Registration Dossier

Administrative data

Description of key information

In an experimental study according to OECD test guideline 439 under GLP conditions, the test substance did not reduce cell viability (96% with an SD of 14.52).

Therefore, it is considered not irritating to skin.

In an experimental study according to OECD test guideline 438 under GLP conditions, the substance scored into ICE class II for corneal opacity and fluorescein retention. It scored into ICE class I for corneal swelling.

In an experimental study according to OECD test guideline 492 under GLP conditions, the test substance substantially reduced the cell viability in two experiments (55.8% and 29.0%).

Therefore, it is considered as eye irritant/inducing 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
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)
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Product no. B4429, Lot no. SLBK5235V
- Expiration date of the lot/batch: 30 April 2018
- Purity test date: na

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no
Test system:
human skin model
Source species:
human
Vehicle:
unchanged (no vehicle)
Details on test system:
TISSUE
- Model used: EPISKIN(TM) Small Model (SM)
- Tissue batch number(s): 16-EKIN-026
- Production date: not specified
- Shipping date: not specified
- Delivery date: not specified
- Date of initiation of testing: 29.06.2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 37°C for 42 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 mL PBS solution used to thoroughly rinse the tissue once
- Observable damage in the tissue due to washing: none
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 2 mL of 0.3 mg/mL MTT per well
- Incubation time: 3 hours +/- 5 min
- Spectrophotometer: not specified
- Wavelength: 570 nm
- Filter: not specified
- Filter bandwidth: not specified
- Linear OD range of spectrophotometer: not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA: secured to batch release quality control

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: no direct MTT interference

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be non-irritating to skin if the viability is greater than or equal to 50%
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg (to moistened skin)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL
- Concentration (if solution): 5% aq.
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:
1
Value:
96
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:

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
- Range of historical values if different from the ones specified in the test guideline: -

In an experimental study according to OECD test guideline 439 under GLP conditions, the test substance did not reduce cell viability (96% with an SD of 14.52).

Therefore, it is considered not irritating to skin.

Interpretation of results:
GHS criteria not met
Executive summary:

The skin irritation potential of the test substance was investigated in an experimental study according to OECD test guideline 439 under GLP conditions using the EPISKINTMSM. The EPISKINTMSM is provided in kits, which are manufactures according to defined quality assurance procedures.

The testing laboratory, which had demonstrated its technical proficiency in conducting the test, received the kit used for testing in good order.

The test substance was applied (10 mg) for 15 minutes to three tissues. After thorough rinsing, the treated tissue were incubated for 42h at 37°C. Then the MTT test was performed, followed by formazan extraction and cell viability measurement with a spectrometer at 570 nm.

Concurrent negative and positive control fulfilled all acceptance criteria, as did the test substance, which did not directly reduce MTT.

With a mean cell viability of 96% (as compared to the negative control), the test substance did only very marginally reduce tissue viability.

Using the standard prediction (with a threshold of 50%) the test substance is considered to be not skin irritating and does not require to be classified (UN GHS 'No Category').

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
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)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Product no. B4429, Lot no. SLBK5235V
- Expiration date of the lot/batch: 30 April 2018
- Purity test date: na

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no
Species:
chicken
Strain:
other: ROSS 308
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Chicken heads for obtained from TARAVIS KFT, 9600 Sárvár, Rábasömjéni út 129, Hungary
- Age at study initiation: eyes for approx. 2 hours old (after head removal)
- Weight at study initiation: not relevant


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): 0.03 g (per eye)


CONTROLS
- Amount(s) applied (volume or weight with unit): 0.03g imidazole; 30 µL NaCl (in saline)
Duration of treatment / exposure:
After 10 seconds the cornea surface was rinsed thoroughly with approx. 20 mL saline solution at ambient temperature
Observation period (in vivo):
30, 75, 120, 180 and 240 minutes after post-treatment rinse (minor variations +/- 5 minutes were considered acceptable)
Cornea thickness and opacity were measured at all time points. Fluorescein retention was determined at baseline (t=0) and at 30 min.
Duration of post- treatment incubation (in vitro):
4 hours
Number of animals or in vitro replicates:
3 eyes (test substance and positive control)
1 eye (negative control)
Details on study design:
SELECTION AND PREPARATION OF ISOLATED EYES:
With the eyes still in the chicken heads, eyelids were carefully cut away with scissors from the chicken eyes. One small drop of fluorescein solution 2% (w/v) was applied onto the cornea surface for a few seconds and then rinsed off with 20 mL isotonic saline. The fluorescein-treated cornea was examined with a hand-held slit lamp or a slit lamp microscope. If the cornea was in good condition, the eyeball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors without cutting off the optical nerve too short. The procedure avoided pressure on the eye, which may lead to corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box to maintain the appropriate humidity.

EQUILIBRATION AND BASELINE RECORDINGS
The prepared eye was placed in a stell clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head), again avoiding too much pressure on the eye. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was applied to fix the eye properly. The clamp was 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 saline solution dripping from a stainless steel tube, at a rate of approx. 3 to 5 drops/min. The door of the chamber was closed, except for manipulations and examination, to maintain temperature and humidity.
Eyes selected for testing were again examined with the slit lamp microscope. Eyes with a high baseline fluorescein staining (i.e. > 0.5) or corneal opacity score (i.e. > 0.5) were rejected.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: NaCl in saline (9 g/L) (n=1)

SOLVENT CONTROL USED: not applicable

POSITIVE CONTROL USED: Imidazole (n=3)

APPLICATION DOSE AND EXPOSURE TIME: 0.03 g per eye for 10 seconds

OBSERVATION PERIOD: 4 hours

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: Thorough rinsing with approx. 20 mL saline solution at ambient temperature
- Indicate any deviation from test procedure in the Guideline: none

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity scores ranging from 0 (no opacity) to 4 (complete opacity; iris invisible) according to the test guideline were measured at all time points
- Damage to epithelium based on fluorescein retention: Fluorescein retention scores ranging from 0 - 3 according to the test guideline were measured at 0 and 30 min.
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Cornea thickness was measured using the depth measuring device on the slit lamp microscope (Haag-Strei BQ 900) with the slit-width set at 9.5, i.e. 0.095mm.
- Macroscopic morphological damage to the surface: not applicable
- Others (e.g, histopathology): not performed

SCORING SYSTEM:
- Mean corneal swelling (%): according to TG
- Mean maximum opacity score: according to TG
- Mean fluorescein retention score at 30 minutes post-treatment: according to TG

DECISION CRITERIA: Decision criteria as indicated in the TG were used.
Irritation parameter:
percent corneal swelling
Value:
3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein leakage
Value:
1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none reported

DEMONSTRATION OF TECHNICAL PROFICIENCY:

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: not applicable

In an experimental study according to OECD test guideline 438 under GLP conditions, the substance scored into ICE class II for corneal opacity and fluorescein retention. It scored into ICE class I for corneal swelling.

With the overall ICE score of 1xI and 2xII, the substance could not be classified (no prediction can be made) according to the UN GHS classification and CLP.

Interpretation of results:
study cannot be used for classification
Executive summary:

The substance was tested in an experimental study under GLP conditions according to OECD TG 438 (Isolated Chicken Eye). Eye (in heads) were obtained from a slaughterhouse and not older than 2 hours at study initiation. Eyes were carefully selected and prepared, especially avoiding pressure on the eye, which may result in opacity.

Three selected eye were treated for 10 seconds with the substance and then thoroughly rinsed.

The eyes were observed 30, 75, 120, 180 and 240 minutes after post-treatment rinse (minor variations +/- 5 minutes were considered acceptable) for cornea thickness and opacity at all time points. Fluorescein retention was determined at baseline (t=0) and at 30 min.

A negative control and a positive control (imidazole) were included, which were valid for all observations.

The substance scored into ICE class II for corneal opacity and fluorescein retention. It scored into ICE class I for corneal swelling.

With the overall ICE score of 1xI and 2xII, the substance could not be classified (no prediction can be made) according to the UN GHS classification and CLP.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Product no. B4429, Lot no. SLBK5235V
- Expiration date of the lot/batch: 30 April 2018
- Purity test date: na

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: no
Species:
human
Details on test animals or tissues and environmental conditions:
- Justification of the test method and considerations regarding applicability: solids are within the applicability domain of the test method
- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live: commercially available EpiOcularTM tissue kit (tissue consist of normal, human-derived keratinocytes) obtained from MatTek In Vitro Life Science Laboratories (Bratislava, Slovakia) (Kit designation: OCL-212-EIT; delivered 26.10.2016; Batch no.: 23742), incl. a certificate of analysis and functionality test results
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): 50.5 and 51.9 mg/tissue (1st experiment) and 51.1 and 50.3 mg/tissue (2nd experiment)
- Concentration (if solution): not applicable

Duration of treatment / exposure:
6h
Duration of post- treatment incubation (in vitro):
1st experiment: 17h and 49 minutes
2nd: experiment: 18h
Number of animals or in vitro replicates:
2 tissues
Details on study design:
- Details of the test procedure used: The test was conducted according to the OCED TG 492 (details below)
- RhCE tissue construct used, including batch number: Kit OCL-212-EIT; Batch no.: 23742
- Doses of test chemical and control substances used: 50.5 and 50.9 mg/tissue
- Duration and temperature of exposure, post-exposure immersion and post-exposure incubation periods (where applicable): Exposure of 6h at approx. 37°C; 25 min post soak at room temperature; and 17h and 49 min post-incubation at approx. 37°C
- Justification for the use of a different negative control than ultrapure H2O (if applicable): not applicable
- Justification for the use of a different positive control than neat methyl acetate (if applicable): not applicable
- Description of any modifications to the test procedure: none
- Indication of controls used for direct MTT-reducers and/or colouring test chemicals (if applicable): In a pre-test, no indication for direct MTT reduction or colouring by the test substance was observed.
- Number of tissue replicates used per test chemical and controls (positive control, negative control, NSMTT, NSCliving and NSCkilled, if applicable): 2
- Wavelength and band pass (if applicable) used for quantifying MTT formazan, and linearity range of measuring device (e.g. spectrophotometer): 570 nm
- Description of the method used to quantify MTT formazan: spectrophotometer
- Description of the qualification of the HPLC/UPLC-spectrophotometry system (if applicable): not applicable
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The evaluation criteria of the OECD TG 492%, i.e. a threshold of 60% to discriminate non eye irritants from eye irritants, were used.
- Reference to historical positive and negative control results demonstrating suitable run acceptance criteria: Both the responses of the negative and the positive controls were within the range of the historical data.
- Complete supporting information for the specific RhCE tissue construct used: see above
- Reference to historical data of the RhCE tissue construct: Acceptable OD range of negative control: 1.000 - 2.024; acceptable relative viability range of the positive control: 14.5 - 49.3%
- Demonstration of proficiency in performing the test method before routine use by testing of the proficiency chemicals: not reported
- Positive and negative control means and acceptance ranges based on historical data: Acceptable OD range of negative control: 1.000 - 2.024; acceptable relative viability range of the positive control: 14.5 - 49.3%
- Acceptable variability between tissue replicates for positive and negative controls: Yes (negative control: 1st exp.: 0.1% < 20%, 2nd exp.: 9.7% < 20%; positive control: 1st exp.: 7.5% < 20%, 2nd exp.: 2.9% < 20%)
- Acceptable variability between tissue replicates for the test chemical: Yes (1st exp.: 10.9% < 20%, 2nd exp.: 0.3% < 20%)
Irritation parameter:
other: % viability
Run / experiment:
Experiment 1
Value:
55.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: % viability
Run / experiment:
Experiment 2
Value:
29
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

In an experimental study according to OECD test guideline 492 under GLP conditions, the test substance substantially reduced the cell viability in two experiments (55.8% and 29.0%).

Therefore, it is considered as eye irritant/inducing serious eye damage.

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Executive summary:

The eye irritation potential of the test substance was investigated in an experimental study according to OECD test guideline 492 under GLP conditions using the EpiOcularTM tissue. The EpiOcularTM tissue is provided in kits, which are manufactures according to defined quality assurance procedures.

The testing laboratory, which had demonstrated its technical proficiency in conducting the test, received the kit used for testing in good order.

The test substance was applied (approx. 50.7 mg) for six hours to two tissues. After thorough rinsing, 25 min post soak at room temperature was performed, before the tissues were post-incubated for 17h and 49 min post-incubation at approx. 37°C. Then the MTT test was performed and cell viability was measured with a spectrometer at 570 nm.

Concurrent negative and positive control fulfilled all acceptance criteria, as did the test substance, which did not directly reduce MTT, nor was colour interfering.

However, the test substance substantially reduced the cell viability in two experiments as compared to the negative control (55.8% and 29.0%).

Therefore, it is considered as eye irritant/inducing serious eye damage and requires to be classified (UN GHS 'Category 1').

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

In an experimental study according to OECD test guideline 439 under GLP conditions, the test substance did not reduce cell viability (96% with an SD of 14.52). Therefore, it is considered as not irritating to skin.

In an experimental study according to OECD test guideline 438 under GLP conditions, the substance scored into ICE class II for corneal swelling and fluorescein retention. It scored into ICE class I for corneal opacity.

In an experimental study according to OECD test guideline 492 under GLP conditions, the test substance substantially reduced the cell viability in two experiments (55.8% and 29.0%).

Therefore, it is considered as eye irritant/inducing serious eye damage.