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Description of key information

Skin irritation/corrosion in vitro: Key study. Method according to OECD 439, GLP study. The mean corrected percent viability of the treated tissues was 93.2 %, 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. Method according to OECD 437, GLP study. According to the overall in vitro classification, no prediction can be made since the combination of the 3 endpoints for the test item was 1 x III, 2 x II.

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 June 6th, 2018 to June 14th, 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
Source species:
human
Cell type:
other: normal human keratinocytes.
Cell source:
other: human adult donor, not specified.
Source strain:
not specified
Details on animal used as source of test system:
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-064
- Delivery date: 12/06/2018
- Expiration date: 18/06/2018
- Date of initiation of testing: 06/06/2018

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 36.2 - 38.0°C, 5% CO2
- Spectrophotometer: ELx800 absorbance microplate reader (BioTek)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: O.D. = 1.1 (CV 2.7%)
- Barrier function: n/a
- 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):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
93.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
(DPBS)
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: 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.
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 1. Summary of results (12/06/2018).

 

Skin

OD

Mean OD/
disc (#)

Mean OD/
product

Viability
%

Mean
viability
%

SD

Conclusion

Negative
control

1

0.936
1.024
1.036

0.999

0.923

108.3

100.0

7.9

-

2

0.852
0.849
0.858

0.853

92.4

3

0.914
0.928
0.905

0.916

99.3

Positive
control

4

0.011
0.013
0.013

0.013

0.012

1.4

1.3

0.1

Irritant

5

0.012
0.012
0.012

0.012

1.3

6

0.012
0.012
0.012

0.012

1.3

Test ítem

PH-16/0272

13

0.836
0.930
0.908

0.892

0.860

96.7

93.2

3.4

Non irritant

14

0.823
0.838
0.829

0.830

90.0

15

0.856
0.848
0.872

0.859

93.1

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

Interpretation of results:
GHS criteria not met
Remarks:
(EU criteria)
Conclusions:
The mean corrected percent viability of the treated tissues was 93.2 %, versus 1.3% 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 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 corrected percent viability of the treated tissues was 93.2%, versus 1.3% 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
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From May 25th, 2018 to May 28th, 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:
no
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
chicken
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: eyes collected from chickens obtained from a slaughterhouse (Etablissement Brun, 33820 Etauliers, France) where they are killed for human consumption.
- Characteristics of donor animals (e.g. age, sex, weight): 7 weeks old. 1.5 - 2.5 kg.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Because eyes were dissected in the laboratory, the intact heads were transported from the slaughterhouse at ambient temperature in plastic boxes humidified with towels moistened with physiological saline.
- Time interval prior to initiating testing: The heads were collected on 28 May 2018 at 8:30 am. The eyes were enucleated at Phycher on 28 May 2018 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
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 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

SOLVENT CONTROL USED: not applicable.

POSITIVE CONTROL USED: 30 mg sodium hydroxide

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 (2 rinses with 10 mL each).
- 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:
2.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE class III
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE class II
Irritation parameter:
percent corneal swelling
Run / experiment:
mean
Value:
7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: ICE class II
Irritation parameter:
morphological effects
Run / experiment:
mean
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
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), 28/05/2018.

Endpoint measured

Eye No.

0

30

75

120

180

240

Corneal opacity

13

0

1

1

1

1

1

14

0

1

1

1

1

1

15

0

1

1

1

1

1

Mean

0.0

1.0

1.0

1.0

1.0

1.0

ICE class

II

Fluorescein

retention

13

0.5

3

-

-

-

-

14

0.5

2

-

-

-

-

15

0.5

2

-

-

-

-

Mean

0.5

2.3

-

-

-

-

ICE class

III

Corneal

thickness

13

0.59

0.59

0.61

0.63

0.63

0.63

14

0.64

0.64

0.66

0.66

0.66

0.66

15

0.59

0.60

0.64

0.64

0.65

0.65

Corneal

swelling (%)

13

-

0

3

7

7

7

14

-

0

3

3

3

3

15

-

2

8

8

10

10

Mean

-

1

5

6

7

7

ICE class

II

Combination of the 3 endpoints

1 x III, 2 x II

Classification

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, 2 x II. 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 (CLP Regulation), no prediction can be made since the combinations of the 3 endpoints for the test item were 1 x III, 2 x II.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

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