Registration Dossier

Administrative data

Description of key information

Eye irritation (in vitro): Weight of Evidence. Test method according to the OECD 438 Guideline with GLP. No prediction can be made for the test item in the ICE test.

Eye irritation (in vivo): Weight of Evidence. Test method according to the OECD 405 Guideline with GLP. Based on the results of an acute eye irritation study performed on New Zealand White rabbits, the test substance is classified as irritating to eyes (category 2).

Skin irritation (in vitro): Key study. Test method according to the OECD 439 Guideline with GLP study. The mean percent viability of the SkinEthic® RHE treated tissues was 1% versus 1.2% in the positive control. Therefore, the test item must be considered as skin irritant (cat 2) or skin corrosive (Cat 1).

Skin corrosion (in vitro): Key study. Test method according to the OECD 431 Guideline with GLP study.Under RHE test method performed in EpiCS® model the test item does not have to be classified as skin corrosive (cat. 1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 March 2017 - 6 April 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Remarks:
SkinEthic RHE® model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: foreskin (number of donors not specified)
Source strain:
not specified
Justification for test system used:
The SkinEthic RHE® model has been validated for irritation testing (Validation study based on the original ECVAM Performance 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)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE® model
- Tissue batch number(s): 17-RHE-039
- Delivery date: 04/04/2017
- Expiration date: 10/04/2017
- Date of initiation of testing: 04/04/2017

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: 3 hours at 37°C, 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: 1.4 (CV = 2.8%) specification OD > 0.7. Historical negative control mean OD range = 0.834-1.574.
- Barrier function: 4.8 h (Specification 0h < ET50< 10h)
- 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
- 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 (32mg/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):
41 hours and 47 minutes.
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
(DPBS)
Positive controls validity:
valid
Remarks:
1.2% viability (5% SDS)
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The mean percent viability of the treated tissues was 1% versus 1.2% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item must be considered as skin irritant (cat 2) or skin corrosive (Cat 1)

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A full demonstration of proficiency was performed for the EpiSkin-SM model, plus a reduced validation with the SkinEthic RHE model. Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, SD of the negative control group was 4.3% (acceptablility criteria, SD ≤ 18%) and OD mean is 1.077 (acceptability criteria, 0.8≤OD≤3).
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes. SD of test item was 0.1% (acceptablility criteria, SD ≤ 18%).

Table 1. Summary of results.

 

Skin

OD

Mean OD /disc (#)

Mean OD / product

Viability

%

Mean viability

%

SD

Viability

Conclusion

Negative

Control

1

0.965

1.026

1.077

95.2

100.0

4.3

 

1.052

1.060

2

1.109

1.117

103.7

1.222

1.121

3

1.090

1.089

101.1

1.076

1.102

Positive

Control

4

0.012

0.012

0.013

1.1

1.2

0.1

Irritant

0.012

0.012

5

0.013

0.013

1.2

0.013

0.013

6

0.013

0.013

1.2

0.012

0.013

Test item

16

0.009

0.010

0.011

0.9

1.0

0.1

Corrosive

or irritant

0.011

0.011

17

0.011

0.011

1.0

0.010

0.011

18

0.012

0.012

1.1

0.011

0.012

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

OD: optical density

*The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol.

Acceptability criteria: SD≤18%.

Interpretation of results:
other: classified as corrosive (Cat 1) or irritant (Cat 2) (CLP Regulation EC no. 1272/2008)
Conclusions:
The mean percent viability of the treated tissues was 1% versus 1.2% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item must be considered as skin irritant (cat 2) or skin corrosive (Cat 1).
Executive summary:

An in vitro skin irritation test of the test item was performed in a reconstructed human SkinEthic RHE® model, according to OECD TG 439 (GLP study). Three epidermis units were treated with 16 mg test item for 42 minutes at room temperature. Exposure of the test item was terminated by rinsing with 25 x 1 mL of DPBS. The epidermis units were then incubated at 37°C for 41 hours 47 minutes in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol during 2 hours under gentle agitation in the dark, and measuring the concentration of formazan by determining the OD at 570 nm, just after dilution of the extracts 1:2 in isopropanol. Under test conditions, the mean percent viability of the treated tissues was 1%, versus 1.2% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item must be considered as skin irritant (cat 2) or skin corrosive (Cat 1).

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 June 2017 - 14 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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 system:
human skin model
Remarks:
EpiCS® model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not specified
Source strain:
not specified
Justification for test system used:
The EpiCS® model (previously named EST-1000) has been validated for corrosion testing (Validation study based on the statement issued by the ECVAM Scientific Advisory Committee (ESAC30) on 12 June 2009) and its use is recommended by the relevant OECD guideline for corrosion testing (OECD No. 431); therefore, it was considered to be suitable for this study.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSC® model
- Tissue batch number(s): 100-AG1038-1
- Production date: 12/06/2017
- Shipping date: 13/06/2017
- Delivery date: 13/06/2017
- Date of initiation of testing: 13/06/2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature for 3 min exposure and 37°C ± 1°C for 1 hour exposure.
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 20 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: 3 hours at 37°C, 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: Not specified. Historical negative control mean OD range = 0.946-1.511 (3 min exposure) and 0.917-1.379 (1 hour exposure).
- Barrier function: 59.8% (test method: MTT, 2 hrs Triton X-100), specification = 50%.
- Morphology: sufficient nº of cornified layers (specification =5) and sufficient nº of vital cell layers (specification =4)
- Contamination: no

NUMBER OF REPLICATE TISSUES: 2

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
- 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%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg (41.7mg/cm2)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL
- Concentration (if solution): N/A
Duration of treatment / exposure:
3 minutes and 60 minutes
Duration of post-treatment incubation (if applicable):
2 hours and 55 minutes (incubation in MTT solution)
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean after 3 min exposure
Value:
107.01
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
(distilled water)
Positive controls validity:
valid
Remarks:
7.44% viability (8N KOH)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean after 1 hour exposure
Value:
18.35
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
(distilled water)
Positive controls validity:
valid
Remarks:
0.93% viability (8N KOH)
Other effects / acceptance of results:
The mean percent viabilities of the epidermis skins treated with the test item were 107.01% (for 3 min exposure) and 18.35% (for 60 min exposure) versus 7.44% and 0.93%, respectively, with the positive control item (potassium hydroxide 8N). Therefore, the test item must be considered as non-corrosive.

- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes. A demonstration of proficiency was performed for the EpiCS® model. Adequate results were obtained for the evaluated chemicals.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes. The mean OD of negative control tissues for treatment time 3 min and 1 hour were 1.063 and 1.186 (acceptability criteria, 0.8≤OD≤2.8 for the EpiCS® model). The extract was diluted at 1:3 isoborneol just before the OD measure, thus the acceptability criteria should be in the range ≥ 0.3 and ≤ 0.9. However, even for values 1.063 or 1.186 the test item remains clearly classified as non-corrosive. This deviation is considered as without any impact on the conclusion and the validity of the study.
- Acceptance criteria met for positive control: yes. Mean viability of the tissue replicates exposed for 1 hour, expressed as % of the negative control, is 0.93% < 20%.
- Acceptance criteria met for variability between replicate measurements: yes. 19% (for 3 min exposure) and 0.9% (for 60 min exposure) < 30%.

Table 1. Individual and average values after 3 minutes exposure

 

Skin

OD

Mean OD/disc (#)

Mean OD/product

Viability %

Mean viability %

Viability difference between replicates %

Negative control

1

1.095

1.094

1.063

102.96

100.00

5.9

1.073

1.114

2

1.030

1.031

97.04

1.012

1.049

Positive control

3

0.076

0.074

0.079

6.96

7.44

0.9

0.073

0.072

4

0.082

0.084

7.91

0.085

0.083

Test item PH-17/0130

9

1.033

1.036

1.137

97.51

107.01

19.0

1.027

1.048

10

1.229

1.238

166.52

1.280

1.203

Table 2. Individual and average values after 1 hour exposure

 

Skin

OD

Mean OD/disc (#)

Mean OD/product

Viability %

Mean viability %

Viability difference between replicates %

Negative control

1

1.117

1.156

1.186

97.51

100.00

5.0

1.182

1.168

2

1.278

1.215

102.49

1.076

1.290

Positive control

3

0.009

0.014

0.011

1.18

0.93

0.5

0.024

0.010

4

0.009

0.008

0.67

0.008

0.008

Test item PH-17/0130

9

0.238

0.223

0.218

18.81

18.35

0.9

0.219

0.213

10

0.221

0.212

17.88

0.222

0.193

Note:

#: mean of 3 values

OD: optical density

As the extract was diluted at 1:3 just before the OD measure, the acceptability criteria should be in the range ≥ 0.3 and ≤ 0.9 for the negative control.

Interpretation of results:
other: non classified as corrosive (Cat 1) (CLP Regulation EC no. 1272/2008)
Conclusions:
Under RHE test method performed in EpiCS® model the test item does not have to be classified as skin corrosive (cat. 1).
Executive summary:

An in vitro skin corrosion test for the test item was performed in a reconstructed human epidermis EpiCS® model, according to OECD TG 431 (GLP study). Two epidermis units were treated with 25 mg test item for 3 minutes at room temperature and for 1 hour, at 37°C, 5% CO2. Exposure of the test item was terminated by rinsing with 20 x 1 mL of DPBS. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol during 2 hours under agitation in the dark, and measuring the concentration of formazan by determining the OD at 570 nm, just after dilution of the extracts 1:3 in isopropanol. Under test conditions, the mean percent viabilities of the treated tissues were 107.01% (for 3 min exposure) and 18.35% (for 60 min exposure) versus 7.44% and 0.93%, respectively, with the positive control item (potassium hydroxide 8N). Therefore, the test item must be considered as non-corrosive.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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:
March 20, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption.
- Characteristics of donor animals (e.g. age, sex, weight): 7 weeks old. 1.5 - 2.5 kg.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):Because eyes were dissected in the laboratory, the 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 heads have been collected on 20 March 2017 at 8: 40 am.The eyes were enucleated at Phycher on 20 March 2017 at 10:00 am.
- 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 of test item
Duration of treatment / exposure:
10 second
Duration of post- treatment incubation (in vitro):
No post-treatment incubation is performed.
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
The eyelids were carefully excised, taking care not to damage the cornea. Then, 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.
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 that the entire cornea was supplied with the physiological saline drip (in the range 0.1 to 0.15 mL/min). The chambers of the superfusion apparatus was temperature controlled between 32.3°C and 32.9°C.
After being placed in the superfusion apparatus, the eyes were examined with a slit-lamp microscope to ensure that they have not been damaged 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. (see table appendix No.4 on "Any other information on results incl. tables")
Once all eyes had been examined and approved, the eyes were incubated between 45 and 64 minutes to equilibrate them to the test system prior to dosing.

EQUILIBRATION AND BASELINE RECORDINGS:
Eyes were incubated between 45 and 64 minutes to equilibrate them to the test system prior to dosing (TG indicates approximately 45 to 60 min. This deviation is considered as without impact on the conclusion of the study)
Following the equilibration period, a zero reference measurement was recorded for corneal thickness and opacity to serve as a baseline (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. 3012316 (one eye)

SOLVENT CONTROL USED: not applicable.

POSITIVE CONTROL USED
Sodium hydroxide – Sigma, Batch No. MKBP7805V - 30 mg (three eyes)

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

OBSERVATION PERIOD:Treated corneas were evaluated pretreatment and starting 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: The test item was rinsed from the eye after 10 seconds of observation with 20 mL of physiological saline at ambient temperature. One additional rinse was performed with 10 mL of physiological saline as the test item remained on the cornea despite the first rinsing.
- Indicate any deviation from test procedure in the Guideline: NO

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: It was calculated by using the area of the cornea that was most densely opacified for scoring. The mean corneal opacity value for all test eyes was calculated for all observation time points (see table 4)
- Damage to epithelium based on fluorescein retention: Fluorescein retention value for all test eyes 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 (Table No.5)
- Swelling: optical pachymeter on a slit-lamp microscope ((HaagStreit BP900 slit-lamp microscope with depth-measuring device no. I). The slit-width was set at 9 1/2 equalling 0.095 mm. The mean percentage of corneal swelling for all test eyes was calculated for all observation time points. Based on the highest mean score for corneal swelling, as observed at any time point, an overall category score was then given for each test item (see table 3).
- Macroscopic morphological damage to the surface: The aim of this evaluation was to determine whether any “pitting” of corneal epithelial cells, “loosening” of epithelium, “roughening” of the corneal surface and “sticking” of the test item to the cornea were visible.These findings can vary in severity and may occur simultaneously.

SCORING SYSTEM:
- Mean corneal swelling: It was expressed as a percentage and was calculated from corneal thickness measurements according to the following formula:
(corneal thickness measurement at time t - corneal thickness at time=0 / corneal thickness at ime=0 )*100

- Mean maximum opacity score:
0 - No opacity,
0.5 -Very faint opacity
1- Scattered or diffuse areas; details of the iris clearly visible
2- Easily discernible translucent area; details of the ris 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: Decision criteria was used as indicated in the TG.
Irritation parameter:
cornea opacity score
Run / experiment:
Mean
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class IV
Irritation parameter:
fluorescein retention score
Run / experiment:
Mean
Value:
2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE class III
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean
Value:
15
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE class II
Irritation parameter:
morphological effects
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: No effects
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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:YES, the combination of the three endpoints for the negative control, physiological saline, was 3xI classified as “No Category”
- Acceptance criteria met for positive control: YES, the combination of the three endpoints for the positive control, sodium hydroxide, was 3 x IV,classified as “Corrosive/Severe Irritant ”

Appendix No. 4: Selected eyes for the performance of the ICE test

Chamber

Flurescein retention

Corneal

opacity

Morphological effects

Corneal thickness (e)

1

0.5

0

N.t.R

0.54

2

0.5

0

N.t.R

0.55

3

0.5

0

N.t.R

0.57

4

0.5

0

N.t.R

0.58

5

0.5

0

N.t.R

0.61

6

0.5

0

N.t.R

0.59

7

0.5

0

N.t.R

0.58

8

0.5

0

N.t.R

0.58

9

0.5

0

N.t.R

0.61

10

0.5

0

N.t.R

0.59

11

0.5

0

N.t.R

0.56

12

0.5

0

N.t.R

0.59

13

0.5

0

N.t.R

0.60

14

0.5

0

N.t.R

0.54

15

0.5

0

N.t.R

0.57

16

0.5

0

N.t.R

0.58

N.t.R: Nothing to report

Table 9: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT

Test item

Endpoint measured

Eye No.

0

30

75

120

180

240

 

 

Corneal opacity

4

0

2

3

3

3

3

5

0

0.5

1

2

2

3

6

0

1

2

2

3

3

Mean

 

0.0

1.2

2.0

2.3

2.7

3

ICE class

 

 

 

 

IV

 

 

 

Fluorescein retention

4

0.5

2

 

 

 

 

5

0.5

2

 

 

 

 

6

0.5

2

 

 

 

 

Mean

 

0.5

2

 

 

 

 

ICE class

 

 

III

 

 

 

 

 

 

 

Corneal thickness

4

0.58

0.62

0.64

0.69

0.73

0.77

5

0.61

0.64

0.64

0.65

0.66

0.68

6

0.59

0.59

0.59

0.59

0.60

0.60

 

Corneal swelling (%)

4

-

7

10

19

26

33

5

-

5

5

7

8

11

6

-

0

0

0

2

2

Mean

 

 

4

5

9

12

15

ICE class

 

 

 

 

II

 

 

Combination of the 3 Endpoints

1 x VI, 1 x III, 1 x II

CLASSIFICATION

No prediction can be made

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

Table 8: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT

Positive control

Endpoint measured

Eye No.

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

0.54

-

-

-

-

-

2

0.55

 -

-

-

-

-

3

0.57

-

-

-

-

-

 

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).

Table 7: INDIVIDUAL AND AVERAGE VALUES FOR EVALUATION OF CORNEAL LESIONS AFTER TREATMENT

Negative control

Endpoint measured

Eye No.

0

30

75

120

180

240

Corneal opacity

16

0.0

0.0

0.0

0.0

0.0

0.0

ICE class

 

 

 

 

I

 

 

Fluorescein retention

16

0.5

0.5

-

-

-

-

ICE class

 

 

I

 

 

 

 

Corneal thickness

16

0.58

0.58

0.58

0.58

0.58

0.58

Corneal swelling (%)

16

-

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.

Interpretation of results:
other: No prediction can be made (CLP Regulation EC no. 1272/2008)
Conclusions:
Under experimental conditions, no prediction can be made for the test item in the ICE test.
Executive summary:

An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 438 under GLP conditions. Eyeballs were isolated from chickens killed for human consumption and after the appropriate preparation were exposed to either 30 mg of the test item, 30 mg of sodium hydroxide (positive control) or 30μL of physiological saline (negative control). Three eyeballs were used in test item and positive groups, and one for the negative control group. 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. Under experimental conditions, no prediction can be made for the test item in the ICE test since the combinations of the 3 endpoints were 1 x VI, 1 x III, 1 x II.

.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
15 November 2017 - 30 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
According to OECD 405 with GLP
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Animal Breeding Facility, Jai Research Foundation
- Age at study initiation:15 to 17 weeks
- Weight at study initiation: Minimum: 2.716 kg, Maximum: 2.921 kg.
- Housing: The animals were individually housed in stainless steel wire meshed cages. Daily, rack was cleaned with cloth, floor of experimental procedure room was swept and all work tops and the floor were mopped with a disinfectant solution.
- Diet (e.g. ad libitum): ad libitum. Teklad Certified Global High Fiber Rabbit Pellet Feed manufactured by Envigo, USA.
- Water (e.g. ad libitum): ad libitum. UV sterilised drinking water filtered through Reverse Osmosis water filtration system.
- Acclimation period: 6 to 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 22 ºC
- Humidity (%): 63 to 65%
- Air changes (per hr): Minimum 15 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 58.2, 58.9 and 57.8 mg of test item for rabbit Nº1, N° 2 and 3, respectively.
- Concentration (if solution): N/A
Duration of treatment / exposure:
24 h until eyes were gently washed with 0.9% normal saline
Observation period (in vivo):
Detailed clinical observations for changes in the cornea, iris, and conjunctiva were performed 1, 24, 48 and 72 hours and 7 days after the application of the test item
Number of animals or in vitro replicates:
3 females
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): gently washed with 0.9% normal saline
- Time after start of exposure: 24 h

SCORING SYSTEM:
Corneal opacity score:
0- No ulceration or opacity
1- Scattered or diffuse areas of opacity (other than slight dulling of normal lustre), details of iris clearly visible
2- Easily discernible translucent area, details of iris slightly obscured
3- Nacreous area, no details of iris visible, size of pupil barely discernible
4- Opaque cornea, iris not discernible through the opacity

Iris score
0-Normal
1-Markedly deepened rugae, congestion, swelling, moderate circumcorneal hyperaemia or injection, iris reactive to light (a sluggish reaction is considered to be an effect)
2-Hemorrhage, gross destruction, or no reaction to light

Conjuntive redness score
0-Normal
1-Some blood vessels hyperaemic (injected)
2-Diffuse, crimson colour, individual vessels not easily discernible
3-Diffuse beefy red

Chemosis score (swelling)
0-Normal
1-Some swelling above normal
2-Obvious swelling, with partial eversion of lids
3-Swelling, with lids about half closed
4-Swelling, with lids more than half closed

TOOL USED TO ASSESS SCORE: fluorescein
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 7d
Remarks on result:
positive indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 7d
Remarks on result:
positive indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24/48/72 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 7d
Remarks on result:
positive indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
ca. 0.33
Max. score:
1
Reversibility:
fully reversible within: 7d
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24/48/72 h
Score:
ca. 0.33
Max. score:
1
Reversibility:
fully reversible within: 7d
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 7d
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
At 1 h post-application, the treated eye revealed conjunctival redness [some blood vessels definitely hyperaemic (injected) in rabbit N° 2 and 3 to diffuse, crimson colour, individual vessels not easily discernible in rabbit N° 1; score of 1 to 2] and conjunctival chemosis [some swelling above normal (includes nictitating membranes); score of 1] in all three rabbits.
At 24 h post-application, the treated eye of all three rabbits revealed conjunctival redness [diffuse, crimson colour, individual vessels not easily discernible; score of 2] and conjunctival chemosis [some swelling above normal (includes nictitating membranes); score of 1].
At 48 and 72 h post-application, the treated eye revealed conjunctival redness [diffuse, crimson colour, individual vessels not easily discernible; score of 2] in all three rabbits and conjunctival chemosis [some swelling above normal (includes nictitating membranes) in rabbit N° 3; score of 1].
On day 7 post-application, the treated eye of all three rabbits recovered completely and appeared normal.
Iritis and corneal opacity were not observed in any of the rabbits throughout the experimental period.
Other effects:
- Lesions and clinical observations:
- Ophthalmoscopic findings: Examination with fluorescein dye and cobalt blue filter [corneal epithelium damage showing as green fluorescein staining] revealed no (area) corneal epithelium damage in all three rabbits at 24 h post-application.
- Histopathological findings: not applicable
- Effects of rinsing or washing: not observed

Table 1: Mean Eye Irritation Scores

Rabbit N°

Mean Score at 24, 48 and 72 Hours

Opacity: Degree of Density

Iris Lesion

Conjunctivae

Redness

Chemosis

1

0.00

0.00

2.00

0.33

2

0.00

0.00

2.00

0.33

3

0.00

0.00

2.00

1.00

Table 2: Individual Scores of Eye Reactions Post-Application

Control eye

Rabbit N°

1

2

3

Site of Application

Left

Left

Left

Reaction

Hour

Day

Hour

Day

Hour

Day

1

24

48

72

7

1

24

48

72

7

1

24

48

72

7

Opacity:

Degree of density

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Iris

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Conjunctivae

(Redness)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Conjunctivae

(Chemosis)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Treated eye

Rabbit N°

1

2

3

Site of Application

Right

Right

Right

Reaction

Hour

Day

Hour

Day

Hour

Day

1

24

48

72

7

1

24

48

72

7

1

24

48

72

7

Opacity:

Degree of density

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Iris

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Conjunctivae

(Redness)

2

2

2

2

0

1

2

2

2

0

1

2

2

2

0

Conjunctivae (Chemosis)

1

1

0

0

0

1

1

0

0

0

1

1

1

1

0

Interpretation of results:
other: classified as irritating to eyes (Cat 2) (CLP Regulation EC no. 1272/2008)
Conclusions:
Based on the results of an acute eye irritation study performed on New Zealand White rabbits, the test substance is classified as irritating to eyes (category 2).


Executive summary:

The eye irritation potential of the test substance was determined in accordance with the OECD guideline 405 with GLP. Three adult female New Zealand White rabbits were given a single ocular application of 0.1 mL test substance in right eye of the rabbit while the contralateral eye remained untreated and served as the control. Animals were observed at 1, 24, 48, 72 h and 7 days after the test item was applied. The corneal opacity score, the iris score and the conjunctive score were recorded. No corneal opacity or iris damage were observed but conjunctival effects were evident at 1, 24, 48 and 72 h post application in all three rabbits. The mean eye irritation scores at 24, 48 and 72 h post-application observations were 0.00 for corneal opacity, 0.00 for iris effects, 2.00 for conjunctival redness and 0.33 to 1.00 for conjunctival chemosis. However, these effects were fully reversible within 7 days. Examination with fluorescein dye and cobalt blue filter at 24 h post application revealed no corneal epithelium damage in all rabbits. The control eye did not show any abnormal reaction during the study. Moreover, there were no signs of systemic toxicity in any animal observed. Based on these results the test substance is classified as irritating to eyes (category 2) according to CLP Regulation.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Eye irritation (in vitro): Weight of Evidence. An in vitro (ex vivo) study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 438 under GLP conditions. Eyeballs were isolated from chickens killed for human consumption and after the appropriate preparation were exposed to either 30 mg of the test item, 30 mg of sodium hydroxide (positive control) or 30μL of physiological saline (negative control). Three eyeballs were used in test item and positive groups, and one for the negative control group. 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. Under experimental conditions, no prediction can be made for the test item in the ICE test since the combinations of the 3 endpoints were 1 x VI, 1 x III, 1 x II.

Eye irritation (in vivo): Weight of Evidence. The eye irritation potential of the test substance was determined in accordance with the OECD guideline 405 with GLP. Three adult female New Zealand White rabbits were given a single ocular application of 0.1 mL test substance in right eye of the rabbit while the contralateral eye remained untreated and served as the control. Animals were observed at 1, 24, 48, 72 h and 7 days after the test item was applied. The corneal opacity score, the iris score and the conjunctive score were recorded. No corneal opacity or iris damage were observed but conjunctival effects were evident at 1, 24, 48 and 72 h post application in all three rabbits. The mean eye irritation scores at 24, 48 and 72 h post-application observations were 0.00 for corneal opacity, 0.00 for iris effects, 2.00 for conjunctival redness and 0.33 to 1.00 for conjunctival chemosis. However, these effects were fully reversible within 7 days. Examination with fluorescein dye and cobalt blue filter at 24 h post application revealed no corneal epithelium damage in all rabbits. The control eye did not show any abnormal reaction during the study. Moreover, there were no signs of systemic toxicity in any animal observed. Based on these results the test substance is classified as irritating to eyes (category 2) according to CLP Regulation.

Skin irritation (in vitro): Key study. An in vitro skin irritation test of the test item was performed in a reconstructed human SkinEthic RHE® model, according to OECD TG 439 (GLP study). Three epidermis units were treated with 16 mg test item for 42 minutes at room temperature. After 42h post-incubation, the viability of each tissue was assessed by incubating the solution with MTT, extracting the precipitated formazan crystals, and determing the OD spectrophotometrically. Under test conditions, the mean percent viability of the treated tissues was 1%, versus 1.2% in the positive control. The test item must be considered as skin irritant (cat 2) or skin corrosive (Cat 1).

Skin corrosion (in vitro): Key study. An in vitro skin corrosion test for the test item was performed in a reconstructed human epidermis EpiCS® model, according to OECD TG 431 (GLP study). Two epidermis units were treated with 25 mg test item for 3 minutes at room temperature and for 1 hour, at 37°C, 5% CO2. Exposure of the test item was terminated by rinsing with 20 x 1 mL of DPBS. The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol during 2 hours under agitation in the dark, and measuring the concentration of formazan by determining the OD at 570 nm, just after dilution of the extracts 1:3 in isopropanol. Under test conditions, the mean percent viabilities of the treated tissues were 107.01% (for 3 min exposure) and 18.35% (for 60 min exposure) versus 7.44% and 0.93%, respectively, with the positive control item (potassium hydroxide 8N). Therefore, the test item must be considered as non-corrosive.

Justification for classification or non-classification

Eye irritation: Based on the available data, the substance is classified as eye irritant (Cat 2) according to CLP Regulation no. 1272/2008.

Skin irritation/corrision: Based on the available data, the substance is classified as skin irritant (Cat 2) according to CLP Regulation no. 1272/2008.