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Administrative data

Description of key information

Skin corrosion: The substance is not corrosive based on absence of skin irritation.

Skin irritation (OECD TG 439): not irritating

Eye irritation (OECD TG 438): irritating

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:
28 September 2016 - 3 October 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)
Version / remarks:
28 July 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
23 July 2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test material information:
Composition 1
Test system:
human skin model
Source species:
other: 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 TM
- Tissue batch number(s): 16-EKIN-039
- Production date/Shipping date: no data
- Delivery date: 27 September 2016
- Date of initiation of testing: 29 September 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure / post-treatment incubation: 37 °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: not specified
- Wavelength: 562 nm (without reference fildeter)
- Filter: no data
- Linear OD range of spectrophotometer: no data

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not required as no MTT colour interference expected (assessment done).

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:
- Applied volume: 10 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:
109.2
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks:
5.9% viability
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: No colour changes observed

DEMONSTRATION OF TECHNICAL PROFICIENCY: No data

ACCEPTANCE OF RESULTS:
-The relative mean tissue viability for the positive control treated tissues was 5.9% relative to the negative control treated tissues and the standard deviation value of the viability was 1.7%. The positive control acceptance criteria were therefore satisfied.
-The mean OD562 for the negative control treated tissues was 1.013 and the standard deviation value of the viability was 2.8%. The negative control acceptance criteria were therefore satisfied.
-The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 12.7%. The test item acceptance criterion was therefore satisfied.

Mean OD562Values and Viabilities for the Negative Control Item, Positive Control Item and Test Item

Item OD562 of
tissues
Mean OD562
of triplicate
tissues
± SD of
OD562
Relative
individual
tissue
viability (%)
Relative
mean
viability (%)
± SD of
Relative
mean
viability (%)
Negative
Control Item
0.981 / 1.031 / 1.028 1.013 0.028 96.8 / 101.8 / 101.5 100* 2.8
Positive
Control Item
0.078 / 0.045 / 0.058 0.060 0.017 7.7 / 4.4 / 5.7 5.9 1.7
Test Item 1.242 / 0.986 / 1.090 1.106 0.129 122.6 / 97.3 / 107.6 109.2 12.7

OD=Optical Density

SD=Standard deviation

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

Interpretation of results:
other: not a skin irritant
Remarks:
in accordance with EU CLP (1272/2008 and its amendments)
Conclusions:
Under the conditions of this test, the relative mean tissue viability for the test item determined to be 109.2%. This value is well above the 50% viability threshold for irritancy. Based on the results obtained, it can be concluded that Muguesia is not an irritant to skin.
Executive summary:

The possible skin irritation potential of Muguesia 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 10 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 5.9% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly. Under the conditions of this test, the relative mean tissue viability for the test item determined to be 109.2%. This value is well above the 50% viability threshold for irritancy. Based on the results obtained, it can be concluded that Muguesia is not an irritant to 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:
12-08-2016 to 21-09-2016
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)
GLP compliance:
yes (incl. certificate)
Remarks:
Triskelion B.V., Utrechtseweg 48, 3700 AV, Zeist
Test material information:
Composition 1
Species:
other: eyes of male or female chickens (ROSS, spring chickens)
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Slaughterhouse v.d. Bor, Nijkerkerveen, The Netherlands
- Characteristics of donor animals: Approximately 7 weeks old, male or female chickens, body weight range approximately 1.5-2.5 kg, were used as eye donors.
- Storage, temperature and transport conditions of ocular tissue: 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.
- Time interval prior to initiating testing: Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus.
- Indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: No
Vehicle:
unchanged (no vehicle)
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using the following procedure: First the eye-lids were carefully removed without damaging the cornea and a small drop of Fluorescein sodium 2.0% w/v was applied to the corneal surface for a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp 900 BP, Haag-Streit AG, Liebefeld-Bern, Switzerland) to ensure that the cornea was not damaged. If undamaged (e.g., fluorescein retention and corneal opacity scores of ≤ 0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was taken to remove the eye-ball from the orbit without cutting off the optical nerve too short. The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus. The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a target rate of 0.10-0.15 mL/min. The chambers of the superfusion apparatus as well as the saline were temperature controlled at approximately 32 °C (water pump set at 36.4 °C). After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged. An accurate measurement was taken at the corneal apex of each eye. Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, eyes showing opacity (score higher than 0.5), or were unacceptably stained with fluorescein (score higher than 0.5) indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced.

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

NUMBER OF REPLICATES
Negative control: 1
Positive control: 3
Test group: 3

NEGATIVE CONTROL USED
Physiological saline

POSITIVE CONTROL USED
Benzalkonium Chloride 5%

APPLICATION DOSE AND EXPOSURE TIME
30 μL for 10 seconds

OBSERVATION PERIOD
240 minutes

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: 20 mL saline. After rinsing, each eye in the holder was returned to its chamber.
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Slit-lamp microscope examination
- Damage to epithelium based on fluorescein retention: Slit-lamp microscope examination
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: set at 0.095 mm
- Others: After the final examination, the test substance treated eyes, the negative and positive control eyes were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at ca 4 μm and stained with PAS (Periodic Acid-Schiff). The microscopic slides were subjected to histopathological examination.

SCORING SYSTEM:
Defined scoring scales were used for each parameter to define the severity of effects into four categories (I-IV).
- Mean corneal swelling (%): According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean maximum opacity score: According to OECD 438 guideline. Examination of the eyes after 0, 30, 75, 120, 180, and 240 minutes
- Mean fluorescein retention score at 30 minutes post-treatment: According to OECD 438 guideline.

DECISION CRITERIA: According to OECD 438 guideline
Irritation parameter:
percent corneal swelling
Run / experiment:
slit-lamp examination
Value:
4
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: maximum mean values
Irritation parameter:
cornea opacity score
Run / experiment:
slit-lamp examination
Value:
2
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: maximum mean values
Irritation parameter:
fluorescein retention score
Run / experiment:
slit-lamp examination
Value:
2
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes
Other effects / acceptance of results:
Slit-lamp examination: The test substance caused corneal effects consisting of very slight corneal swelling (mean of 4%), moderate opacity (mean score of 2.0) and moderate fluorescein retention (mean score of 2.0).
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 severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants.

Microscopic examination of the corneas treated with the test substance revealed very slight or slight erosion, slight or moderate necrosis, and very slight or slight vacuolation 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 or severe erosion and slight or moderate vacuolisation of the epithelium, the epithelium partly detached from the basement membrane (two corneas), and endothelial necrosis (two corneas).
Interpretation of results:
other: Category 2 (irritating to eyes)
Remarks:
in accordance with EU-CLP (1272/2008 and its amendments)
Conclusions:
Under the test conditions (OECD 438 and GLP) the test substance is considered to be an eye irritant.
Executive summary:

In accordance to 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 very slight corneal swelling (mean of 4%), moderate opacity (mean score of 2.0) and moderate fluorescein retention (mean score of 2.0). 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 severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance revealed very slight or slight erosion, slight or moderate necrosis, and very slight or slight vacuolisation 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 or severe erosion and slight or moderate vacuolation of the epithelium, the epithelium partly detached from the basement membrane (two corneas), and endothelial necrosis (two corneas). Under the test conditions (OECD 438 and GLP), the test substance is considered to be an eye irritant.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

Skin irritation in vitro

The possible skin irritation potential of Muguesia 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 10 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 5.9% after 15 minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 18%, indicating that the test system functioned properly. Under the conditions of this test, the relative mean tissue viability for the test item determined to be 109.2%. This value is well above the 50% viability threshold for irritancy. Based on the results obtained, it can be concluded that Muguesia is not an irritant to skin.

Eye irritation in vitro

In accordance to 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 very slight corneal swelling (mean of 4%), moderate opacity (mean score of 2.0) and moderate fluorescein retention (mean score of 2.0). 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 severe corneal effects and demonstrated the ICE test valid to detect severe eye irritants. Microscopic examination of the corneas treated with the test substance revealed very slight or slight erosion, slight or moderate necrosis, and very slight or slight vacuolisation 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 or severe erosion and slight or moderate vacuolation of the epithelium, the epithelium partly detached from the basement membrane (two corneas), and endothelial necrosis (two corneas). Under the test conditions (OECD 438 and GLP), the test substance is considered to be an eye irritant.

Justification for classification or non-classification

Based on the results found in the EpiSkin test, Muguesia is not considered to be a skin irritant and should therefore not need to be classified as such in accordance with the criteria outlined in EU-CLP (1272/2008/EC and its amendments).

Based on the positive results found in the in vitro eye irritation test, Muguesia is considered to cause eye irritation. In accordance with the criteria outlined EU-CLP Regulation (1272/2008/EC and its amendments), this results in a Category 2 classification for this endpoint (H319 / Causes serious eye irritation).