Registration Dossier

Administrative data

Description of key information

Skin corrosion: Skin corrosion is not anticipated in view of absence of acidic and base groups in the chemical structure of the substance. In addition, no skin corrosion was seen in the LLNA up to 50%.

Skin irritation (OECD TG 439): irritating

Eye irritation (OECD TG 438): irritating

Respiratory irritation: not 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:
16 Jan 2017 to 10 Feb 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
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:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Envigo CRS GmbH, In den Leppsteinswiesen 19, 64380 Rossdorf, Germany
Test material information:
Composition 1
Test system:
human skin model
Source species:
human
Cell type:
other: EPISKIN™ in vitro Reconstructed Human Epidermis (RHE) Model Kit
Justification for test system used:
Because systemic reactions play a minor role in modulating local skin toxicity potential of chemicals, skin irritation potential may be predicted by in vitro systems, provided they are sufficiently complex to mimic human skin barrier and cell reactivity. In an international validation study performed by ECVAM, the in vitro skin irritation test using the human skin models EpiSkin™ and EpiDerm and measurement of cell viability by dehydrogenase conversion of MTT into a blue formazan salt have turned out as a sufficiently promising predictor for skin irritancy potential.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ in vitro Reconstructed Human Epidermis (RHE) Model Kit
- Tissue batch number(s): 17-EKIN-006
- Supplier: SkinEthic Laboratories (69007 Lyon, France)
- The EpiSkin™ tissues (surface 0.38 cm²) are cultured on specially prepared cell culture inserts.
- EpiSkin™ tissues were shipped at ambient temperature on medium-supplemented agarose gels in a 12-well plate
- Delivery date: 07 February 2017.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1.5 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
The tissues were gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the inserts and blotting the bottom with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- 2 mL assay medium containing 0.3 mg/mL MTT per well.
- Incubation time: 3hrs at 37±1.5°C
- Spectrophotometer: Microplate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, version 4.7.1)
- Wavelength: 570 nm
- After MTT-loading a biopsy of each epidermis was made and placed into micro tubes containing isopropanol containing 0.04 N HCl for extraction of formazan crystals out of the MTT-loaded tissues.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL AND CONTROLS
10 µL
Duration of treatment / exposure:
15 min.
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
15 min exposure with 10 µL undiluted substance
Value:
9.4
Vehicle controls valid:
not applicable
Negative controls valid:
yes
Positive controls valid:
yes

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference). Also its intrinsic colour was not intensive. Consequently, additional tests with freeze-killed or viable tissues were not necessary.

Interpretation of results:
other: Skin irritant category 2
Remarks:
in accordance with CLP (1272/2008 and its updates)
Conclusions:
Under the conditions of this study the test item was considered to be irritating to the skin, because the relative mean tissue viability was below 50% after 15 min exposure.
Executive summary:

The skin irritation potential of the test substance was tested in vitro using the EPISKIN™ after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The study procedures in the study were according to OECD TG 439 and GLP principles. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test substance by means of the colorimetric MTT reduction assay. Triplicate tissues were treated with 10 µL undiluted test item for an exposure period of 15 minutes. Concurrent positive (5% SLS solution in deionized water) and negative (deionized water) controls were included. After MTT-loading a biopsy of each epidermis was made and placed into micro tubes containing isopropanol containing 0.04 N HCl for extraction of formazan crystals out of the MTT-loaded tissues. The optical density was measured at 570 nm. Data are presented in the form of relative viability (%) (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 9.4%. Under the conditions of this study the test item was considered to be irritating to the skin, because the relative mean tissue viability was below 50% after 15 min exposure.

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:
30 Nov 2016 to 25 Jan 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
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)
Version / remarks:
26 July 2013
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)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
30 µL
Duration of treatment / exposure:
10 seconds
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 0.9%

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:
6
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:
1.5
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:
1.5
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 or slight corneal swelling (mean of 6%), slight-to-moderate opacity (mean score of 1.5) and slight-to-moderate fluorescein retention (mean score of 1.5). 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 erosion, very slight necrosis (one cornea) and very slight vacuolation (two corneas) of the epithelium. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities other than very slight erosion of the epithelium, which is considered an acceptable background finding. Microscopic examination of the corneas treated with the positive control BAC 5% revealed moderate erosion and very slight (two corneas) or slight (one cornea) vacuolation of the epithelium, the epithelium partly detached from the basement membrane, and endothelial necrosis (two corneas).
Interpretation of results:
other: Irritating to eyes (Category 2)
Remarks:
Based on CLP criteria (EC 1272/2008 and its updates)
Conclusions:
Under the test conditions (OECD 438, GLP) the test substance is considered irritating to eyes (Category 2) in accordance with the criteria outlined in Annex I of the CLP regulations (1272/2008/EC).
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 or slight corneal swelling (mean of 6%), slight-to-moderate opacity (mean score of 1.5) and slight-to-moderate fluorescein retention (mean score of 1.5).  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 erosion, very slight necrosis (one cornea) and very slight vacuolation (two corneas) of the epithelium. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities other than very slight erosion of the epithelium, which is considered an acceptable background finding. Microscopic examination of the corneas treated with the positive control BAC 5% revealed moderate erosion and very slight (two corneas) or slight (one cornea) vacuolation of the epithelium, the epithelium partly detached from the basement membrane, and endothelial necrosis (two corneas). Based on these results, the test substance should be classified as irritating to eyes.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Respiratory irritation

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

Additional information

Skin corrosion: Skin corrosion is not anticipated in view of absence of acidic and base groups in the chemical structure of the substance. In addition, no skin corrosion was seen in the LLNA up to 50%.

In vitro skin irritation

The skin irritation potential of the test substance was tested in vitro using the EPISKIN™ after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The study procedures in the study were according to OECD TG 439 and GLP principles. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures following topical exposure to the test substance by means of the colorimetric MTT reduction assay. Triplicate tissues were treated with 10 µL undiluted test item for an exposure period of 15 minutes. Concurrent positive (5% SLS solution in deionized water) and negative (deionized water) controls were included. After MTT-loading a biopsy of each epidermis was made and placed into micro tubes containing isopropanol containing 0.04 N HCl for extraction of formazan crystals out of the MTT-loaded tissues. The optical density was measured at 570 nm. Data are presented in the form of relative viability (%) (MTT reduction in the test item treated tissues relative to negative control tissues). The relative mean viability of the test item treated tissues was 9.4%. Under the conditions of this study the test item was considered to be irritating to the skin, because the relative mean tissue viability was below 50% after 15 min exposure.

In vitro eye irritation

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 or slight corneal swelling (mean of 6%), slight-to-moderate opacity (mean score of 1.5) and slight-to-moderate fluorescein retention (mean score of 1.5).  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 erosion, very slight necrosis (one cornea) and very slight vacuolation (two corneas) of the epithelium. Microscopic examination of the cornea treated with the negative control (saline) did not reveal any abnormalities other than very slight erosion of the epithelium, which is considered an acceptable background finding. Microscopic examination of the corneas treated with the positive control BAC 5% revealed moderate erosion and very slight (two corneas) or slight (one cornea) vacuolation of the epithelium, the epithelium partly detached from the basement membrane, and endothelial necrosis (two corneas). Based on these results, the test substance should be classified as irritating to eyes.

Respiratory irritation:

For respiratory irritation mostly human data are used for the assessment because no suitable in vitro or in vivo tests are available that can identify respiratory irritation (REACH guidance R.7.2.3). There are no human data such as indicated in R7.2.3 of the ECHA guidance that indicate respiratory reactions of the substance e.g. from consumer experience or occupational exposure. Due to its low vapour pressure (4.2 Pa, insufficient air concentration will reach the lung during handling) the inhalation concentration will be low and because the substance is not corrosive or severely irritating respiratory irritation is unlikely minimising the respiratory irritation hazard (REACH guidance: 7.2.1.2).

Justification for classification or non-classification

According to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 the substance does not need to be classified for skin corrosion and respiratory irritation but has to be classified as a skin and eye irritant Category 2 and the following H phrases need to be assigned: H315 and 319, respectively.