Registration Dossier

Administrative data

Description of key information

- Not irritating to skin (GLPs and OECD 431 and OECD 439 compliant studies)

- No irritating to eyes (GLPs and OECD 437 compliant study)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 31 August 2015 to 20 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: MR92143651
- Expiration date of the lot/batch: 01 January 2017
- Purity: 86.4% (No correction was made for purity of the test substance)
- Purity test date: 18 June 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Yes, maximum temperature: 50°C, maximum duration: unknown (until liquefaction).
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was heated to 37°C, to obtain a homogeneous liquid sample.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A
Test system:
human skin model
Remarks:
Supplier: MatTek Corporation, Ashland MA, U.S.A.
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue batch numbers: 22660 and 22268
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 31 August 2015

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0°C
- Temperature of post-treatment incubation: N/A

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the exposure period, the tissues were washed with phosphate buffered saline.
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD value = 1.699 and 1.590 in batches 22660 and 22268, respectively (acceptance criteria: 1- Barrier function: ET-50 = 7.77 hrs and 8.71 hrs in batchs 22660 and 22268, respectively (acceptance criteria: 4.77 hrs- Morphology: not specified
- Contamination: No contamination in either batches.
- Reproducibility: not specified

NUMBER OF REPLICATE TISSUES: 4 tissues per test item together with a negative control and positive control.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues: freeze-killed tissues
- Procedure used to prepare the killed tissues (if applicable): Living epidermis was transferred to a freezer (≤-15°C), thawed, and then again transferred to (≤-15°C). The freeze-killed epidermis was stored at ≤ -15°C until use. Freeze-killed tissues were thawed by placing them for 1 hour at room temperature in a 6 well plate on 0.9 ml DMEM medium.
- N. of replicates : 2 tissues treated with test item and 2 negative control treated tissues.
- Method of calculation used: The net OD of the treated freeze-killed tissues was subtracted from the ODs of the test item treated viable tissues.
- Test for colour interference by the test item: 1-Phenyldecane-1,3-dione was checked for possible colour interference before the study was started. Some non-coloured test items may change into coloured items in aqueous conditions and thus stain the skin tissues during the 1-hour exposure. To assess the colour interference, 50 μl of 1-Phenyldecane-1,3-dione or 50 μl Milli-Q water as a negative control were added to 0.3 ml Milli-Q water. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0°C in the dark. At the end of the exposure time the mixture was shaken and it was checked if a blue / purple colour change was observed.
- Test for reduction of MTT by the test item: 1-Phenyldecane-1,3-dione was checked for possible direct MTT reduction before the study was started. To assess the ability of the test item to reduce MTT, 50 μl of 1-Phenyldecane-1,3-dione was added to 1 ml MTT (Sigma, Zwijndrecht, The Netherlands) solution (1 mg/ml) in phosphate buffered saline. The mixture was incubated for approximately 1 hour at 37.0 ± 1.0ºC. A negative control, sterile Milli-Q water was tested concurrently.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
- Justification for the selection of the cut-off point(s) if different than recommended in TG 431 and 439: N/A
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50µL
- Concentration (if solution): as such

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): as such

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL
- Concentration (if solution): as such
Duration of treatment / exposure:
Two tissues were used for a 3-minute exposure to 1-Phenyldecane-1,3-dione and two for a 1-hour exposure.
Duration of post-treatment incubation (if applicable):
N.A
Number of replicates:
2 per duration of treatment
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Relative mean tissue viability obtained after the 3-minute treatment
Value:
99
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Relative mean tissue viability obtaine after the 1-hour treatment
Value:
78
Vehicle controls valid:
not specified
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No specified
- Direct-MTT reduction: In addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each timepoint. The non-specific reduction of MTT by 1-Phenyldecane-1,3-dione was 0.6% and 4% of the negative control tissues after 3 minutes and 1 hour respectively.
- Colour interference with MTT: 1-Phenyldecane-1,3-dione was checked for colour interference in aqueous conditions and for possible direct MTT reduction by adding the test item to MTT medium. Because a colour change was observed by adding MTT-medium it was concluded that 1-Phenyldecane-1,3-dione did interact with the MTT endpoint.

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: The maximum inter-tissue variability in viability between two tissues treated identically was less than 26% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was less than 15% for the negative control and test item. For the positive control, the maximum inter-tissue variability in viability between two tissues treated identically was up to 31% and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues was up to 19%, however since the viabilities were below 20% the acceptability criteria were met. It was therefore concluded that the test system functioned properly.
- Range of historical values if different from the ones specified in the test guideline: N/A
Interpretation of results:
GHS criteria not met
Conclusions:
It is concluded that this test is valid and that 1-Phenyldecane-1,3-dione is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.
Executive summary:

This report describes the ability of 1-Phenyldecane-1,3-dione to induce skin corrosion on a human three dimensional epidermal model (EpiDerm (EPI-200)). The possible corrosive potential of 1-Phenyldecane-1,3-dione was tested through topical application for 3 minutes and 1 hour.

The study procedures described in this report were based on the most recent OECD guideline no. 431 and EC guideline B.40 Bis.

Batch MR92143651 of 1-Phenyldecane-1,3-dione is a Reddish solid below 25°C / liquid above 25°C with a purity of 86.4%. 1-Phenyldecane-1,3-dione was applied undiluted (50 μl) directly on top of the skin tissue.

The positive control had a mean relative tissue viability of 7% after 3 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The acceptability criteria for the maximum inter-tissue variability in viability between two tissues treated identically and the maximum difference in percentage between the mean viability of two tissues and one of the two tissues were met, indicating that the test system functioned properly.

1-Phenyldecane-1,3-dione did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal 3-minute and 1-hour procedure, two freeze-killed tissues treated with test item and two freeze-killed negative control treated tissues were used for the cytotoxicity evaluation with MTT at each timepoint. The non-specific reduction of MTT by 1-Phenyldecane-1,3-dione was 0.6% and 4% of the negative control tissues after 3 minutes and 1 hour respectively. The net OD of the treated freeze-killed tissues was subtracted from the ODs of the test item treated viable tissues.

Skin corrosion is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 3-minute and 1-hour treatments with 1-Phenyldecane-1,3-dione compared to the negative control tissues was 99% and 78%, respectively. Because the mean relative tissue viability for 1-Phenyldecane-1,3-dione was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment, 1-Phenyldecane-1,3-dione is considered to be not corrosive.

Finally, it is concluded that this test is valid and that 1-Phenyldecane-1,3-dione is not corrosive in the in vitro skin corrosion test under the experimental conditions described in this report.

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 13 October to 20 November 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
yes
Remarks:
The standard deviation of three tissues treated identically with the test item was 22% which is above the acceptance criteria of 18%. Since the test item shows a clearly negative result, this does not affect the study outcome.
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: MR92143651
- Expiration date of the lot/batch: 01 January 2017
- Purity: 86.4% (No correction was made for purity of the test substance)
- Purity test date: 18 June 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Yes, maximum temperature: 50°C, maximum duration: unknown (until liquefaction).
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was heated to 40 - 50°C until complete melting of the crystallized part before being used in liquid form.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A
Test system:
human skin model
Remarks:
EPISKIN Small ModelTM
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: donors (tissue not specified)
Source strain:
not specified
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM
- Tissue batch numbers: 15-EKIN-041, 15-EKIN-027 and 15-EKIN-030.
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 13 October 2015

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the tissues were washed with phosphate buffered saline to remove residual test item
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml
- Incubation time: 3 hours
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: not specified
- Barrier function: evaluated by the determination of IC50. IC50 = 2.1 , 2.3 and 2.0 mg/mL for batches 15-EKIN-041, 15-EKIN-027 and 15-EKIN-030 (specification >= 1.5 mg/mL)
- Morphology: satisfactory (Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
- Contamination: none (absence of bacteria, fungus and mycoplasma
- Reproducibility: not specified

NUMBER OF REPLICATE TISSUES: 3 tissues per test item together with negative and positive controls

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
1-Phenyldecane-1,3-dione was checked for possible direct MTT reduction and colour interference in the Skin corrosion test using EpiDerm as a skin model (project 510207). Because solutions did turn blue / purple and/or a blue / purple precipitate was observed it was concluded that 1-Phenyldecane-1,3-dione did interfere with the MTT endpoint.
- Fresh tissues / killed tissues: yes
- Procedure used to prepare the killed tissues: Living epidermis was transferred to 12 well plates and incubated with 2 ml Milli-Q for 48 ± 1 hours. After incubation, killed epidermis was stored at ≤ -15°C. Killed tissues were thawed by placing them for 1 hour at room temperature in 12 well plates on 2 ml maintenance medium.
- N. of replicates : 3
- Method of calculation used: The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.

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

PREDICTION MODEL / DECISION CRITERIA:
A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL, NEGATIVE AND POSITIVE CONTROLS:
- Amount(s) applied (volume or weight with unit): 25µL
- Concentration (if solution): as such
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Value:
105
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: The non-specific reduction of MTT by 1-Phenyldecane-1,3-dione was 14% of the negative control tissues.
- Colour interference with MTT: yes. Because solutions did turn blue / purple and/or a blue / purple precipitate was observed it was concluded that 1-Phenyldecane-1,3-dione did interfere with the MTT endpoint

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The in vitro skin irritation test is considered acceptable if it meets the following criteria:
a) The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18.
b) The mean relative tissue viability of the positive control should be ≤50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18.
c) The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18.
d) The non-specific MTT reduction should be ≤ 30% relative to the negative control OD.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range
- Acceptance criteria met for positive control: Yes: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 12%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was less than 14% for the negative and positive control. The standard deviation value of the percentage viability of three tissues treated identically with the test item was less than 22%, however since the test item was clearly negative this does not influence the study outcome (see deviation).
Interpretation of results:
GHS criteria not met
Conclusions:
It is concluded that this test is valid and that 1-Phenyldecane-1,3-dione is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate the ability of 1-Phenyldecane-1,3-dione to induce skin irritation on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SMTM)). The possible skin irritation potential of 1-Phenyldecane-1,3-dione was tested through topical application for 15 minutes.

The study procedures described in this report were based on the most recent OECD guideline no. 439 and EC guideline B.46.

Batch MR92143651 of 1-Phenyldecane-1,3-dione was a reddish solid below 25°C / liquid above 25°C with a purity of 86.4%. 1-Phenyldecane-1,3-dione was applied undiluted (25 μl) directly on top of the skin tissue for 15 ± 0.5 minutes. After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment.

1-Phenyldecane-1,3-dione did interact with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). In addition to the normal procedure, three killed tissues treated with test item and three killed non-treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by 1-Phenyldecane-1,3-dione was 14% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.

Skin irritation is expressed as the remaining cell viability after exposure to the test item. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with 1-Phenyldecane-1,3-dione compared to the negative control tissues was 105%. Since the mean relative tissue viability for 1-Phenyldecane-1,3-dione was above 50% after 15 ± 0.5 minutes treatment 1-Phenyldecane-1,3-dione is considered to be non-irritant.

The positive control had a mean cell viability of 12% after 15 ± 0.5 minutes exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was less than 14% for the negative and positive control, indicating that the test system functioned properly.

Finally, it is concluded that this test is valid and that 1-Phenyldecane-1,3-dione is non-irritant in the in vitro skin irritation test under the experimental conditions described in this report.

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:
From 8 September to 26 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: MR92143651
- Expiration date of the lot/batch: 01 January 2017
- Purity: 86.4% (No correction was made for purity of the test substance)
- Purity test date: 18 June 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Yes, maximum temperature: 50°C, maximum duration: unknown (until liquefaction).
- Solubility and stability of the test substance in the solvent/vehicle: N/A
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Before use the test substance was warmed in a 45°C water bath to obtain a homogeneous sample..
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands)
- Number of animals: not specified
- Characteristics of donor animals (e.g. age, sex, weight): not specified
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing: The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)) and incubated for the minimum of 1 hour at 32 +/- 1°C.
- indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
- Indication of any antibiotics used: no
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL, NEGATIVE AND POSITIVE CONTROLS:
- Amount(s) applied (volume or weight with unit): 750 µl
- Concentration (if solution): as such

Duration of treatment / exposure:
10 +/- 1 minutes at 32 +/- 1°C
Duration of post- treatment incubation (in vitro):
120 +/- 10 minutes at 32 +/- 1°C in fresh cMEM medium.
Number of animals or in vitro replicates:
triplicates
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1°C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1°C.
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations will be performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea will be read against a cMEM filled chamber, and the initial opacity reading thus determined will be recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

QUALITY CHECK OF THE ISOLATED CORNEAS: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: physiological saline (Eurovet Animal Health, Bladel, The Netherlands)

SOLVENT CONTROL USED (if applicable): N/A

POSITIVE CONTROL USED: 10% (w/v) Benzalkonium Chloride (Merck KGaA, Darmstadt, Germany) [CAS Number 63449-41-2] solution prepared in physiological saline.

APPLICATION DOSE AND EXPOSURE TIME: 750 µL for 10 +/- 1 minutes at 32 +/- 1°C

TREATMENT METHOD: not specified

POST-INCUBATION PERIOD: yes. If YES please specify duration: 120 +/- 10 minutes at 32 +/- 1°C

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Eagle’s Minimum Essential
Medium, Life Technologies) and thereafter with cMEM .

- POST-EXPOSURE INCUBATION: see above

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microplate reader (TECAN Infinite® M200 Pro Plate Reader) (OD490)
- Others (e.g, pertinent visual observations, histopathology): Each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used: Yes
Irritation parameter:
in vitro irritation score
Value:
>= -0.3 - <= 1.4
Vehicle controls valid:
not examined
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (IVIS ranged from -2.8 to 0.9 so < 3). The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas.
- Acceptance criteria met for positive control: yes (IVIS ranged from 108 to 143 so > 55). The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 120 and was within two standard deviations of the current historical positive control mean.
- Range of historical values if different from the ones specified in the test guideline: Negative control: IVIS ranged from -3.1 to 2.3; Positive control: IVIS ranged from 80.5 to 186.1.
Interpretation of results:
GHS criteria not met
Conclusions:
1-Phenyldecane-1,3-dione induced an IVIS ≤ 3 in this BCOP test therefore no classification is required for eye irritation or serious eye damage.
Executive summary:

The aim of this study was to evaluate the eye hazard potential of 1-Phenyldecane-1,3-dione using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the potency of 1-Phenyldecane-1,3-dione to induce serious eye damage after topical application for 10 minutes on isolated bovine corneas. . The study procedures described in this report were based on OECD guideline no. 437 and EC guideline B.47 and performed in compliance with Good Laboratory Practices.

Batch MR92143651 of 1-Phenyldecane-1,3-dione was a reddish solid below 25°C / liquid above 25°C with a purity of 86.4%. The test item was applied as it is (750 μl) directly on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (10% (w/v) Benzalkonium Chloride) was 120 and was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

1-Phenyldecane-1,3-dione did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score (IVIS) of 0.6 after 10 minutes of treatment.

Since 1-Phenyldecane-1,3-dione induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage according to Globally Harmonized System of Classification and Labelling of Chemicals (GHS) and Regulation (EC) No 1272/2008.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Two Klimisch score 1 studies are available to assess the skin corrosive and irritating potential and were used as key studies study:

In the first one, performed according to OECD guideline no. 431 and in compliance with GLPs, the possible corrosive potential of 1-Phenyldecane-1,3-dione was tested through topical application for 3 minutes and 1 hour on a human three dimensional epidermal model (EpiDerm (EPI-200)) .

The relative mean tissue viability obtained after 3-minute and 1-hour treatments with 1-Phenyldecane-1,3-dione compared to the negative control tissues was 99% and 78%, respectively. Because the mean relative tissue viability for 1-Phenyldecane-1,3-dione was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment, 1-Phenyldecane-1,3-dione is considered to be not corrosive.

In the second study, performed according to OECD guideline no. 439 and in compliance with GLPs, the possible irritating potential of 1-Phenyldecane-1,3-dione was tested through topical application for 15 minutes and 1 hour on a human three dimensional epidermal model (EPISKIN Standard model (EPISKIN-SMTM)).

The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with 1-Phenyldecane-1,3-dione compared to the negative control tissues was 105%. Since the mean relative tissue viability for 1-Phenyldecane-1,3-dione was above 50% after 15 ± 0.5 minutes treatment, 1-Phenyldecane-1,3-dione is considered to be non-irritant.

 

One Klimisch score 1 study is available to assess the eye corrosive and irritating potential of the registered substance and was used as key study:

In this study, performed according to OECD guideline no. 437 (BCOP test) and in compliance with GLPs, 1-Phenyldecane-1,3-dione was applied for 10 minutes as it is (750 μl) on isolated bovine corneas. 1-Phenyldecane-1,3-dione did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score (IVIS) of 0.6 after 10 minutes of treatment. Since 1-Phenyldecane-1,3-dione induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

Justification for classification or non-classification

In an in vitro skin corrosion (OECD 431) and in vitro skin irritation (OECD 439) tests performed using a reconstructed human epidermis model, the mean relative cell viability following exposure with the registered substance was above the threshold of 50% compared to the negative control, respectively, indicating that the substance is not corrosive nor irritant to skin.

In addition, in a Bovine Corneal Opacity and Permeability test (OECD 437), the mean In Vitro Irritancy Score was below the threshold of 3 indicating that the substance is not corrosive nor irritant to eyes.

In conclusion the registered substance is not classified for skin and eye irritation/corrosion according to CLP and GHS-UN regulations.