Registration Dossier

Administrative data

Description of key information

Skin irritation. Key study. Method according to OECD 439, GLP study. The mean corrected percent viability of the SkinEthic™ RHE treated tissues was 66.7 %, versus 1.0% in the positive control. Therefore, the test item is not irritant to the skin.

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

Weight of evidence. Method according to OECD 438, GLP study. According to the overall in vitro classification, no prediction can be made, since the combinations of the 3 endpoints for the test item were 1 x III, 1 x II, 1 x I.

Weight of evidence. Method according to OECD 437, GLP study. The mean IVIS score for the test item was found to be -0.14, while the values for the negative and positive controls were 2.33 and 160.99, respectively. Therefore, the test item is not irritating to the eye (no category), not requiring classification or serious eye damage.

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:
From November 16th to December 1st, 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
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 material information:
Composition 1
Test system:
human skin model
Remarks:
SkinEthic™ RHE model
Source species:
human
Cell type:
other: normal human keratinocytes.
Cell source:
other: human adult donor, 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): 16 MPE 128
- Delivery date: 29/11/2016
- Expiration date: 05/12/2016
- Date of initiation of testing: 16/11/2016

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:

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: O.D. = 1.3 (CV 1.3%)
- Barrier function: 4.3 h
- 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 12 minutes.
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
66.7
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Remarks:
(DPBS)
Positive controls valid:
yes
Remarks:
1.0% viability (5% SDS)
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The mean corrected percent viability of the treated tissues was 66.7 %, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is not irritant to the skin.

- 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. The standard deviation of the negative control group was 21.7%, instead of ≤ 18% as initially scheduled. However, considering the results obtained, this deviation is considered as without impact on the conclusion of the study.
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1. Summary of results.

 

Skin

OD

Mean OD /disc (#)

Mean OD / product

Viability

%

Mean viability

%

SD

Viability

Conclusion

Negative

Control

1

1.541

1.619

1.316

123.1

100.0

21.7

 

1.650

1.668

2

1.268

1.276

97.0

1.271

1.290

3

1.032

1.052

80.0

1.044

1.082

Positive

Control

4

0.013

0.014

0.013

1.1

1.0

0.1

Irritant

0.016

0.013

5

0.015

0.013

1.0

0.013

0.013

6

0.012

0.011

0.8

0.011

0.011

Test item

21

0.814

0.856

0.877

65.1

66.7

2.0

Non-irritant

0.878

0.877

22

0.868

0.869

66.1

0.878

0.862

23

0.897

0.907

68.9

0.905

0.921

 

 

Interpretation of results:
GHS criteria not met
Remarks:
EU criteria (not irritant).
Conclusions:
The mean corrected percent viability of the treated tissues was 66.7 %, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is not irritant to the 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 12 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 corrected percent viability of the treated tissues was 66.7 %, versus 1.0% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is 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:
weight of evidence
Study period:
November 14th, 2016.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
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)
Test material information:
Composition 1
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 were collected on 14 November 2016 at 8:15 am. The eyes were enucleated at Phycher on 14 November 2016 at 9:35 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
Duration of treatment / exposure:
10 seconds
Number of animals or in vitro replicates:
3 replicates per group (test item, positive control, negative control)
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES: In order to control the quality of the procedure, the eyeballs used for the purpose of the experiment were assessed for potential damage. 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. Corneal thickness was also measured at this time at the corneal apex using the depth measuring device on the slit-lamp microscope. Eyes with; (i), a fluorescein retention score of > 0.5; (ii) corneal opacity > 0.5; or, (iii), any additional signs of damage were replaced. For eyes that are not rejected based on any of these criteria, individual eyes with a corneal thickness deviating more than 10% from the mean value for all eyes are to be rejected.

EQUILIBRATION AND BASELINE RECORDINGS: Once all eyes had been examined and approved, the eyes were incubated between 45 and 65 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. 3012316.

SOLVENT CONTROL USED: not applicable.

POSITIVE CONTROL USED: 30 mg sodium hydroxide – Sigma, Batch No. MKBP7805V.

APPLICATION DOSE AND EXPOSURE TIME: 30 mg test item, exposure 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 with 20 mL of physiological saline at ambient temperature. As test item remained on the cornea despite the rinsing, 2 additional rinses were performed with 10 mL of physiological saline. A cotton swab moistened with physiological saline was used to gently remove all the test item from the cornea. A last rinse was performed with 10 mL of physiological saline.
- Indicate any deviation from test procedure in the Guideline: no.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: corneal opacity 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. Based on the highest mean score for corneal opacity, as observed at any time point, an overall category score was then given for each test or control item.
- Damage to epithelium based on fluorescein retention: The mean 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.
- Swelling: measured with optical pachymeter on a slit-lamp microscope (HaagStreit BP900 slit-lamp microscope with depth-measuring device no. I); slit-width setting: the slit-width was set at 9½, equalling 0.095 mm. The mean percentage of corneal swelling for all test eyes was calculated for all observation time points. The value was determined from corneal thickness measurements. 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.
- Macroscopic morphological damage to the surface: qualitative assessment. 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.
- Others (e.g, histopathology): no.

SCORING SYSTEM:
- Mean corneal swelling (%) was calculated as follows: [(corneal thickness at time t - corneal thickness at time 0)/corneal thickness at time 0] x 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 iris are slightly obscured
3 Severe corneal opacity; no specific details of the iris are visible; size of the pupil is barely discernible
4 Complete corneal opacity; iris invisible

- Mean fluorescein retention score at 30 minutes post-treatment:
0 No fluorescein retention
0.5 Very minor single cell staining
1 Single cell staining scattered throughout the treated area of the cornea
2 Focal or confluent dense single cell staining
3 Confluent large areas of the cornea retaining fluorescein

DECISION CRITERIA: as indicated in the TG.
Irritation parameter:
fluorescein retention score
Run / experiment:
mean
Value:
2
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: ICE class III
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
0.8
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: ICE class II
Irritation parameter:
percent corneal swelling
Run / experiment:
mean
Value:
2
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: ICE class I
Irritation parameter:
morphological effects
Run / experiment:
mean
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no. No morphological effects were noted, whatever the examination time.
- Other observations: the test item required additional rinsing to be removed from the cornea.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes.
- Acceptance criteria met for positive control: yes.
- Range of historical values if different from the ones specified in the test guideline: no.

Table 1. Test item results (30 mg), 14/11/2016.

Endpoint measured

Eye No.

Time (min)

0

30

75

120

180

240

Corneal opacity

13

0.5

1

1

1

1

1

14

0

0.5

0.5

0.5

1

1

15

0

0.5

0.5

0.5

0.5

0.5

Mean

0.2

0.7

0.7

0.7

0.8

0.8

ICE class

II

Fluorescein retention

13

0.5

2

-

-

-

-

14

0.5

2

-

-

-

-

15

0.5

2

-

-

-

-

Mean

0.5

2.0

-

-

-

-

ICE class

III

Corneal thickness

13

0.62

0.63

0.63

0.63

0.63

0.63

14

0.63

0.65

0.65

0.65

0.65

0.65

15

0.60

0.60

0.60

0.60

0.60

0.60

Corneal swelling (%)

13

-

2

2

2

2

2

14

-

3

3

3

3

3

15

-

0

0

0

0

0

Mean

-

2

2

2

2

2

ICE class

I

Classification

1 x III, 1 x II, 1 x I

No prediction can be made

No morphological effects were noted, whatever the examination time.

 

Interpretation of results:
study cannot be used for classification
Remarks:
EU criteria
Conclusions:
The combinations of the 3 endpoints for the test item were 1 x III, 1 x II, 1 x I. Therefore, no prediction can be made.
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 30 µL of physiological saline (negative control). Three eyeballs were used in each 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. According to the overall in vitro classification (UN GHS), no prediction can be made since the combinations of the 3 endpoints for the test item were 1 x III, 1 x II, 1 x I.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
From April 6th to April 9th, 2017.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Deonar Abattoir slaughter house, Mumbai, Maharashtra.
- Characteristics of donor animals (e.g. age, sex, weight): 1-5 years of age.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): transported under cold condition in Hank's Balanced Salt Solution containing antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL).
- Time interval prior to initiating testing: eyes were used within 24 h from the slaughtering.
- indication of any existing defects or lesions in ocular tissue samples: no.
- Indication of any antibiotics used: yes.
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration (if solution): 20% (w/v).

VEHICLE
- Amount(s) applied (volume or weight with unit): 750 μL
- Concentration (if solution): 0.9% w/v sodium chloride (normal saline).
Duration of treatment / exposure:
4 h ± 5 min
Number of animals or in vitro replicates:
3 replicates.
Details on study design:
SELECTION AND PREPARATION OF CORNEAS: All eyes were examined prior to use, corneas free from defects were dissected to a 2-3 mm rim and transferred to a container with Hank's Balanced Salt Solution. Corneas from eyes free of visible defects and with an opacity value inferior than 7 were used. The selected corneas were mounted on the corneal holders with the endothelial side against the O-ring of the posterior half of the holder, the anterior half of the holder was then placed on top of the cornea and fixed with screws. Both chambers were filled with pre-warmed phenol red free Eagle's Minimum Essential Medium (EMEM) and equilibrated at 32 ± 1ºC for at least 1h.

QUALITY CHECK OF THE ISOLATED CORNEAS: Following the equilibration period, the medium was removed from both chambers and baseline opacity readings were taken for each cornea.

NUMBER OF REPLICATES: 3

SOLVENT CONTROL USED: yes. Normal saline (0.9% w/v sodium chloride), source: Axa Parenterals Ltd, lot: A161342.

POSITIVE CONTROL USED: yes. Imidazole (20% w/v solution), source: Sigma Aldrich, lot: SLBP2962V.

APPLICATION DOSE AND EXPOSURE TIME: 750 μL of 20% (w/v) test item solution in normal saline, 4h exposure.

TREATMENT METHOD: closed chamber.

POST-INCUBATION PERIOD: no.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: the corneal epithelium was washed until no visual evidence of test item was observed using EMEM (containing phenol red) and, once the medium was free of test item, a final rinse with phenol red-free EMEM was performed.
- POST-EXPOSURE INCUBATION: no.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: corneal opacity was measured with an opacitometer BASF-OP3.0 (Duratec, Germany)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: as indicated in the TG.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
-0.14
Vehicle controls valid:
yes
Remarks:
2.33
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks:
160.99
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
-0.52
Vehicle controls valid:
yes
Remarks:
2.28
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks:
86.39
Irritation parameter:
other: permeability
Run / experiment:
mean
Value:
0.025
Vehicle controls valid:
yes
Remarks:
0.003
Negative controls valid:
not applicable
Positive controls valid:
yes
Remarks:
4.973
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes

ACCEPTANCE OF RESULTS:
- A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean
- Acceptance criteria met for negative control: yes (historical value range: -1.68 - 2.52, SD: 1.19)
- Acceptance criteria met for positive control: yes (historical value range: 63.28 - 257.23, SD: 64.49; mean = 148.9, 2*SD = 128.99)

Table 1. In vitro Irritation Score.

 

Group : Normal Saline, 0.75 mL (Adopted from JRF Study N° 530-1-01-16969)

Cornea Holder N°

Io

 (LUX)

I (Initial)

(LUX)

Initial Opacity Value

I

(Post Treatment)

(LUX)

Post Treatment Opacity

Value

Corr. Opacity

Value

OD490

Value

Corr. OD490

Value

IVIS

3

1133

1000

5.72

954

7.90

2.18

0.075

0.012

2.36

4

1121

1088

1.63

1032

3.86

2.23

0.057

-0.006

2.14

9

1137

1070

2.92

1012

5.34

2.42

0.067

0.004

2.48

Mean

2.28

-

0.003

2.33

SD

0.13

-

0.009

0.17

 

 

Group : Imidazole at 20% (w/v) in normal saline, 0.75 mL (Adopted from JRF Study N° 530-1-01-16969)

Cornea holder N°

Io

 (LUX)

I (Initial)

(LUX)

Initial Opacity Value

I

(Post

Treatment)

(LUX)

Post Treatment Opacity

Corr. Opacity

Final Opacity

OD490

Corr. OD490

Final OD490

IVIS

Score

6

1133

1006

5.45

397

74.28

68.83

66.55

7.042

6.979

6.976

171.19

7

1162

1131

1.51

346

94.38

92.87

90.59

3.420

3.357

3.354

140.90

11

1162

1097

2.78

316

107.08

104.30

102.02

4.656

4.593

4.590

170.87

Mean

86.39

-

4.976

4.973

160.99

SD

18.10

-

1.841

1.841

17.40

Group : Naringin (suspension) at 20% (w/v) in Normal Saline, 0.75 mL

 

Cornea holder N°

Io

 (LUX)

I (Initial)

(LUX)

Initial Opacity Value

I

(Post

Treatment)

(LUX)

Post Treatment Opacity

Value

Corr. Opacity

Value

Final Opacity

Value

OD490

Value

Corr. OD490

Value

Final OD490

Value

IVIS

 

2

1141

1074

2.91

1019

5.19

2.28

0.00

0.07

0.007

0.004

0.06

 

14

1145

1032

4.78

1002

6.11

1.33

-0.95

0.087

0.024

0.021

-0.64

 

15

1128

1069

2.62

1028

4.30

1.68

-0.60

0.117

0.054

0.051

0.17

 

Mean

-0.52

-

0.028

0.025

-0.14

 

SD

0.48

-

0.024

0.024

0.44

 

Keys: IVIS = In Vitro Irritation Score, Io =Baseline Reading (With medium but without cornea), I = LUX. Reading with Medium and Cornea, OD490= Optical Density at 490 Wave Length, - = Not Applicable, Corr. = Corrected. Blank OD490value = 0.063.

- Initial Opacity Value = [((Io⁄I)-b)/a)], Where I = Initial LUX, Io = Baseline Reading, Note: a (0.0251) and b (0.9894) are constant.

- Post Treatment Opacity Value = [((Io⁄I)-b)/a)], Where, I = Post Treatment LUX, Io = Baseline Reading.

- Corr. Opacity Value = Post Treatment Opacity Value - Initial Opacity Value

- Final Opacity Value = Corr. Opacity Value – Mean Corr. Opacity Value of Control (Group I)

- Corr. OD490Value = OD490Value – Blank OD490Value, Where Blank Value for this trial was 0.063

- Final OD490Value = Corr. OD490Value –Mean Corr.OD490Value of Control (Group I)

- IVIS (Control) = Corr. Opacity Value + (15 x Corrected OD490)

- IVIS (Treatment) = Final Opacity Value + (15 x Final OD490)

Interpretation of results:
GHS criteria not met
Conclusions:
The mean IVIS score for the test item was found to be -0.14. Therefore, the test item is not irritating to the eye (no category).
Executive summary:

An in vitro (ex vivo) Bovine Corneal Opacity and Permeability study was conducted in order to determine the potential severe eye damaging effects of the test item according to the OECD guideline 437 under GLP conditions. Sets consisting of three corneas each were tested: the first set was the positive control, and was treated with 750 μL of 20% (w/v) imidazole in normal saline; the second set was treated with 750 μL of 20% (w/v) of test item suspension in normal saline for 4h at 32ºC; a negative control was carried out in parallel with one cornea, treated with 750 μL normal saline. After exposure, opacity of the corneas was measured. Then, to determine permeability, 1 mL fluorescein solution (5 mg/mL) was applied on the anterior surface of the corneas, while fresh EMEM (phenol red-free) was added to the posterior chamber and, after 90 min incubation at 32ºC, the OD (490 nm) of the medium in the posterior chamber was measured. The mean corneal opacity for the test item treated corneas was -0.52, and the mean permeability was 0.025. The mean IVIS score for the control was 2.33, the mean score for the positive control was 160.99, and the mean IVIS score for the test item was found to be -0.14. Therefore, the test item is not irritating to the eye (no category).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation. Key study. An in vitro skin irritation test of the test item was performed in a reconstructed human SkinEthic™ RH 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. After 42h post-incubation, the viability of each tissue was assessed by incubating the solution with MTT, extracting the precipitated formazan crystals, and determining the OD spectrophotometrically. Under test conditions, the mean corrected percent viability of the treated tissues was 66.7 %, versus 1.0% in the positive control. Therefore, the test item is not irritant to the skin.

Eye irritation. Weight of evidence. First, an in vitro (ex vivo) study was conducted according to OECD guideline 438 (GLP study). Three eyeballs per group were isolated from chickens and, after 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). According to the overall in vitro classification (UN GHS), no prediction can be made since the combinations of the 3 endpoints for the test item were 1 x III, 1 x II, 1 x I.

Due to this inconclusive result, a second in vitro study was considered for this endpoint. An in vitro (ex vivo) Bovine Corneal Opacity and Permeability study was conducted according to OECD guideline 437 (GLP study), in order to determine the potential severe eye damaging effects of the test item. Three corneas per group were exposed to 750 μL normal saline (negative control), 750 μL of 20% (w/v) imidazole in normal saline (positive control) or 750 μL of 20% (w/v) of test item suspension in normal saline for 4h at 32ºC. After exposure, opacity of the corneas was measured. Then, to determine permeability, 1 mL fluorescein solution (5 mg/mL) was applied on the anterior surface of the corneas, while fresh EMEM (phenol red-free) was added to the posterior chamber and, after 90 min incubation at 32ºC, the OD (490 nm) of the medium in the posterior chamber was measured. The mean corneal opacity for the test item treated corneas was -0.52, and the mean permeability was 0.025. The mean IVIS score for the control was 2.33, the mean score for the positive control was 160.99, and the mean IVIS score for the test item was found to be -0.14. Therefore, the test item is not irritating to the eye (no category).

Justification for classification or non-classification

Skin irritation: Based on the available information (66.7 % tissue viability), the test item is not classified for skin irritation/corrosion, in accordance with CLP Regulation (EU) No. 1272/2008.

Eye irritation: Based on the available information (IVIS score of 0), the substance does not cause serious eye damage (Category 1) according to CLP Regulation (EC) No. 1272/2008.