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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 1.2% versus 1.5% in the positive control. Therefore, the test item must be considered at least as skin irritant (cat 2).

Eye irritation (in vitro): Key study. Test method according to the OECD Guideline 438 with GLP. The test item was determined to not cause severe damage or irrritation for eyes.

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:
15/02/2018 - 22/02/2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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-013
- Delivery date: 20/02/2018
- Expiration date: 26/02/2018
- Date of initiation of testing: 20/02/2018

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=1.3 (CV = 2.4%) 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: 4.6 h (Specification 4.0h < ET50< 10.0h)
- Morphology: 7 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:
1.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
(DPBS)
Positive controls validity:
valid
Remarks:
1.5% viability (5% SDS)
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The mean percent viability of the treated tissues was 1.2% versus 1.5% 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 3.3% (acceptablility criteria, SD ≤ 18%) and OD mean was 1.194 (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 0.1% (acceptablility criteria, SD ≤ 18%) and mean viability was 1.5% which is much lower than 50%.
- Acceptance criteria met for variability between replicate measurements: yes. SD of test item was 0.1% (acceptablility criteria, SD ≤ 18%).

Table 1. Summary of results.

 

Skin

OD

Mean OD /disc (#)

Mean OD / product

Viability

%

Mean viability

%

SD

Viability

Conclusion

Negative

Control

1

1.157

  

1.228

1.194

102.9

100.0

3.3

 

1.257

1.271

2

1.153

1.151

96.4

1.149

1.150

3

1.182

1.202

100.7

1.206

1.217

Positive

Control

4

0.018

0.019

0.018

1.6

1.5

0.1

Irritant

0.020

0.019

5

0.017

0.017

1.4

0.017

0.016

6

0.018

0.017

1.4

0.018

0.016

Test item

7

0.016

0.016

0.015

1.3

1.2

0.1

Irritant

or corrosive

0.016

0.017

8

0.016

0.015

1.3

0.015

0.015

 

9

0.014

0.013

1.1

0.013

0.013

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

OD: optical density

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

Acceptability criteria: SD≤18%.

Interpretation of results:
other: classified at least as 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 1.2% versus 1.5% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item must be considered at least as skin irritant (cat 2).
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 1.2%, versus 1.5% 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 conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
19 February 2018
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.
- 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 19 February 2018 at 8:20 am. The eyes were enucleated at Phycher on 19 February 2018 at 9:40 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 33.0ºC and 33.5ºC.
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 - Cooper Batch No. 19LF27GB (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:
0.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE Class I
Irritation parameter:
fluorescein retention score
Run / experiment:
Mean
Value:
1.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE class II
Irritation parameter:
percent corneal swelling
Run / experiment:
Mean
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
ICE class I
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 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

4

0

0

0

0

0.5

0.5

5

0

0

0.5

0.5

0.5

0.5

6

0

0

0.5

0.5

0.5

0.5

Mean

 

0.0

0.0

0.3

0.3

0.5

0.5

ICE class

 

 

 

 

I

 

 

 

Fluorescein retention

4

0.5

2

 

 

 

 

5

0.5

0.5

 

 

 

 

6

0.5

1

 

 

 

 

Mean

 

0.5

1.2

 

 

 

 

ICE class

 

 

II

 

 

 

 

 

 

 

Corneal thickness

4

0.59

0.59

0.59

0.59

0.59

0.59

5

0.62

0.62

0.62

0.62

0.62

0.62

6

0.57

0.57

0.57

0.57

0.57

0.57

 

Corneal swelling (%)

4

-

0

0

0

0

0

5

-

0

0

0

0

0

6

-

0

0

0

0

0

Mean

 

 

0

0

 0

0

0

ICE class

 

 

 

 

I

 

 

Combination of the 3 Endpoints

2 x I, 1 x II

CLASSIFICATION

No category

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

0.58

0.61

0.63

0.72

0.74

0.82

2

0.58

0.68

0.68

0.72

0.76

0.81

3

0.56

0.63

0.65

0.68

0.70

0.75

 

Corneal swelling (%)

1

-

5

9

24

28

41

2

-

17

17

24

31

40

3

-

13

16

21

25

34

Mean

 

-

12

14

23

28

38

ICE class

 

 

 

 

IV

 

 

Combination of the 3 Endpoints

3 x IV

CLASSIFICATION

Category 1 : Corrosive/Severe irritant

Note: Blisters on skin 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

0.5

0.5

0.5

ICE class

 

 

 

 

I

 

 

Fluorescein retention

16

0.5

0.5

-

-

-

-

ICE class

 

 

I

 

 

 

 

Corneal thickness

16

0.61

0.61

0.61

0.61

0.61

0.61

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: Negative (CLP Regulation EC no. 1272/2008)
Conclusions:
The test item was determined to not cause severe damage or irrritation for eyes 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. According to CLP Regulation EC no. 1272/2008 the test item does not require classification for eye irritation and serious eye damage as defined by the UN GHS (No category), since the combinations of the 3 endpoints for the test item were 2x I and 1xII.

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 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 3.2%, 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).

Eye irritation (in vitro): Key study. 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. According to CLP Regulation EC no. 1272/2008 the test item does not require classification for eye irritation and serious eye damage as defined by the UN GHS (No category), since the combinations of the 3 endpoints for the test item were 2x I and 1xII.

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 damage/irritation: Although the result of the in vitro test OECD TG 438 does not trigger the need for classification, it was decided as a precautionary principle to classify the substance as eye irritation (cat. 2) too, taking into account that is has been determined to be a skin irritant (cat. 2), which is somewhat an inconsistent result if considering that the eyes should be more sensitive than the skin.