteria met for negative control: After treatment with the negative control (DPBS-buffer) the mean OD was 2.515 (standard deviation: 2.85%) and, thus, higher than the historically established boundary of 1.383.
- Acceptance criteria met for positive control: After treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) the mean viability value was 0.83% (standard deviation: 2.77%) and, thus, lower than the historically established boundary of 3.60%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation of the negative control and the positive control was <=18%, respectively. The standard deviation of the three tissues treated with the test item was 7.97% and, thus, <= 18%.

A table of the results is attached under 'Attached background material'.

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

The tissue viability after treatment with the test item was higher than 50% (mean viability: 78.43%). Therefore, the test item is not considered to possess an irritant potential to skin.

Executive summary:

This in vitro study was performed according to OECD Guideline 439 (Reconstructed Human Epidermis Test) following GLP. The test consisted of a topical exposure of the test item to a human reconstructed skin model followed by a cell viability test. Cell viability was quantitatively measured by dehydrogenase conversion of MTT into a blue formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE™-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before adding the solid test item 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. Afterwards, 16 mg of the test item were applied to the tissues.

After treatment with the negative control (DPBS-buffer) the mean OD was 2.515. Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 0.83%. Therefore, the study fulfilled the validity criteria. The tissue viability after treatment with the test item was 78.43% and, thus, higher than 50%, i.e. according to UN GHS classification the test item is considered as non-irritant to skin.

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:

22 OCT 2014 - 08 JAN 2015

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:

26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Unwelt, Energie, Landwirtschaft und Verbraucherschutz, Mainzer Straße 80, D-65189 Wiesbaden (18 November 2014)

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Characteristics of donor animals (e.g. age, sex, weight):
age: 18 - 26 months
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
transport medium: HBSS (Hanks' Balanced Salt Solution)
- indication of any existing defects or lesions in ocular tissue samples: All eyes were carefully examined macroscopically for defects. In addition, an opacity measurement before treatment was performed. Any corneas that showed macroscopic tissue damage (e.g. scratches, pigmentation, neovascularization) or an opacity of >7 opacity units were discarded.
- Indication of any antibiotics used: Streptomycin and Penicillin was added for the transport (5 mL/500 mL HBSS)

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 150 mg/750 µL (20 % (w/v))

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL
- Concentration (if solution): 0.9 % sodium chloride

Duration of treatment / exposure:

240 min

Number of animals or in vitro replicates:

3

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
The corneas were prepared immediately after delivery of the eyes to the laboratory. All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity or scratches were discarded. The corneas were carefully removed from the eyes using scalpel and rounded scissors. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneas used in the study were collected in incubation medium (prewarmed at 32 ± 1°C) and the corneal diameter of each cornea was measured and recorded.
Each cornea was mounted in a cornea holder (CiToxLAB, Veszprem, Hungary) with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring without stretching the cornea. Afterwards, the anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex form.

QUALITY CHECK OF THE ISOLATED CORNEAS
Any corneas that showed macroscopic tissue damage (e.g. scratches, pigmentation, neovascularization) or an opacity >7 opacity units were discarded.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
solvent control serves as negative control

SOLVENT CONTROL USED (if applicable)
yes, 0.9% sodium chloride solution

POSITIVE CONTROL USED
yes, 20% (w/v) Imidazole solution (in 0.9% sodium chloride solution)

APPLICATION DOSE AND EXPOSURE TIME
50 mg/750 µL (20 % (w/v)) for 240 min

TREATMENT METHOD: closed chamber for positive and negative control, open chamber for test item treatment

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: To remove the test item from the epithelium, the window locking-ring and the glass window from the anterior chamber were removed. Using a syringe, the corneas treated with the test item were gently rinsed with wash medium. Before measurement of the opacity value after treatment, fresh incubation medium was replaced in both compartments.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity was determined with an opacitometer (BASF-OP2.0, BASF SE, Ludwigshafen, Germany). The opacitometer measured the light transmission passing through the corneas and displayed a lux value. This value was recorded in a table and converted into an opacity value. The opacitometer was calibrated as described in the manual and the opacity of each cornea was determined by reading each holder placed in the photoreceptor compartment of the opacitometer.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)
- Others (e.g, pertinent visual observations, histopathology): visual inspection of the corneas after treatment

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria as indicated in the OECD Guideline 437 were applied. The IVIS cut-off values for identifying test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category) are given in the following:
≤ 3 No Category (UN GHS)
< 3; ≤ 55 No prediction can be made
> 55 Category 1 (UN GHS)

Irritation parameter:

in vitro irritation score

Run / experiment:

mean

Value:

2.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No observations (e.g. tissue peeling, residual test chemical, non-uniform opacity patterns) were seen in a visually inspection of the corneas after treatment.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Technical proficiency was demonstrated in a study with 2 of the 13 proficiency chemicals recommended in OECD Guideline 437. The IVIS obtained after treatment with Phenylbutazone was 0.9 and, thus, lower than 3. Therefore, the test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category). The IVIS obtained after treatment with (-)-Dibenzoyl-L-tartaric acid was 158.8 and, thus, higher than 55. Therefore, the test item requires classification for serious eye damage (UN GHS classification: Category 1).

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.1 and, thus, within three standard deviations of the current historical mean of the negative control (IVIS: -1.3 - 3.3).
- Acceptance criteria met for positive control: After treatment with the positive control (20% Imidazole) the calculated IVIS was 98.0 and, thus, also within two standard deviations of the current historical mean of the positive control (IVIS 74.7 - 138.3).

A table of the results is attached under 'Attached background material'.

Interpretation of results:

other: EU GHS criteria not met

Conclusions:

The IVIS obtained after treatment with the test item was 2.4 and, thus, lower than 3. Therefore, test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category).

Executive summary:

A BCOP (Bovine Corneal Opacity and Permeability Assay) according to OECD Guideline 437 following GLP was performed with the test item. The induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20% (w/v) suspension in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) imidazole was used. Three corneas were used per group (negative control, positive control or test item group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the suspended test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS).

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.1. Treatment with the positive control (20% Imidazole) revealed an IVIS of 98.0. Therefore, the study fulfilled the validity criteria. The IVIS obtained after treatment with the test item was 2.4 and, thus, lower than 3, i.e. according to UN GHS classification the test item is not requiring classification for eye irritation or serious eye damage.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

An in vitro study was performed according to OECD Guideline 439 (Reconstructed Human Epidermis Test) following GLP. The test consisted of a topical exposure of the test item to a human reconstructed skin model followed by a cell viability test. Cell viability was quantitatively measured by dehydrogenase conversion of MTT into a blue formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential. Triplicates of the human skin RHE™-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before adding the solid test item 10 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. Afterwards, 16 mg of the test item were applied to the tissues.

After treatment with the negative control (DPBS-buffer) the mean OD was 2.515. Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 0.83%. Therefore, the study fulfilled the validity criteria. The tissue viability after treatment with the test item was 78.43% and, thus, higher than 50%, i.e. according to UN GHS classification the test item is considered as non-irritant to skin.

Eye irritation

A BCOP (Bovine Corneal Opacity and Permeability Assay) according to OECD Guideline 437 following GLP was performed with the test item. The induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20% (w/v) suspension in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) imidazole was used. Three corneas were used per group (negative control, positive control or test item group). After a first opacity measurement of the untreated bovine corneas, 750 µL of the suspended test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS).

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.1. Treatment with the positive control (20% Imidazole) revealed an IVIS of 98.0. Therefore, the study fulfilled the validity criteria. The IVIS obtained after treatment with the test item was 2.4 and, thus, lower than 3, i.e. according to UN GHS classification the test item is not requiring classification for eye irritation or serious eye damage.

Justification for classification or non-classification

Based on the results of the key studies for skin and eye irritation, no classification for irritation is triggered in accordance with Regulation (EC) No 1272/2008.