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Description of key information

The test substance was tested for skin and eye irritation according to OECD TG 439 and TG 437. Both studies were carried out under GLP and both studies did not identify any irritation potential.

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:
2016-2-24 to 2016-8-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
adopted 28 July 2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Remarks:
; the study used EpiDerm™ from MatTek
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
This test used the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and represents the target organ of the species of interest in vitro and closely mimics the biochemical and physiological properties of the upper parts of human skin, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™
- Tissue batch number(s): 23316 (main experiment) and 23339 (NSC evaluation)
- Production date: 2016-2-24 and 2016-6-1
- Date of initiation of testing: 2016-2-23

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C for 35 ± 1 min and room temperature for 25 ± 1 min
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were washed by filling and emptying the inserts 15 times with DPBS using a constant stream in about 1.5 cm distance from the tissue surface, subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item.
- Observable damage in the tissue due to washing: -
- Modifications to validated SOP: -

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/ml
- Incubation time: 3 h ± 5 min
- Spectrophotometer: plate reader
- Wavelength: 570 nm
- Filter bandwidth: maximum ± 30 nm at 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: 1.551 ± 0.125 and 1.57 ± 0.057
- Barrier function: 7.25 h and 6.51 h
- Contamination: Sterile and Sterile


NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Viable tissues
- N. of replicates: 2
- Method of calculation used: Interference [%] = [OD(additional test item treated living tissues without MTT staining)/OD(additional negative control treated living tissue with MTT staining)]*100

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement):
-as recommended in TG 439: The test item is considered to be irritant to skin in accordance with regulation EC 1272/2008 (UN GHS “Category 2”) if the tissue viability after exposure and post-incubation is less or equal to 50%.
Control samples:
yes, concurrent no treatment
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

VEHICLE
- Amount(s) applied (volume or weight with unit): tissues were pre-wetted with 25 μL of sterile DPBS before applying solid test substance

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL DPBS
- Concentration (if solution):

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 μL
- Concentration (if solution): 5% SDS solution
Irritation / corrosion parameter:
% tissue viability
Value:
99
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:
The mixture of 25 mg test item with 1 ml MTT medium did not result in blue/purple colouring and the test item, consequently, did not directly reduce MTT.

The mixture of 25 mg of the test item with 300 μL isopropanol showed colouring detectable by unaided eye-assessment and absorbed light in the relevant range.
For quantitative correction of results, the non-specific colour was determined by using additional viable tissues incubated with 25 mg of the test item and not subjected to MTT-staining and tissues treated as the control and subsequently incubated with MTT. The non-specific colour was calculated to amount 2.3%, which is well below 5% and, therefore, no correction of the determined tissue viability was necessary.

Please see attached file for results.

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item showed no irritant effects. The relative mean tissue viability after 60 min of exposure and 42 h post-incubation was > 50% (99 ± 16%). The test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.
Executive summary:

A valid test for skin irritation according to OECD Guideline and under GLP was carried out with the test substance and did not identify any signs of skin irritation.

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:
2016-3-2 to 2016-7-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The Bovine Corneal Opacity and Permeability (BCOP) test method is an in vitro test method that can be used to classify substances as “ocular corrosives / severe irritants” and “non-irritants”. The BCOP is recommended for use as part of a tiered-testing strategy for regulatory classification and labelling within a specific applicability domain. Test substances can be classified as ocular corrosives / severe irritants or non-irritants without further testing in rabbits.
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability 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
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Details on test animals or tissues and environmental conditions:
The assay used isolated corneas obtained as a by-product from animals freshly slaughtered at the abattoir A. Moksel AG, Buchloe, Germany.
On the test day, fresh eyes were collected from the slaughterhouse and were transported in HBSS containing Pen/Strep on ice to the laboratories. Immediately after arrival of the eyes, cornea preparation was initiated.
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): 750 mg

Duration of treatment / exposure:
4 hours ± 5 minutes
Number of animals or in vitro replicates:
3 corneas per test group
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
The eyes were carefully examined for defects and any defective eyes were discarded.

The tissue surrounding the eyeball was carefully pulled away and the cornea was excised leaving a 2 to 3 mm rim of sclera. The isolated corneas were stored in a petri dish containing HBSS. Before the corneas were mounted in corneal holders (Duratec GmbH) with the endothelial side against the O-ring of the posterior chamber, they had been visually examined for defects and any defective cornea had been discarded. The anterior chamber was then positioned on top of the cornea and tightened with screws. The chambers of the corneal holder were then filled with RPMI (without phenol red) containing 1% FBS and 2 mM L-glutamine (complete RPMI). The posterior chamber was always filled first. The corneas were incubated for one hour at 32 ± 1 °C.

QUALITY CHECK OF THE ISOLATED CORNEAS
Only corneas that had an initial illuminance reading I > I(0)/1.1651 lux were used for the assay.

NUMBER OF REPLICATES:
3 corneas per test group

NEGATIVE CONTROL USED
750 μl physiological saline 0.9% NaCl

SOLVENT CONTROL USED (if applicable)
not applicable

POSITIVE CONTROL USED
750 μl 20% imidazole in physiological saline 0.9% NaCl

APPLICATION DOSE AND EXPOSURE TIME
750 mg of the solid test substance was applied and moistened with physiological saline 0.9%. For controls, 750 μl of PBS or the control substanceswas introduced into the anterior chamber. Exposure time was 4 hours ± 5 minutes.

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 4 at least


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
The opacitometer (BASF-OP3.0, Duratec GmbH) was switched on at least 15 min before starting the calibration procedure. The filter holder was placed into the opacitometer and the readout was adjusted to 1000 lux ± 10 lux using the “Calibrate”-turning knob. For calibration the glass filter F2 was introduced into the filter holder. The readout lied in the range between 540-560 lux. To test the linearity of the measurement, two additional calibration filters, glass filter F3 and glass filter F4, were measured. For these glass filters, the opacitometer displayed values between 300-310 lux and between 95-105 lux. The calibration procedure was performed before the test and is documented in the raw data.

- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)
After the illuminance measurement was performed, the medium was removed from both chambers of the holder. The posterior chamber was refilled with fresh complete RPMI. 1 ml of a 5 mg/ml sodium fluorescein solution was added to the anterior chamber and the corneas were incubated for 90 minutes at 32 ± 1 °C. Then the medium from the posterior chamber was removed and its optical density at 490 nm (OD490) was determined, using a spectrophotometer (Jenway 6405 UV/VIS).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: the decision criteria as indicated in TG 437 were used (please see attached file for results and classification criteria).
Irritation parameter:
in vitro irritation score
Value:
2.06
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid

Please see attached file for results.

Interpretation of results:
GHS criteria not met
Conclusions:
The following mean in vitro irritation score was calculated: 2.06
Therefore the test item was classified into UN GHS No Category.
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

The mean relative tissue viability (% negative control) was > 50% (99 ± 16%) in a valid test for skin irritation according to OECD TG 439 and under GLP. The test item was therefore classified as “non-irritant” in accordance with UN GHS “No Category”. A mean in vitro irritation score of 2.06 was determined in a valid test for eye irritation according to OECD TG 437 and under GLP. According to the evaluation criteria the test item is accordingly classified into UN GHS “No Category”.