Registration Dossier

Administrative data

Description of key information

Skin irritation (in vitro): Key study. Test method according to the OECD Guideline 439 with GLP study. The mean percent viability of the SkinEthic® RHE treated tissues was 10.1% versus 2.3% in the positive control. Therefore, the test item should be considered as skin irritant (cat 2) or skin corrosive (Cat 1).

Skin corrosion (in vitro): Key study. Test method according to the OECD Guideline 431 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).

Eye irritation (in vitro): Weight of evidence. Test method according to the OECD Guideline 438 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 rabbits,

the test substance is not classified for eye irritation according to CLP Regulation.

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:
16 May 2018 - 31 May 2018
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): 18-RHE-057
- Delivery date: 29/05/2018
- Expiration date: 04/06/2018
- Date of initiation of testing: 29/05/2018

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: not specified.

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD=1.2 (CV = 3.0%), specification OD > 0.7. Historical negative control mean OD range = 0.653-1.194 (measured after a 1:2 dilution of the extracts in isopropanol).
- Barrier function: 4.0 h (Specification 4.0h < ET50< 10.0h)
- 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 (or corrosive) if the viability after 42 minutes exposure and 42 hours of post-treatment incubation 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 and 42 hours of post-treatment incubation 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 μL (32 μL/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
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
10.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
(distilled water)
Positive controls validity:
valid
Remarks:
2.3% viability (5% SDS)
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The mean percent viability of the treated tissues was 10.1% versus 2.3% 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 10.1% (acceptablility criteria, SD ≤ 18%) and OD mean was 0.822 (measured after a 1:2 dilution of the formazan extracts in isopropanol; the acceptability criteria
should be in the range ≥ 0.4 and ≤1.5).
- Acceptance criteria met for positive control: yes, SD of the positive control group was 0.2% (acceptablility criteria, SD ≤ 18%) and mean viability was 2.3% which is much lower than 50%.
- Acceptance criteria met for variability between replicate measurements: yes. SD of test item was 1.7% (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

0.876

  

0.871

0.822

106.0

100.0

10.1

 

0.877

0.858

2

0.695

0.726

88.4

0.748

0.734

3

0.850

0.868

105.6

0.875

0.878

Positive

Control

4

0.020

0.020

0.019

2.4

2.3

0.2

Irritant

0.020

0.019

5

0.020

0.019

2.3

0.018

0.018

6

0.016

0.017

2.1

0.017

0.017

Test item

13

0.099

0.099

0.083

12.0

10.1

1.7

Irritant

or corrosive

0.098

0.098

14

0.083

0.078

9.5

0.076

0.075

 

15

0.075

0.073

8.9

0.073

0.070

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

OD: optical density

Acceptability criteria:

SD ≤ 18%

Negative control: OD value of the 3 replicates in the range ≥ 0.8 and ≤ 3.0. The optical density was measured after a 1:2 dilution of the formazan extracts in isopropanol; the acceptability criteria should be in the range ≥ 0.4 and ≤1.5 for the negative control.

Interpretation of results:
other: classified as corrosive (Cat 1) or irritant (Cat 2) (CLP Regulation EC no. 1272/2008)
Conclusions:
In the in vitro skin irritation RHE method, the mean percent viability of the treated tissues was 10.1% versus 2.3% 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 μL 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 42 hours 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 10.1%, versus 2.3% 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:
24 August 2018 - 05 September 2018
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-AH1620-1
- Production date: 03/09/2018
- Shipping date: 04/09/2018
- Delivery date: 04/09/2018
- Date of initiation of testing: 05/09/2018

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 (measured after 1:3 dilution in isopropanol) = 0.719-1.374 (3 min exposure) and 0.735-1.395 (1 hour exposure).
- Barrier function: ET50 = 3 hr 22 min (test method with Triton X-100), specification = 2-5 hr.
- 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): 50 μL (83.3 μL/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):
3 hours (incubation in MTT solution)
Number of replicates:
2 replicates for each exposure time.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean after 3 min exposure
Value:
76.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
(distilled water)
Positive controls validity:
valid
Remarks:
0.31% viability (8N KOH)
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean after 1 hour exposure
Value:
92.55
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
(distilled water)
Positive controls validity:
valid
Remarks:
0.88% viability (8N KOH)
Other effects / acceptance of results:
The mean percent viabilities of the epidermis skins treated with the test item were 76.10% (for 3 min exposure) and 92.55% (for 60 min exposure) versus 0.31% and 0.88%, 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 0.649 and 0.685 (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. 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 0.88% < 20%.
- Acceptance criteria met for variability between replicate measurements: yes. 7.9% (for 3 min exposure) and 11.1% (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

0.559

0.570

0.649

87.90

100.00

24.2

0.551

0.600

2

0.659

0.727

112.10

0.738

0.785

Positive control

3

0.002

0.001

0.002

0.15

0.31

0.3

0.001

0.001

4

0.004

0.003

0.46

0.003

0.003

Test item PH-18/0234

5

0.498

0.519

0.494

80.03

76.10

7.9

0.517

0.542

6

0.438

0.468

72.17

0.493

0.474

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

13

0.707

0.694

0.685

101.31

100.00

2.6

0.687

0.688

14

0.687

0.676

98.69

0.672

0.668

Positive control

15

0.009

0.007

0.006

1.02

0.88

0.3

0.007

0.006

16

0.006

0.005

0.73

0.005

0.005

Test item PH-18/0234

17

0.576

0.596

0.634

87.01

92.55

11.1

0.592

0.621

18

0.665

0.672

98.10

0.675

0.677

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 50 µL 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 76.10% (for 3 min exposure) and 92.55% (for 60 min exposure) versus 0.31% and 0.88%, 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:
04 June 2018
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:
yes
Remarks:
The eyes were incubated between 45 and 64 min instead of 45-60 min. As the results obtained with the negative control (longest time) were conformed to what was expected, the deviation is considered as without impact on the conclusion of the study.
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
yes
Remarks:
Same as with OECD TG 438
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 04 June 2018 at 8: 25 am. The eyes were enucleated at Phycher on 04 June 2018 at 10:15 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 μL 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 32.0ºC and 32.2ºC.
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.
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.
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. 3013132 (one eye)

SOLVENT CONTROL USED: not applicable.

POSITIVE CONTROL USED
5% (w/v) Benzalkonium chloride –Sigma–Batch No. BCBQ9761V - 30 μL (three eyes)

APPLICATION DOSE AND EXPOSURE TIME
30 μL 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.
- 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:
1.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class III
Irritation parameter:
fluorescein retention 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:
percent corneal swelling
Run / experiment:
Mean
Value:
18
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, 5% (w/v) Benzalkonium chloride, was 3 x IV,classified as “Corrosive/Severe Irritant ”

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

7

0

1

1

1

1

1

8

0

2

2

2

2

2

9

0

2

2

2

2

2

Mean

 

0.0

1.3

1.7

1.7

1.7

1.7

ICE class

 

 

 

 

III

 

 

 

Fluorescein retention

7

0.5

3

 

 

 

 

8

0.5

3

 

 

 

 

9

0.5

3

 

 

 

 

Mean

 

0.5

3.0

 

 

 

 

ICE class

 

 

IV

 

 

 

 

 

 

 

Corneal thickness

7

0.61

0.65

0.69

0.69

0.69

0.69

8

0.62

0.66

0.71

0.75

0.75

0.75

9

0.61

0.64

0.70

0.73

0.74

0.74

 

Corneal swelling (%)

7

-

7

13

13

13

13

8

-

6

15

21

21

21

9

-

5

15

20

21

21

Mean

 

 

6

14

 18

18

18

ICE class

 

 

 

 

II

 

 

Combination of the 3 Endpoints

1 x IV, 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

3

3

3

3

3

2

0

3

3

3

3

3

3

0

3

3

3

3

3

Mean

 

0.0

3.0

3.0

3.0

3.0

3.0

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

0.60

0.69

0.75

0.78

0.84

0.89

2

0.59

0.66

0.71

0.79

0.82

0.85

3

0.61

0.71

0.75

0.82

0.86

0.87

 

Corneal swelling (%)

1

-

15

25

30

40

48

2

-

12

20

34

39

44

3

-

16

23

34

41

43

Mean

 

-

14

23

33

40

45

ICE class

 

 

 

 

IV

 

 

Combination of the 3 Endpoints

3 x IV

CLASSIFICATION

Category 1 : Corrosive/Severe irritant

Note: Blisters on the cornea noted from 30 minutes post-dose in eyes No. 1, No. 2 and No. 3.

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

0.5

-

-

-

-

ICE class

 

 

I

 

 

 

 

Corneal thickness

16

0.60

0.60

0.60

0.60

0.60

0.60

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 μL of the test item, 30 μL of 5% Benzalkonium chloride (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 IV, 1 x III, 1 x II.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
24 September 2018 - 16 October 2018
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:
yes
Remarks:
(A relative humidity lower than 30% was registered on two days during the study. As no effect was noted on the health of the animals, this deviation was considered as without impact on the conclusion of the study)
GLP compliance:
yes (incl. certificate)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: C.E.G.A.V (La Passerie, F-61350 Saint Martin d’Egrenne) and Granja San Bernardo (Tulebras, Navarra - Spain)
- Age at study initiation: 10 and 11 weeks old
- Weight at study initiation: 2.19 to 2.34 kg.
- Housing: The animals were individually housed.
- Diet (e.g. ad libitum): ad libitum. ENVIGO – 2030C
- Water (e.g. ad libitum): ad libitum. Drinking water (tap-water from public distribution system). Microbiological and chemical analyses of the water were carried out once every six months by Bureau Veritas – Eurofins (FRANCE).
- Acclimation period: ≥ 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20ºC ± 3ºC
- Humidity (%): 30 to 70%
- Air changes (per hr): Minimum 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light (07.00 to 19.00)/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): 0.1 mL
- Concentration (if solution): N/A
Duration of treatment / exposure:
One application of test item and not removed throughout the test.
Observation period (in vivo):
Clinical observations for changes in the cornea, iris and conjunctivae were performed 1, 24, 48 and 72 hours following treatment. If no reaction was observed 72 hours after instillation, the study was terminated. In case of persistent reactions, additional observations were carried out from D7 to D21 in order to determine the reversible character of the lesions observed.
Number of animals or in vitro replicates:
3
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): No
- Time after start of exposure: N/A

SCORING SYSTEM:

CHEMOSIS (A):
0-No swelling
1-Slight swelling, including the nictitating membrane
2-Swelling with eversion of the eyelid
3-Swelling with eyelid half-closed
4-Swelling with eyelid more than half-closed

DISCHARGE (B):
0-No discharge
1-Slight discharge (normal slight secretions in the inner corner not to be taken into account)
2-Discharge with moistening of the eyelids and neighbouring hairs
3-Discharge with moistening of the eyelids and large areas around the eye

REDNESS (C):
0-Blood vessels normal
1-Vessels significantly more prominent than normal
Vessels individually distinguishable with difficulty
2-Generalised red coloration
3-Generalised deep red coloration

IRIS (D):
0-Normal
1-Iris significantly more wrinkled than normal, congestion, swelling of the iris which continues to react to light, even slowly
2-No reaction to light, haemorrhage, significant damage (any or all of these characteristics)

CORNEA: DEGREE OF OPACITY (E):
0- No modification visible either directly or after instillation of fluorescein (no loss of glint or polish)
1- Translucent areas (diffuse or disseminated), iris details clearly visible
2- Easily identifiable translucent area, iris details slightly obscured
3- Opalescent area, no iris details visible, pupil outline scarcely distinguishable
4- Total corneal opacity, completely obscuring the iris and pupil

CORNEA: EXTENT OF OPACITY (F):
1- Opaque area present but covering one quarter or less
2- Between one quarter and half
3- Between half and three quarters
4- Between three quarters and the entire surface


TOOL USED TO ASSESS SCORE: not specified
Irritation parameter:
cornea opacity score
Basis:
animal #1
Remarks:
A7289
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
Remarks:
A7292
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
Remarks:
A7295
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
Remarks:
A7289
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
Remarks:
A7292
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
Remarks:
A7295
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
Remarks:
A7289
Time point:
24/48/72 h
Score:
0.3
Max. score:
1
Reversibility:
fully reversible within: 48 h
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #2
Remarks:
A7292
Time point:
24/48/72 h
Score:
0.7
Max. score:
1
Reversibility:
fully reversible within: 3 days
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Basis:
animal #3
Remarks:
A7295
Time point:
24/48/72 h
Score:
1.3
Max. score:
2
Reversibility:
fully reversible within: 21 days
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #1
Remarks:
A7289
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #2
Remarks:
A7292
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
animal #3
Remarks:
A7295
Time point:
24/48/72 h
Score:
0
Max. score:
0
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
-at the conjunctivae level: a slight to moderate redness noted 1 hour after the test item instillation in all animal and totally reversible between Days 2 and 21. This reaction was associated with a moderate chemosis noted 1 hour after the test item instillation in all animals and totally reversible on Day 1.

Table 1. Assessment of acute eye irritation. Individual and mean scores of conjunctivae, iris and cornea

TEST ITEM: (+)-PIN-2(3)-ENE

Instillation: 0.1 mL of the test item

Instillation dates (D0): 24 September 2018 (A7289) & 25 September 2018 (A7292 & A7295)

Animal n°
Weight (kg)

Time after
treatment

CONJUNCTIVAE

IRIS

CORNEA

CHEMOSIS (A)

REDNESS ( C)

LESION (D)

OPACITY ( E)

A7289

24 hours

0

1

0

0

48 hours

0

0

0

0

72 hours

0

0

0

0

Start: 2.34

TOTAL

0

1

0

0

End: 2.49

Mean

0.0

0.3

0.0

0.0

A7292

24 hours

0

1

0

0

48 hours

0

1

0

0

72 hours

0

0

0

0

Start: 2.35

TOTAL

0

2

0

0

End: 2.51

Mean

0.0

0.7

0.0

0.0

A7295

24 hours

0

2

0

0

48 hours

0

1

0

0

72 hours

0

1

0

0

Start: 2.19

TOTAL

0

4

0

0

End: 3.11

Mean

0.0

1.3

0.0

0.0

CLASSIFICATION
in accordance with the
CLP regulation

According to the calculated means,
the test item does not have to be classified

Table 2. Total and individual scores of ocular irritation. Animal No.: A7289

Observation
time

CONJUNCTIVAE

IRIS

CORNEA

Individual
irritation
index

A

B

C

(A+B+C)x2

D

Dx5

E

F

ExFx5

 

 

 

 

X=

 

Y=

 

 

Z=

X+Y+Z=

1 Hour (D0)

2

2

2

12

0

0

0

0

0

12

24 Hours (D1)

0

0

1

2

0

0

0

0

0

2

48 Hours (D2)

0

0

0

0

0

0

0

0

0

0

72 Hours (D3)

0

0

0

0

0

0

0

0

0

0

Table 3. Total and individual scores of ocular irritation. Animal No.: A7292

Observation
time

CONJUNCTIVAE

IRIS

CORNEA

Individual
irritation
index

A

B

C

(A+B+C)x2

D

Dx5

E

F

ExFx5

 

 

 

 

X=

 

Y=

 

 

Z=

X+Y+Z=

1 Hour (D0)

2

3

2

14

0

0

0

0

0

14

24 Hours (D1)

0

0

1

2

0

0

0

0

0

2

48 Hours (D2)

0

0

1

2

0

0

0

0

0

2

72 Hours (D3)

0

0

0

0

0

0

0

0

0

0

Table 4. Total and individual scores of ocular irritation. Animal No.: A7295

Observation
time

CONJUNCTIVAE

IRIS

CORNEA

Individual
irritation
index

A

B

C

(A+B+C)x2

D

Dx5

E

F

ExFx5

 

 

 

 

X=

 

Y=

 

 

Z=

X+Y+Z=

1 Hour (D0)

2

3

2

14

0

0

0

0

0

14

24 Hours (D1)

0

0

2

4

0

0

0

0

0

4

48 Hours (D2)

0

0

1

2

0

0

0

0

0

2

72 Hours (D3)

0

0

1

2

0

0

0

0

0

2

Day 7 (D7)

0

0

1

2

0

0

0

0

0

2

Day 14 (D14)

0

0

1

2

0

0

0

0

0

2

Day 21 (D21)

0

0

0

0

0

0

0

0

0

0

Interpretation of results:
other: Not classified for eye irritation (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 not classified for eye irritation.


Executive summary:

The eye irritation potential of the test substance was determined in accordance with the OECD guideline 405 with GLP. Three young adult albino New Zealand rabbits were given a single ocular application of 0.1 mL test substance in one 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 also from D7 to D21 in case of persistent reactions. The corneal opacity score, the iris score and the conjunctive score were recorded. The mean eye irritation scores at 24, 48 and 72 h post-application observations for each animal were 0.0, 0.0 and 0.0 for corneal opacity, 0.0, 0.0 and 0.0 for iris effects, 0.3, 0.7 and 1.3 for conjunctival redness and 0.0, 0.0 and 0.0 for conjunctival chemosis. Effects observed for redness were fully reversible between days 2 and 21. Based on these results the test substance is not classified for eye irritation according to CLP Regulation.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation (in vitro): Key study. An in vitro skin irritation test of the test item was performed in a reconstructed humanSkinEthic RHE®model, according to OECD TG 439 (GLP study). Three epidermis units were treated with16 μLtest item for 42 minutes at room temperature. Exposure of the test item was terminated by rinsing with25 x 1 mL of DPBS. The epidermis units were then incubated at 37°C for 42 hours 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 10.1%, versus 2.3% 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).

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 50 µL 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 76.10% (for 3 min exposure) and 92.55% (for 60 min exposure) versus 0.31% and 0.88%, respectively, with the positive control item (potassium hydroxide 8N). Therefore, the test item must be considered as non-corrosive.

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 μL of the test item, 30 μL of 5% Benzalkonium chloride (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 IV, 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 young adult albino New Zealand rabbits were given a single ocular application of 0.1 mL test substance in one 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 also from D7 to D21 in case of persistent reactions. The corneal opacity score, the iris score and the conjunctive score were recorded. The mean eye irritation scores at 24, 48 and 72 h post-application observations for each animal were 0.0, 0.0 and 0.0 for corneal opacity, 0.0, 0.0 and 0.0 for iris effects, 0.3, 0.7 and 1.3 for conjunctival redness and 0.0, 0.0 and 0.0 for conjunctival chemosis. Effects observed for redness were fully reversible between days 2 and 21. Based on these results the test substance is not classified for eye irritation according to CLP Regulation.

Justification for classification or non-classification

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

Eye irritation: Based on the available data, the substance is not classified for eye irritation according to CLP Regulation no. 1272/2008.