Registration Dossier

Administrative data

Description of key information

Skin irritation (in vitro): Key study. Test method according to OECD 439, GLP study. The mean corrected percent viability of the treated tissues was 112.6 %, versus 1.3% in the positive control (5% SDS). Therefore, the test item is not irritant to the skin.

Eye irritation (in vitro): Key study. Test method according to OECD 438, GLP study. No prediction can be made for the test item in the ICE test.

Eye irritation (in vivo): Key study. Test method according to the OECD 405, GLP study. Based on the results of an acute eye irritation study performed on New Zealand White rabbits, the test substance is classified as eye damage (category 1).

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 September 2019 - 17 September 2019
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:
yes
Remarks:
Standard deviation between the 2 NSCkilled replicates treated with the test item was 35.4% instead of 18% at the maximum as defined in OECD test guideline. This deviation is considered as without impact on the conclusion of the study (see reasoning below)
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)
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): 19-RHE-156 (living epidermis); 17-RHE-124, 18-RHE-002, 18-RHE-142 and 19-RHE-096 (killed epidermis).
- Production date: N/A
- Shipping date: 17/09/2019
- Delivery date: 17/09/2019
- Expiration date: 23/09/2019
- Date of initiation of testing: 17/09/2019

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 37ºC, 5% CO2
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: O.D. = 1.4 (CV = 2.7%) specification OD > 0.7. Historical negative control mean OD range = 0.489-1.217 (OD measured after 1:2 dilution in isopropanol; acceptability criteria should be in the range ≥ 0.4 and ≤1.5)
- Barrier function: 5.8 h (Specification 4.0h < ET50< 10.0h)
- Morphology: 8 Cell layers (specification > 4.), highly differentiated epidermis consisting of organized basal, spinous and granular layers and a multilayered stratum corneum.
- Contamination: No.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE:
- Fresh tissues / killed tissues: 2 killed tissues ( NSMTT control); 2 living tissues ((NSCliving control) and 2 additional killed tissues (NSCkilled control).
- N. of replicates : 2
- Method of calculation used: True viability % = [(OD of living tissues exposed to test item - OD of killed tissues exposed to test item - OD of living tissues exposed to test item incubated with medium without MTT + OD of killed tissues exposed to test item incubated with medium without MTT) / OD of living tissues exposed to negative control] x 100

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

PREDICTION MODEL / DECISION CRITERIA
- The test item is considered as non-irritant to skin if the mean percent viability after 42 minutes exposure and 42 hours of post-treatment incubation is > 50%.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2) if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and the result of a skin corrosion test is “non corrosive”.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after 42 minutes exposure and 42 hours of post-treatment incubation is ≤ 50% and in absence of information on a skin corrosion test.




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 15 minutes
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
112.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
(distilled water)
Positive controls validity:
valid
Remarks:
1.3% viability (5% SDS)
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: assessed by NSMTT controls (see table below).
- Colour interference with MTT: assessed by NSCliving controls and NSCkilled controls (see table below).

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, mean OD = 0.999 (OD measured after 1:2 dilution in isopropanol; acceptability criteria should be in the range ≥ 0.4 and ≤1.5).
- Acceptance criteria met for positive control: yes, mean viability = 1.3% (acceptability criteria should be < 40%).
- Acceptance criteria met for variability between replicate measurements: Standard deviation between the 2 NSCkilled control replicates treated with the test item was 35.4% instead of 18% at the maximum as defined in OECD test guideline. This deviation is considered as without impact on the conclusion of the study because regardless of the addition of the viability obtained by the NSCkilled control, the viability obtained with the test item remains higher than 50% and does not change the classification of the product.

Table 1. Table of results

Well ID

OD

Mean OD /
disc (#)

Mean OD /
product

Viability
%

Mean viability
%

SD

Conclusion

Negative
control

SPL 1

0.964

0.955

0.999

95.6

100.0

4.5

 

0.945

0.956

SPL 2

1.044

1.045

104.6

1.046

1.045

SPL 3

0.981

0.997

99.8

1.017

0.992

Positive
control

SPL 4

0.012

0.013

0.013

1.3

1.3

0.1

Irritant

0.012

0.013

SPL 5

0.013

0.013

1.3

0.013

0.013

SPL 6

0.011

0.012

1.2

0.011

0.012

Test item
PH-19/0483

SPL 13

1.080

1.087

0.995

108.8

99.6

9.4

 

1.080

1.100

SPL 14

0.985

1.000

100.1

1.017

0.998

SPL 15

0.888

0.899

90.0

0.919

0.889

Test item
PH-19/0483
NSC Control
living tissues

SPL 18

0.054

0.055

0.069

5.5

6.9

1.9

0.057

0.052

SPL 19

0.083

0.082

8.2

0.083

0.078

Test item
PH-19/0483
NSC Control
killed tissues

SPL 20

0.021

0.020

0.270

2.0

27.0

35.4

0.020

0.019

SPL 21

0.514

0.520

52.1

0.513

0.531

Test item
PH-19/0483
killed tissues
MTT control

SPL 16

0.060

0.062

0.072

6.2

7.2

1.4

0.062

0.062

SPL 17

0.082

0.082

8.2

0.082

0.080

Test item
PH-19/0483
corrected

 

112.6

 

Non irritant

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

Interpretation of results:
other: Not classified (CLP Regulation EC no. 1272/2008)
Conclusions:
In the vitro human reconstructed epidermis test the mean corrected percent viability of the treated tissues was 112.6%. Therefore, the test item can be considered as not irritant to skin.
Executive summary:

An in vitro skin irritation test of the test item was performed in a reconstructed human SkinEthic™ RHE epidermis 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 and 15 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. Additionally, 2 killed Human skin model surfaces were treated (SkinEthic RHE® model) in the same manner in order to generate non-specific MTT reduction. Moreover, 2 living and 2 killed Human skin model surfaces were treated in the same manner but they were incubated in culture medium instead of MTT solution in order to generate non-specific living and killed colour controls. Under the test conditions, the mean corrected percent viability of the treated tissues was 112.6%, versus 1.3% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is considered as not irritant to the skin.

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:
key study
Study period:
16 September 2019
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)
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.
- Number of animals: Not specified.
- 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 16 September 2019 at 8: 26 am.The eyes were enucleated at Phycher on 16 September 2019 at 10:05 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 seconds
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 at 31.9°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 were 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 were 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. 3013524 (one eye)

SOLVENT CONTROL USED: not applicable.

POSITIVE CONTROL USED
Sodium hydroxide – Fisher Scientific, Batch No. 1550248 - 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 eyes were rinsed twice with 10 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 (see table 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:
Highest mean
Value:
0.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class II
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:
Highest mean
Value:
14
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

0.55

2

0.5

0

N.t.R

0.52

3

0.5

0

N.t.R

0.50

4

0.5

0

N.t.R

0.52

5

0.5

0

N.t.R

0.56

6

0.5

0

N.t.R

0.50

7

0.5

0

N.t.R

0.52

8

0.5

0

N.t.R

0.51

9

0.5

0

N.t.R

0.50

10

0.5

0

N.t.R

0.50

11

0.5

0

N.t.R

0.52

12

0.5

0

N.t.R

0.53

13

0.5

0

N.t.R

0.52

14

0.5

0

N.t.R

0.52

15

0.5

0

N.t.R

0.48

16

0.5

0

N.t.R

0.51

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

10

0

1

1

1

1

1

11

0

1

1

1

1

1

12

0

0.5

0.5

0.5

0.5

0.5

Mean

 

0.0

0.8

0.8

0.8

0.8

0.8

ICE class

 

 

 

 

II

 

 

 

Fluorescein retention

10

0.5

2

 

 

 

 

11

0.5

2

 

 

 

 

12

0.5

2

 

 

 

 

Mean

 

0.5

2

 

 

 

 

ICE class

 

 

III

 

 

 

 

 

 

 

Corneal thickness

10

0.54

0.54

0.56

0.56

0.60

0.60

11

0.52

0.53

0.53

0.54

0.60

0.60

12

0.53

0.56

0.56

0.56

0.56

0.56

 

Corneal swelling (%)

10

-

8

12

12

20

20

11

-

2

2

4

15

15

12

-

6

6

6

6

6

Mean

 

 

5

7

7

14

14

ICE class

 

 

 

 

II

 

 

Combination of the 3 Endpoints

2 x II, 1 x III

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

0.55

-

-

-

-

-

2

0.52

 -

-

-

-

-

3

0.50

-

-

-

-

-

 

Corneal swelling (%)

1

(-)

(-)

(-)

(-)

(-)

(-)

2

(-)

(-)

(-)

(-)

(-)

(-)

3

(-)

(-)

(-)

(-)

(-)

(-)

Mean

 

-

-

-

-

-

-

ICE class

 

 

 

 

IV

 

 

Combination of the 3 Endpoints

3 x IV

CLASSIFICATION

Category 1 : Corrosive/Severe irritant

Note:

(-): Severe loosening of the corneal epithelium 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

0.5

0.5

-

-

-

-

ICE class

 

 

I

 

 

 

 

Corneal thickness

16

0.51

0.53

0.53

0.53

0.53

0.53

Corneal swelling (%)

16

-

4

4

4

4

4

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 III, 2 x II.

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
04 November 2019 - 08 November 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
According to OECD 405 with GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Granja San Bernardo (Tulebras, Navarra – Spain).
- Age at study initiation:15 weeks old
- Weight at study initiation: 3.17 kg.
- Housing: The animal was kept in an individual box installed in conventional air conditioned animal husbanding.
- Diet (e.g. ad libitum): ad libitum. ENVIGO – 2030C.
- Water (e.g. ad libitum): ad libitum. Drinking water (tap-water from public distribution system).
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23 ºC
- Humidity (%): 30 to 70%. A relative humidity higher than the maximum recommended value was registered on 04 November 2019 (81%). As no effect was noted on the health of the animals, this deviation is considered as without impact on the conclusion of the study.
- Air changes (per hr): ≥ 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): 100 mg.
- Concentration (if solution): N/A
Duration of treatment / exposure:
Residual test item was noted at the reading time of 1 hour after treatment, thus the eye was rinsed with physiological saline.
Observation period (in vivo):
Ocular examinations were performed on both eyes (treated and control) 1 hour, 24, 48 and 72 hours following treatment.
Number of animals or in vitro replicates:
1 animal.
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Washing (if done): yes, with physiological saline because residual test item remained in the eye affter one hour.
- Time after start of exposure: 1 h

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
Time point:
24/48/72 h
Score:
3.7
Max. score:
4
Reversibility:
not reversible
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.7
Max. score:
2
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24/48/72 h
Reversibility:
not reversible
Remarks on result:
not determinable
Remarks:
(black coloration preventing the redness quotation)
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24/48/72 h
Score:
3
Max. score:
3
Reversibility:
not reversible
Irritant / corrosive response data:
The ocular reactions observed during the study were important to severe:
- at the conjunctivae level: a black coloration of palpebral and bulbar conjunctivae preventing quotation of redness. A moderate chemosis noted 24 hours after the test item instillation and remaining on the last day of the test (Day 4) with same intensity (grade 3).
- at the iris level: no reaction to light was noted on Day 1.
- at the corneal level: a moderate opacity noted 24 hours after the test item instillation and remaining on the last day of the test (Day 4) with severe intensity (grade 4).
Considering the severity of the reactions, the animal was euthanized on day 4 in accordance with the principles of animal welfare as the study was stopped.

Tables of results. Assessment of acute eye irritation. Individual and mean scores of conjunctivae, iris and cornea

TEST ITEM: Violet LCC6D

Instillation: 0.1 g of the test item

Instillation dates (D0): 04 November 2019

Animal Nº: A7755

Table 1

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

*

Not calculable

*

Not calc.

*

*

Not calc.

Not calculable

24 Hours

(D1)

3

1

*

Not calculable

2

10

3

4

60

Not calculable

48 Hours

(D2)

3

1

*

Not calculable

0

0

4

3

60

Not calculable

72 Hours

(D3)

3

1

*

Not calculable

0

0

4

3

60

Not calculable

Day 4 (D4)

3

1

*

Not calculable

0

0

4

3

60

Not calculable

Notes:

(*) black coloration of palpebral and bulbar conjunctivae preventing quotation of redness, corneal opacity reading and the iris observation.

Considering the severe reactions observed, the animal was euthanized on day 4.

Table 2

Animal n°
Weight (kg)

Time after
treatment

CONJUNCTIVAE

IRIS

CORNEA

CHEMOSIS (A)

REDNESS ( C)

LESION (D)

OPACITY ( E)

A7755

24 hours

3

*

2

3

48 hours

3

*

0

4

72 hours

3

*

0

4

Start: 3.17

TOTAL

9

Not calc.

2

11

End: 3.16

Mean

3.0

Not calc.

0.7

3.7

CLASSIFICATION
in accordance with the CLP regulation

Taking into account the severity of the reactions the test item has to be classified in Category 1 "Irreversible effects on the eyes" with the hazard statement H318 "Causes serious eye damage"

Notes:

(*) black coloration of palpebral and bulbar conjunctivae preventing quotation of redness.

Interpretation of results:
other: classified as serious eye damage (Cat 1) (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 has to be classified in category 1 (serious eye damage) as irreversible effects were found on the eye.


Executive summary:

The eye irritation potential of the test substance was determined in accordance with the OECD guideline 405 with GLP. One adult New Zealand White rabbit was given a single ocular application of 0.1 g test substance in one eye while the contralateral eye remained untreated and served as the control. Animal was observed at 1, 24, 48, 72 h after the test item was applied. The corneal opacity, iris and the conjunctivae (redness and chemosis) scores were recorded. The individual eye irritation scores at 24, 48 and 72 h post-application observations were 3, 4 and 4 for corneal opacity, 2, 0 and 0 for iris effects, 3, 3 and 3 for conjunctival chemosis. Conjunctival redness could not be measured due to a black coloration of palpebral and bulbar conjunctivae. Considering the severity of the reactions, the animal was euthanized on day 4 in accordance with the principles of animal welfare as the study was stopped. The moderate chemosis and opacity noted 24 hours after the test item instillation remained on the last day of the test (Day 4) with intensity of grade 3 and 4 respectively. Based on these results, the test substance has to be classified in category 1 (serious eye damage) according to CLP Regulation.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Serious eye damage/eye irritation: In vitro studies were first performed in order to decide on the classification of the substance. Results from in vitro test were inconclusive thus an acute eye irritation/corrosion test in accordance with OECD Guideline 405 was started. Irreversible effects were found on the eye treated with the test substance and it was concluded that the test substance should be classified in category 1 (serious eye damage) according to CLP Regulation.

Justification for classification or non-classification

Skin irritation/corrosion. Based on the available information, the substance is not classified for skin irritation/corrosion in accordance with CLP, Regulation (EC) No. 1272/2008.

Eye irritation/serious eye damage: Based on the available data, the substance is classified as serious eye damage (Cat 1) according to CLP Regulation no. 1272/2008.