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Administrative data

Description of key information

Skin corrosion: Substance is not skin irritating and therefore also not corrosive

Skin irritation (OECD TG 439): Not skin irritating

Eye irritation (OECD TG 438): 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:
14 March 2016 - 21 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
in accordance with GLP conditions
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
(2015)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
(2012)
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes
Cell source:
other: SkinEthic Laboratories, Lyon, France.
Source strain:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM
- Tissue batch number: 16-EKIN-011
- Production / shipping / delivery date: No data
- Date of initiation of testing: 14 March 2016

PRE-TEST PROCEDURE:
- Pre-incubation: On day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed Maintenance Medium for approximately 22 hours at 37°C.
- Test item colour interference: To assess colour interference, 10 μL of Peomosa (mono-constituent) was added to 90 μL Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μL Milli-Q water was tested concurrently. At the end of the shaking period a colour check was performed.
- Test item MTT reduction: To assess the ability of the test item to reduce MTT, 25 μL of the test item was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently. At the end of the incubation period a colour check was performed.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure / post-treatment incubation: 36.3 - 37.3 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
Tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml in PBS
- Incubation time: 3 hours at 37 °C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: No data
- Linear OD range of spectrophotometer: No data

DECISION CRITERIA
- A test substance is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test substance is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Test material
- Applied volume: 25 μL
Duration of treatment / exposure:
15 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Relative mean tissue viability compared to the negative control tissues (100%)
Value:
74
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:
- Direct-MTT reduction: No colour changes observed
- Colour interference with MTT: Not colour changes observed

DEMONSTRATION OF TECHNICAL PROFICIENCY: No data

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 44%. The positive control meets the validity criterion meets the validity criterion even though it is just outside the historical control range, which has not affected the result of the results for the test substance.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was less than 17%, indicating that the test system functioned properly.

Mean OD570 Values and Percentage Viabilities for the Negative Control Item, Positive Control Item and Test Item:

Item

OD570 of

tissues

Mean OD562

of triplicate

tissues

± SD of

OD570

Relative

individual

tissue

viability (%)

Relative

mean

viability (%)

Negative

Control Item

1.066

1.047

0.022

100

1.052

1.024

Positive Control Item

0.398

0.462

0.078

37

44

0.549

52

0.437

43

Test Item

0.796

0.770

0.163

75

74

0.595

57

0.919

90

SD = Standard deviation

*The mean viability of the negative control tissues is set at 100%.

Interpretation of results:
other: not skin irritating
Remarks:
According to EU CLP 1272/2008 and its amendments.
Conclusions:
Under the conditons of this test, the relative mean tissue viability for the test item determined to be 74%. Based on this result, the substance is considered to be non-irritant and does not need to be classified for skin irritation in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC) and its amendments.
Executive summary:

The possible skin irritation potential of Peomosa was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 µL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 44% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 5%, indicating that the test system functioned properly. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 74%. Based on this result, the substance is considered to be non-irritant and does not need to be classified for skin irritation in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC)

and its amendments

.
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:
22 March 2016 - 4 April 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
in accordance with GLP conditions
Reference:
Composition 1
Qualifier:
according to
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Species:
chicken
Strain:
other: ROSS, spring chickens
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Number of animals: No data
- Characteristics of donor animals: male/female, approximately 7 weeks of age and 1.5-2.5 kg
- Storage, temperature and transport conditions of ocular tissue: Transport in small plastic boxes on tissues moistened with isotonic saline at ambient temperature
- Time interval prior to initiating testing: Within 2 hours after kill
- Indication of any existing defects or lesions in ocular tissue samples: Only undamaged eyes used
- Indication of any antibiotics used: No data
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 30 µL
Duration of treatment / exposure:
10 seconds
Observation period (in vivo):
240 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
- Heads of the animals were cut off immediately after sedation of the animals by electric shock and incision of the neck for bleeding, and before they reached the next station on the process line. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.
- The preparation and validation of the eyes prior to the ICE-test were all according to OECD guideline 438.

EQUILIBRATION AND BASELINE RECORDINGS
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. After an equilibration period of 45-60 minutes, the corneal thickness of the eyes was measured to determine the zero reference value for corneal swelling calculations.

OBSERVATION PERIOD
Examination of the eyes was performed after 0, 30, 75, 120, 180 and 240 minutes

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The eyes were rinsed with 20 mL saline after 10 seconds exposure

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity, fluorescein retention and swelling: slit-lamp microscope (Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland)
- Macroscopic morphological damage to the surface: microscope
- Others: histopathology (preserved with 4% formaldehyde and stained with PAS)

SCORING SYSTEM:
- Mean corneal swelling (%), mean maximum opacity score and mean fluorescein retention score at 30 minutes post-treatment: According to criteria specified in OECD TG 438

DECISION CRITERIA: According to OECD TG 438.
Irritation parameter:
percent corneal swelling
Run / experiment:
Slit-lamp examination
Value:
13
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Maximum mean score
Irritation parameter:
cornea opacity score
Run / experiment:
Slit-lamp examination
Value:
3
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: Maximum mean score
Irritation parameter:
fluorescein retention score
Run / experiment:
Slit-lamp examination
Value:
3
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: Microscopic examination of the corneas treated with Peomosa revealed very slight or slight erosion, moderate necrosis (two corneas) and moderate and severe vacuolation (two corneas) of the epithelium. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities. Microscopic examination of the corneas treated with the positive control BAC 5% revealed slight, moderate or severe erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane (one cornea), and endothelial necrosis (one cornea).

DEMONSTRATION OF TECHNICAL PROFICIENCY: This OECD TG was developed at this CRO and therefore the test has been performed for many years.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate.
- Acceptance criteria met for positive control: The positive control BAC 5% caused moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.
Interpretation of results:
other: Category 1 (serious eye damage)
Remarks:
According to EU CLP 1272/2008 and its amendments.
Conclusions:
Under the test conditions (OECD 438 and GLP), the test substance is considered to cause serious damage to the eye and should be classified as such (Eye Dam. 1 / H318) in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC) and its amendments.
Executive summary:

In accordance with OECD guideline 438 and GLP, the test substance was examined for its in vitro eye irritating potential using the Isolated Chicken Eye (ICE) Test. In the ICE test, 3 eyes were exposed to 30 µL test substance for 10 seconds. In addition, one negative control eye (30 µL saline) and three positive control eyes (30 µL Benzalkonium Chloride (BAC)) were tested. After the exposure the eyes were rinsed with 20 mL saline and were examined at approximately 0, 30, 75, 120, 180, and 240 minutes after treatment. The test substance caused corneal effects consisting of slight corneal swelling (mean of 13%), severe opacity (mean score of 3.0) and severe fluorescein retention (mean score of 3.0). In addition, loosening of epithelium was observed. Microscopic examination of the corneas revealed very slight or slight erosion, moderate necrosis (two corneas) and moderate and severe vacuolation (two corneas) of the epithelium. The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. Microscopic examination did not reveal any abnormalities. The positive control BAC 5% caused moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination revealed slight, moderate or severe erosion and very slight or slight vacuolation of the epithelium, the epithelium partly detached from the basement membrane (one cornea), and endothelial necrosis (one cornea). Based on these results, the test substance is considered to cause serious damage to the eye and should be classified as such (Eye Dam. 1 / H318) in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC)

and its amendments

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

Additional information

In vitro skin irritation

The skin irritation potential of Peomosa was tested in vitro using a human skin model in accordance with OECD TG 439 under GLP conditions. Reliable negative and positive controls were included. The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was found to be 74%. Based on this result, the substance is considered to be non-irritant.

In vitro eye irritation

The eye irritating potential of Peomosa was tested in an Isolated Chicken Eye (ICE) Test in accordance with OECD TG 438 and under GLP conditions. The negative control eye did not show any corneal effect and demonstrated that the general conditions during the tests were adequate. The positive control BAC 5% caused moderate or severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. The test substance caused corneal effects consisting of slight corneal swelling (mean of 13%), severe opacity (mean score of 3.0) and severe fluorescein retention (mean score of 3.0). In addition, loosening of epithelium was observed. Microscopic examination of the corneas revealed very slight or slight erosion, moderate necrosis (two corneas) and moderate and severe vacuolation (two corneas) of the epithelium. Based on these results, the test substance is considered to cause serious damage to the eye.

Justification for classification or non-classification

Based on the negative result found in the in vitro skin irritation test, the substance does not need to be classified as a skin irritant in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC) and its amendments.

Based on the positive result found in the ICE test the substance is considered to cause serious eye damage. In accordance the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC) and its amendments, this results in a Category 1 classification for this endpoint (H318: Causes serious eye damage).