Registration Dossier

Administrative data

Description of key information

Skin irritation: Key study: OECD 439 and EU method B.46. GLP study. The mean corrected percent viability of the treated tissues was 55.9% versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item has to be considered as non-irritant to skin.

Eye irritation: Weight of evidence. Based on the available information, the test item is not irritating to the eye.

Weight of evidence: OECD 438 and EU B.48. GLP study. The combination of the 3 endpoints for the test item was 1 x III, 2 x I, according to OECD 438. Therefore, no prediction can be made.

Weight of evidence: OECD 437. GLP study. Under the experimental conditions of this study, the test item has been identified as not irritant to eye (i.e. IVIS was 2), not requiring classification for eye irritation or serious eye damage (UN GHS No Category).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Remarks:
Reconstructed Human Epidermis Test Method
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 1, 2017 - February 16, 2017
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes
Test system:
human skin model
Remarks:
SkinEthic™ RHE model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
not specified
Justification for test system used:
The SkinEthic™ RHE model has been validated for irritation testing (Validation study bsed on the original ECVAM Performace Standards (21) in 2008) and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439), therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Remarks:
The test item was applied as supplied
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE model
- Tissue batch number(s): 17-RHE-017
- Delivery date: 14 February 2017
- Expiration date: 20 February 2017
- Date of initiation of testing: 14 February 2017

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µl of a MTT solution at 1.0 mg/mL
- Incubation time: 2 h 58 min at 37ºC, 5% CO2
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm
- Linear OD range of spectrophotometer: The linearity range of optical density measured is validated for an optical density between 0 and 2.0.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD = 1.3 (CV=4.1%) specification OD > 0.7. Historical negative control mean OD range = 0.834 - 1.574
- Barrier function: 4.7 h (Specification 4 h < ET50 < 10 h)
- Morphology: 5.5 cell layers, absence of significant histological abnormalities, well differentiated epidermis (Specification > 4)
- Contamination: no

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE. no interference

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 (32 mg/cm2)

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 µL
- Concentration (if solution): 5% SDS
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
42 hours and 05 minutes.
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
55.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
1.5% tissue viability
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
77.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
1.5% tissue viability
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
45.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
1.5% tissue viability
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
44.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
1.5% tissue viability
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The mean corrected percent viability of the treated tissues was 55.9%, versus 1.5% in the positive control (5% Sodium Dodecyl sulfate). However, the results obtained on the three epidermises treated with the test item were heterogeneous: 77.6%, 45.3% and 44.9% (two epidermises classified as irritant and one epidermis classified as non irritant, leading to a mean viavility at 55.9% with a standard deviation at 18.8%).

- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: A cloudy yellow solution was observed after 3 hours of incubation between 36.3ºC and 37.8ºC, 5% CO2. Therefore, there is no direct interaction between the test item and MTT.
- Colour interference with MTT:
In water: A colourless solution at the bottom of the well was obtained after 3 hours of incubation between 36.3ºC and 37.8ºC, 5% CO2
In isopropanol: A colourless solution was obtained after 2 hours of incubation at room temperature. Therefore, the test item will not interfere with the MTT assay and there is no need to add nonspecific coloration controls to the study.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes,a full demonstration of proficiency was performed with Episkin-SM model, plus a reduced validation with SkinEthic RHE model. Adequate results were obtained for the evaluated chemicals. Summary of proficiency chemicals tested according to OECD 439 criteria included in the report.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the standard deviation of the cell viability for the epidermises treated with the test item was 18.8%, instead of 18% as initially scheduled. This deviation is considered as without impact on the conclusion of the test. The mean OD of the negative control is 0.819 and the acceptability criteria is OD>0.6x<1.5
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: the results are heterogeneous on the three epidermises (two classified as irritant and one classified as non-irritant). A second run should be performed to infirm or confirm the results.

The results were expressed as a viability percentage compared with the negative control:

 

Viability %= ODtest item/ ODnegative controlx 100

 

The OD values obtained for each test simple were used to calculate a percentage of viability relative to the negative control, which was arbitrarily set at 100%.

 

Table 1. Individual and average values of OD after 42 minutes exposure

 

 

Skin

OD

Mean OD / disc (#)

Mean OD / product

Viability %

Mean viability %

SD

Conclusion

Negative control

1

0.827

0.811

0.819

99.0

100.0

1.1

 

0.800

0.806

2

0.789

0.829

101.2

0.843

0.857

3

0.812

0.818

99.8

0.823

0.820

Positive control

4

0.015

0.015

0.013

1.8

1.5

0.3

Irritant

0.015

0.015

5

0.012

0.011

1.3

0.012

0.011

6

0.012

0.012

1.5

0.013

0.012

Test item PH-16/0670

25

0.612

0.636

0.458

77.6

55.9

18.8

Non irritant

0.677

0.621

26

0.373

0.371

45.3

0.378

0.364

27

0.366

0.368

44.9

0.376

0.364

# mean of 3 values (triplicate of the same extract)

OD: optival density

Acceptability criteria: SD18%

 

Notes:

           

- If the viability obtained for the test item is greater than 50%, the test item has to be considered as non irritant.

- If the viability obtained for the test item is less than or equal to 50%, the test item has to be considered as irritant.

Interpretation of results:
GHS criteria not met
Conclusions:
The mean corrected percent viability of the treated tissues was 55.9% versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item has to be considered as non-irritant to skin.
Executive summary:

The evaluation of the possible irritating effects of the test item has been tested after topical application on in vitro human reconstructed epidermis (SkinEthic RHE model) in accordance with OECD 439 and EU method B.46, following GLP. The test item was applied, as supplied, at a dose of 16 mg to 3 living reconstructed Human epidermis during 42 minutes, followed by a rinse with 25 mL of DPBS and a 42 h and 5 min post-incubation period at 37ºC, 5% CO2. Cell viability was measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. Positive and negative controls were run in parallel. All acceptability criteria were met. The mean corrected percent viability of the treated tissues was 55.9%, versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate). However, the results obtained on the three epidermises treated with the test item were heterogeneous: 77.6%, 45.3% and 44.9% (two epidermises classified as irritant and one epidermis classified as non irritant, leading to a mean viability at 55.9% with a standard deviation at 18.8%).

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
July 3, 2017 - August 24, 2017
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes
Species:
cattle
Strain:
other: Bos taurus (bovine cattle)
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source:freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
- Characteristics of donor animals: up to 12 months old, typically, 5 to 8 months old (French Authorities avoid the use of any organs from the head of bovines aged more than 12 months).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Transported at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.
- Time interval prior to initiating testing: Upon arrival to the laboratory, preparation and selection was performed immediately.The corneas were used within a maximum of 24 hours.
- indication of any existing defects or lesions in ocular tissue samples:No - A macroscopic examination was performed on all eyes to detect the presence of any defects.
- Indication of any antibiotics used:Antibiotic used during transport in buffered Hanks medium [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)].
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 (± 8 µL) applied on each cornea.
- Concentration (if solution) :20% (w/v) in 0.9% NaCl (w/v)

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 µL (± 8 µL)

Duration of treatment / exposure:
4 hours (± 5 minutes)
Duration of post- treatment incubation (in vitro):
As the test item is solid no post-exposure incubation was required.
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
Any eyes with defects were discarded. The examination of defects was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. The tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared. The corneas were washed for 15 minutes, three times, in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature. The corneas were used within a maximum of 24 hours. As the corneas were not used immediately, they were stored after washing. Each cornea was stored individually in 12 mL of M199 medium containing 5% dextran, plus penicillin/streptomycin, at +4°C, for a maximum of 24 hours before use.

QUALITY CHECK OF THE ISOLATED CORNEAS: A pre-incubation was performed before treatment aplication.Both chambers of the corneal holder were filled with MEM culture media supplemented with 1% fetal bovine serum plus penicillin/streptomycin (cMEM) at room temperature. After making sure that no air bubbles were present within the holder, it was immersed in a water bath, horizontally (cornea positioned vertically), up to approximately three quarters of its height. The holders were pre incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C (± 1°C). At the end of the pre-incubation period, the corneas were examined macroscopically through the holder to detect the presence of any defects. Then, the opacity of the cornea was measured to obtain OPT0. Corneas that showed any macroscopic defect or an OPT0 value over 7 were discarded.

NUMBER OF REPLICATES:3

SOLVENT CONTROL USED (if applicable): Yes, NaCl 0.9%

POSITIVE CONTROL USED: Yes, 20% imidazole solution in 0.9% NaCl (w/w).

APPLICATION DOSE AND EXPOSURE TIME: 750 µL (± 8 µL) of 20% (w/w) in 0.9% NaCl (w/w), 4 hours exposure.

TREATMENT METHOD: The open-chamber method was used for the test item to ensure uniform spreading of the test item formulation over the corneas. The closed chamber was used for the vehicle/positive controls.
The medium of the anterior chamber was removed and the each item was applied onto the epithelium of the cornea. The treatment time of each series of three corneas was carefully measured with a chronometer. After application of the items, the holders were incubated vertically (cornea positioned horizontally with the treated side uppermost) in a water bath at +32°C (± 1°C), for 4h.

POST-INCUBATION PERIOD:No

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: The test item formulation was removed with a cotton bud and a pipette of heated cMEM (32°C).The corneas were rinsed four times with pre-warmed cMEM containing phenol red until it was visually confirmed by observing the transparency and the colour changing of the rinsing medium (containing phenol red).Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.

- POST-EXPOSURE INCUBATION:No

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: An opacitometer (OPKIT opacitometer and calibrators (MC2, Clermont-Ferrand, France)) was used to measure light transmission (the level of opacity) through the center of each mounted cornea. The change in opacity for each cornea treated with test item, vehicle control or positive control was calculated by subtracting the initial baseline opacity measurement (OPT0) from the post-treatment opacity reading (OPT2). The average change in opacity for the corneas treated with the vehicle control was calculated and this value was subtracted from the change in opacity for each cornea treated with test item formulation or positive control to obtain a corrected opacity value (cOPT). When the average change in opacity for the corneas treated with the vehicle control was negative, it is considered equal to 0. The mean cOPT value of each series of three corneas was calculated from the individual corrected opacity values.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of UV/VIS spectrophotometry Cary 100 (OD490). The corrected OD490 nm value (cOD490 nm) for each cornea treated with either test item or positive control was calculated subtracting the average vehicle control cornea OD490 nm value from the original OD490 nm value of each cornea. The mean cOD490 nm value of each series of three corneas was calculated from the individual cOD490 nm values.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS). The following formula was used to determine the In Vitro Irritancy Score (IVIS): IVIS = cOPT + (15 x cOD490 nm)
The IVIS was calculated for each test item and positive control cornea. The mean IVIS for each series of three corneas was calculated from the individual scores.

DECISION CRITERIA: as indicated in the TG.
IVIS UN GHS
If the test item induces an IVIS ≤ 3 No category
If the test item induces an 3 < IVIS ≤ 55 No prediction can be made
If the test item induces an IVIS > 55 Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
ca. 2
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
Mean
Value:
ca. 1.7
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No.
- Fluorescein fixation was observed on the three test item-treated corneas.

DEMONSTRATION OF TECHNICAL PROFICIENCY:Yes, full validation dossier is available in laboratory GLP archive for review or inspection purpose.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Criteria were fulfilled, mean OD490 nm of the vehicle control (OD490 = 0.039) fall within the established upper limit of historical data (mean OD490 <=0.043). The mean opacity (1.7) fall within the acceptable range: opacity <= 4.

- Acceptance criteria met for positive control: Yes, the mean In Vitro Irritancy Score (IVIS) of the positive control corneas (163) should fall within two standard deviations of the historical mean (mean+2SD=168.85, acceptable range 113-169).

Due to that the test item induces an IVIS of 2.0 (this is ≤ 3), the test item has no irritation potential to eye. Please see "Any other information on results incl. tables" for more details about individual values.

Table 1. Results

Vehicle control

OPACITY

PERMEABILITY

Holder

OPT0

OPT2

OPT2-OPT0

Neg correction

OD 490 nm

Neg correction

87

2

1

-1

0

0.023

0.023

73

2

1

-1

0

0.041

0.041

51

2

1

-1

0

0.054

0.054

Mean

 

 

 

0.0

 

0.039

SD

 

 

 

0.0

 

0.016

 

Test item

OPACITY

PERMEABILITY

Holder

OPT0

OPT2

OPT2-OPT0

Neg correction

cOPT

OD 490 nm

Neg correction

cOD 490 nm

IVIS

65

1

2

1

1

1

0.026

0.026

0.000

1

79

2

4

2

2

2

0.029

0.029

0.000

2

72

2

4

2

2

2

0.035

0.035

0.000

2

Mean

 

 

 

 

1.7

 

 

0.000

2

SD

 

 

 

 

0.6

 

 

0.000

0.6

 

Positive control

OPACITY

PERMEABILITY

Holder

OPT0

OPT2

OPT2-OPT0

Neg correction

cOPT

OD 490 nm

Neg correction

cOD 490 nm

IVIS

85

1

116

115

115

115

3.276

3.276

3.237

164

53

1

109

108

108

108

3.400

3.400

3.361

158

74

2

84

82

82

82

5.680

5.680

5.641

167

Mean

 

 

 

 

101.7

 

 

4.079

163

SD

 

 

 

 

17.4

 

 

1.354

4.1

 

np- not performed

OD: optical density

cOD-optical density corrected by mean OD value of vehicle control

cOPT: corneal opacity corrected by mean opacity value of vehicle control

OPT0: corneal opcity before treatment

OPT2:corneal opacity after treatment

Neg. correction: all individual values below 0 are set at 0

SD: standard deviation

IVIS: in vitro irritation score

Vehicle control: 0.9% NaCl

Positive control: 20% imidazole solution in 0.9% NaCl

Interpretation of results:
GHS criteria not met
Remarks:
No irritating
Conclusions:
Under the experimental conditions of this study, the test item has been identified as not irritant to eye (IVIS was 2), not requiring classification for eye irritation or serious eye damage (UN GHS No Category).
Executive summary:

An in vitro (ex vivo) Bovine Corneal Opacity and Permeability study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 437, following GLP. Corneas were obtained from freshly slaughtered calves. A single experiment was performed using three corneas for each treated series (test item, positive control and vehicle control). Before treatment, opacity measurements were conducted. Then, the test item was applied at the concentration 20% (w/v) in the vehicle (0.9% NaCl), using a treatment time of 4 hours and the open-chamber method. Vehicle and positive controls were applied using the same treatment time and using the closed-chamber method. After exposure, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. Then the opacity cornea was measured. On the other hand, for measureing the permeability a fluorescein solution was used and the holders were incubated. After this procedure, the OD490 nm was measured and the corneas were examined for irregularities. The mean corneal opacity and the mean IVIS for the test item was 2. Fluorescein fixation was observed on the three test item-treated corneas. All validity criteria were fulfilled and the study was considered valid. Therefore test item is considered not irritating to eye (no category).

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 1, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye 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
Qualifier:
according to guideline
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: yes
Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Slaughterhouse (Etablissement Brun, 33820 Etauliers, France), the chickens were killed for human consumption
- Characteristics of donor animals: spring chickens (approximately 7 weeks old, 1.5 - 2.5 kg)
- Storage, temperature and transport conditions of ocular tissue: Heads were removed immediately after sedation of the chickens by electric shock, and incision of the neck for bleeding. Intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline.
- Time interval prior to initiating testing: The head collection was at 8:35 am and arrived at 9:45 am at the laboratory.
- indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: No
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg
Duration of treatment / exposure:
10 seconds
Duration of post- treatment incubation (in vitro):
4 hours
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
Dissection of the eyes: the eyelids were carefully excised and the eye was further dissected from the skull, taking care not to damage the cornea. The eyeball was pulled from the orbit by holding the nictitating membrane firmly with surgical forceps, and the eye muscles were cut with a bent, blunt-tipped scissor. When the eye is removed from the orbit, a visible portion of the optic nerve should be left attached. Once removed from the orbit, the eye was placed on an absorbent pad and the nictitating membrane and other connective tissue were cut away.
Selection of the eyes: The enucleated eye was mounted in a stainless steel clamp with the cornea positioned vertically. The clamp was then transferred to a chamber of the superfusion apparatus. The clamps were positioned in the superfusion apparatus such the entire cornea was supplied with the pysiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 32.3ºC and 32.7ºC. After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damage during the dissection procedure using sodium fluorescein. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of >0.5; (ii) corneal opacity>0.5, or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.

EQUILIBRATION AND BASELINE RECORDINGS
The eyes examined and approved were incubated between 45 and 60 minutes to equilibrate them to the test system prior to dosing. Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baselin (i.e. time = 0). The fluorescein score determined at dissection was used as the baseline measurement for that endpoint.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 30 µL Physiological saline - Dutscher Batch No. 3011922 (1 replicate)

POSITIVE CONTROL USED: 30 mg Sodium hydroxide - Sigma Batch No. MKBP7805V (3 replicates)

APPLICATION DOSE AND EXPOSURE TIME
30 mg of the test item was applied for 10 seconds

OBSERVATION PERIOD
Pretreatment and at 30, 75, 120, 180, and 240 minutes (± 5 minutes) after the post-treatment rinse.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL of physiological saline at ambient temperature. As test item remained on the cornea despite the rinsing, one additional rinse was performed with 10 mL of physiological saline.
- Indicate any deviation from test procedure in the Guideline: no

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: It was measured using a HaagStreit BP900 slit-lamp microscope with depth-measuring device no. I. It was calculated using the area of the cornea that was most densely opacified for scoring.
- Damage to epithelium based on fluorescein retention: It was calculated for the 30-minute observation time point only, which was used for the overall category score given for each test or control item.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: It was measured using a HaagStreit BP900 slit-lamp microscope with depth-measuring device no. I, the slit-width was set at 91/2, equalling 0.095 mm. The mean percentage of corneal swelling was calculated for all observation time points.
- Macroscopic morphological damage to the surface: include "pitting" of corneal epithelial cells, "loosening" of epithelium, "roughening" of the corneal surface and "sticking" of the test item to the cornea. The classification of these findings is subjective according to the interpretation of the investigator.

SCORING SYSTEM:
- Mean corneal swelling (%): (corneal thickness at time t -corneal thickness at time=0/corneal thickness at time=0)x100.
- Mean maximum opacity score: 0= No opacity; 0.5= Very faint opacity; 1= Scattered or diffuse areas, details of the iris clearly visible; 2= Easlily discernible translucent area, details of the iris are slightly obscured; 3= Severe corneal opacity, no specific details of the iris are visible, size of the pupil is barely discernible; 4= Complete corneal opacity, iris invisible.
- Mean fluorescein retention score at 30 minutes post-treatment: 0= No fluorescein retention; 0.5= Very minor single cell staining; 1= Single cell staining scattered throughout the treated area of the cornea; 2= Focal or confluent dense single cell staining; 3= Confluent large areas of the cornea retaining fluorescein.

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used. All endpoints were evaluated separately to generate an ICE class for each endpoint and then combined to generate an Irritancy Classification for the test item. The ICE classes were assigned based on a predetermined range.
ICE classification criteria for corneal thickness: 0 to 5 = class I; >5 to 12= class II; >12 to 18 (>75 min after treatment)=class II; >12 to 18 (≤75 min after treatment)=class III;>18 to 26= class III; >26 to 32 (>75 min after treatment)= class III; >26 to 32 (≤75 min after treatment)= class IV; >32=class IV.
ICE classification criteria for opacy: 0.0-0.5 = class I; 0.6-1.5= class II; 1.6-2.5 =class III; 2.6-4.0=class IV.
ICE classification criteria for mean fluorescein retention: 0.0-0.5 = class I; 0.6-1.5= class II; 1.6-2.5 =class III; 2.6-3.0=class IV.

Overall in vitro classifications:
No category= 3 x I / 2 x I, 1 x II
No prediction can be made= Other combinations
Category 1 (Corrosive/Severe Irritant)= 3 x IV / 2 x IV, 1 x III / 2 x IV, 1 x II / 2 x IV, 1 x I / Corneal opacity ≥ 3 at 30 min (in at least 2 eyes) / Corneal opacity = 4 at any time point (in at least 2 eyes) / Severe loosening of the epithelium (in at least 1 eye)
Irritation parameter:
cornea opacity score
Run / experiment:
Maximal mean
Value:
ca. 0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE calss I
Irritation parameter:
fluorescein retention score
Run / experiment:
Mean
Value:
ca. 2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE class III
Irritation parameter:
percent corneal swelling
Run / experiment:
Maximal mean
Value:
ca. 2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE class I
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No. No morphological effects were noted, whatever the examination time.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Yes, 12 of the 13 chemicals tested obtained the same category as OECD 438. There was a borderline chemical (DMSO) over-classified ("No prediction can be made" vs. "No Category") in three separated tests. It should be tested with other adequately validated in vitro test.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the combination of the 3 endpoints was 3 x I and classified as "No category"
- Acceptance criteria met for positive control: Yes, the combination of the 3 endpoints was 3 x IV and classified as "Corrosive/Severe Irritant"

Table 1. Individual and average values for evaluation of corneal lesions after treatment with the negative control (Physiological saline)

Endpoint measured

Eye No.

Time (min)

0

30

75

120

180

240

Corneal opacity

13

0.0

0.0

0.0

0.0

0.0

0.0

ICE class

I

Fluorescein retention

13

0.5

0.5

-

-

-

-

ICE class

 

I

-

-

-

-

Corneal thickness

13

0.60

0.60

0.60

0.60

0.60

0.60

Corneal swelling (%)

13

-

0

0

0

0

0

ICE class

I

Combination of the 3 endpoints

3 x I

CLASSIFICATION

No Category

Note: No morphological effects were noted, whatever the examination time.

 

 

Table 2. Individual and average values for evaluation of corneal lesions after treatment with the positive control (5% Benzalkonium chloride)

 

Endpoint measured

Eye No.

Time (min)

0

30

75

120

180

240

Corneal opacity

1

0

4

4

4

4

4

2

0

4

4

4

4

4

3

0

4

4

4

4

4

Mean

0.0

4.0

4.0

4.0

4.0

4.0

ICE class

IV

Fluorescein retention

1

0.5

3

-

-

-

-

2

0.5

3

-

-

-

-

3

0.5

3

-

-

-

-

Mean

0.5

3.0

-

-

-

-

ICE class

 

IV

-

-

-

-

Corneal thickness

1

0.59

(-)

(-)

(-)

(-)

(-)

2

0.56

(-)

(-)

(-)

(-)

(-)

3

0.56

(-)

(-)

(-)

(-)

(-)

Corneal swelling (%)

1

(-)

(-)

(-)

(-)

(-)

(-)

2

(-)

(-)

(-)

(-)

(-)

(-)

3

(-)

(-)

(-)

(-)

(-)

(-)

Mean

-

-

-

-

-

-

ICE class

IV

Combination of the 3 endpoints

3 x IV

CLASSIFICATION

Category 1: Corrosive/Severe irritant

Note:

 (-): evaluation of corneal swelling not possible (Corneal opacity=4 at each examination time, leading to a marked refraction of the light preventing from the evaluation of the corneal swelling with the biomicroscope).

Severe loosening of the corneal epithelium noted from 30 minutes post-dose in eyes No. 1, 2 & 3.

 

 

Table 3. Individual and average values for evaluation of corneal lesions after treatment with the test item.

 

Endpoint measured

Eye No.

Time (min)

0

30

75

120

180

240

Corneal opacity

7

0

0

0

0

0

0

8

0

0

0

0

0

0

9

0

0

0

0

0

0

Mean

0.0

0.0

0.0

0.0

0.0

0.0

ICE class

I

Fluorescein retention

7

0.5

2

-

-

-

-

8

0.5

2

-

-

-

-

9

0.5

2

-

-

-

-

Mean

0.5

2.0

-

-

-

-

ICE class

 

III

-

-

-

-

Corneal thickness

7

0.57

0.58

0.58

0.58

0.58

0.58

8

0.59

0.59

0.60

0.62

0.62

0.62

9

0.60

0.60

0.60

0.60

0.60

0.60

Corneal swelling (%)

7

-

2

2

2

2

2

8

-

0

2

5

5

5

9

-

0

0

0

0

0

Mean

-

1

1

2

2

2

ICE class

I

Combination of the 3 endpoints

1 x III, 2 x I

CLASSIFICATION

No prediction can be made

Note: No morphological effects were noted, whatever the examination time.

Interpretation of results:
study cannot be used for classification
Remarks:
No prediction can be made
Conclusions:
The combination of the 3 endpoints fot the test item was 1 x III, 2 x I, according to OECD 438. Therefore, no prediction can be made.
Executive summary:

The eye irritation of the test item has been evaluated using the tests Isolated Chicken Eye test method, according to OECD 438 and EU method B.48, following GLP. The test item was applied, as supplied, at the dose of 30 mg to 3 enucleated chicken eyes during 10 seconds. Then the eyes were rinsed three times with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control (sodium hydroxide) and one eye with a negative control (physiological saline). Damages by the tests item were assessed by determination of corneal swelling, opacity, and fluorescein retention were evaluated pretreatment and at 30, 75, 120, 180 and 240 minutes post-dose. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438. Positive control and negative control results were valid. The mean corneal opacity for the test item treated corneas was 0, corresponding to ICE category class I; the mean score of fluoroscein retention was 2, corresponding to ICE category class III; and the mean corneal swelling was 2%, corresponding to ICE class I. The combination of the three endpoints for the test item was 1 x III, 2 x I. Therefore, no prediction can be made.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation: Key study. The evaluation of the possible irritating effects of the test item has been tested after topical application on in vitro human reconstructed epidermis (SkinEthicRHE model) in accordance with OECD 439 and EU method B.46, following GLP. The test item was applied, as supplied, at a dose of 16 mg to 3 living reconstructed Human epidermis during 42 minutes, followed by a rinse with 25 mL of DPBS and a 42 h and 5 min post-incubation period at 37ºC, 5% CO2. Cell viability was measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues. Positive and negative controls were run in parallel. All acceptability criteria were met. The mean corrected percent viability of the treated tissues was 55.9%, versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate). However, the results obtained on the three epidermises treated with the test item were heterogeneous: 77.6%, 45.3% and 44.9% (two epidermises classified as irritant and one epidermis classified as non irritant, leading to a mean viability at 55.9% with a standard deviation at 18.8%).

Eye irritation: A first study was conducted with the test item according to Isolated Chicken Eye Test Method and the results indicate that no prediction can be made.

Weight of evidence: The eye irritation of the test item has been evaluated using the tests Isolated Chicken Eye test method, according to OECD 438 and EU method B.48, following GLP. The test item was applied, as supplied, at the dose of 30 mg to 3 enucleated chicken eyes during 10 seconds. Then the eyes were rinsed three times with 10 mL of physiological saline. Three eyes were treated in the same manner with a positive control (sodium hydroxide) and one eye with a negative control (physiological saline). Damages by the tests item were assessed by determination of corneal swelling, opacity, and fluorescein retention were evaluated pretreatment and at 30, 75, 120, 180 and 240 minutes post-dose. Fluorescein retention, corneal opacity and corneal swelling were evaluated, then the results of each endpoint were assigned to ICE classes according to OECD guideline 438. Positive control and negative control results were valid. The mean corneal opacity for the test item treated corneas was 0, corresponding to ICE category class I; the mean score of fluorescein retention was 2, corresponding to ICE category class III; and the mean corneal swelling was 2%, corresponding to ICE class I. The combination of the three endpoints for the test item was 1 x III, 2 x I. Therefore, no prediction can be made.

Due to the inconclusive results from the first test performed, another in vitro study for this endpoint has been considered. The Bovine Corneal Opacity and Permeability study has been conducted.

Weight of evidence: An in vitro (ex vivo) Bovine Corneal Opacity and Permeability study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 437, following GLP. Corneas were obtained from freshly slaughtered calves. A single experiment was performed using three corneas for each treated series (test item, positive control and vehicle control). Before treatment, opacity measurements were conducted. Then ,the test item was applied at the concentration 20% (w/v) in the vehicle (0.9% NaCl), using a treatment time of 4 hours and the open-chamber method. Vehicle and positive controls were applied using the same treatment time and using the closed-chamber method. After exposure, all items were removed from the front opening of the anterior chamber and the epithelia were rinsed. Then the opacity cornea was measured. On the other hand, for measuring the permeability a fluorescein solution was used and the holders were incubated. After this procedure, the OD490 nm was measured and the corneas were examined for irregularities. The mean corneal opacity and the mean IVIS for the test item was 2. Fluorescein fixation was observed on the three test item-treated corneas. All validity criteria were fulfilled and the study was considered valid. Therefore test item is considered not irritating to eye (no category).

Justification for classification or non-classification

Skin irritation. Based on the available information, mean tissue viability >50% in the OECD 439 test, the substance is non-irritant to the skin according to the CLP Regulation (EC) no. 1272/2008.

Eye irritation: Based on the available information, the in vitro irritation score was < 3 in the OECD 437 test, the substance does not cause serious eye damage according to CLP Regulation (EC) no. 1272/2008.