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Administrative data

Description of key information

Under the experimental conditions reported, the test item is not irritating to the skin according to OECD 439.

The structural analogue substance caused severe eye damage according to OECD Guideline 437.

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:
August 24, 2011 - August 26, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2009
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
other: Skinethic Skin irritation test -42bis Standard operating procedure (SOP) 2009
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
To reduce animal testing, this alternative in vitro method was used. The human skin RHE-model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model
- Tissue batch number(s): 11 022A 0802
- Date of initiation of testing: August 24, 2011

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation: 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: minimum volume of 25 mL PBS using a multi pipette


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: spectrophotometer
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES:
3

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION:
One experiment in triplicate

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the viability after exposure is less than 50% or equal to 50 %.
- The test substance is considered to be non-irritant to skin if the viability after exposure is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 16 mg

NEGATIVE CONTROL
- Amount applied: 16 µL

POSITIVE CONTROL
- Amount applied: 16 µL
Duration of treatment / exposure:
42 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 1/experiment 1
Value:
1.681
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 2/experiment 1
Value:
1.79
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Tissue 3/experiment 1
Value:
1.798
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no

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

Results

 Dose group

 treatment interval  optical density tissue1  optical density tissue 2  optical density tissue 3  MEAN optical density  mean relative viability [%] Standard deviation 
 negative control  42 min  1.768  1.598  1.878  1.748  100  8.06
 positive control  42 min  0.057  0.049  0.057  0.054  3.11  8.32
 test item  42 min  1.681  1.790  1.798  1.756  100.47  3.74
Interpretation of results:
GHS criteria not met
Conclusions:
The test substance is to be considered as non-irritating to the skin under the conditions of this experiment.
Executive summary:

In an in vitro skin irritation model, substance was tested. The applied cellline (RHE (Reconstructed Human Epidermis) is produced by SkinEthic by culturing human adult keratinocytes on a polycarbonate filter in such a quality that a functional stratum corneum is available and thermal differentation takes place. The test was conducted under GLP and according to OECD guideline 439, the EU Method B.46 and the Standard Operating Procedure of SkinEthic's (Skin irritation test -42bis).

Each test is performed in triplicates. 16µl of controls were directly applied to the tissue. Prior 16mg of test substance were applied, 10 µl of water were sprayed to the tissue. After treatment of (treatment time 42minutes at roomtemperature) substances were removed and tissues were incubated for 424 hours.

For testing validity of the Skin model, a positive control (5% sodiumdodecylsulfate in deionized water) and a negative control (PBS buffer) were used.

After topical exposure of test substance, positive control, or negative control cell viability is measured in a MTT assay. In this assay cell viability is measured by conversion of MTT to a formazan salt by a dehydrogenase enzyme.

As the test item has a very high cell viability of (100%) passing the threshold value of 50%, this item is to be considered as non-irritant. The test is valid since the criteria for the positive control (cell viability < 40%; 3,1% in this test) and the negative control (optical density between 1.2 and 2.5; 1.75 in this itest) are met.

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
Study period:
2015-11-20 to 2015-11-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Vehicle:
physiological saline
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
750 µL (i.e. 150mg/750µL) of test item, negative or positive control
Duration of treatment / exposure:
240 minutes
Number of animals or in vitro replicates:
in vitro: triplicate design
Irritation parameter:
in vitro irritation score
Run / experiment:
run 1
Value:
113.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
run 1
Value:
62.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein leakage
Run / experiment:
run 1
Value:
3.45
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

Opacity
Permeability
IVIS
per cornea
per group
(mean value)
SD
Negative Control
0.9 % NaCl Solution
-0.062
-0.001
-0.082
0.8
0.9
1.816
-0.001 1.801
0.706
-0.001 0.696
Positive Control
20 % Imidazole  solution
70.793
2.142
102.928
106.6
4.1
71.315
2.303
105.860
79.270
2.119
111.060
Test material
20% suspension
66.507
3.447
118.217
113.9
16.4
72.337
3.699
127.827
47.660
3.206
95.780



Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Under the given experimental conditions the test material the test material has ocular corrosive potential.
Executive summary:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 437. The test material was corrosive in this in vitro assay.

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
For this endpoint information from a structural similar compound is available. This study for this similar compound was performed according to GLP and the methods applied are fully compliant with OECD TG 437. See chapter 13 report for a more detailed justification.
Reason / purpose:
read-across source
Irritation parameter:
in vitro irritation score
Run / experiment:
run 1
Value:
113.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation parameter:
cornea opacity score
Run / experiment:
run 1
Value:
62.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein leakage
Run / experiment:
run 1
Value:
3.45
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In vitro skin irritation study

In an in vitro skin irritation model, substance was tested. The applied cell line (RHE (Reconstructed Human Epidermis) is produced by SkinEthic by culturing human adult keratinocytes on a polycarbonate filter in such a quality that a functional stratum corneum is available and thermal differentation takes place. The test was conducted under GLP and according to OECD guideline 439, the EU Method B.46 and the Standard Operating Procedure of SkinEthic's (Skin irritation test -42bis).

Each test is performed in triplicates. 16µl of controls were directly applied to the tissue. Prior 16 mg of test substance were applied, 10 µl of water were sprayed to the tissue. After treatment of (treatment time 42 minutes at room temperature) substances were removed and tissues were incubated for 42 hours.

For testing validity of the Skin model, a positive control (5% sodiumdodecylsulfate in deionized water) and a negative control (PBS buffer) were used.

After topical exposure of test substance, positive control, or negative control cell viability is measured in a MTT assay. In this assay cell viability is measured by conversion of MTT to a formazan salt by a dehydrogenase enzyme.

As the test item has a very high cell viability of (100%) passing the threshold value of 50%, this item is to be considered as non-irritant. The test is valid since the criteria for the positive control (cell viability < 40%; 3,1% in this test) and the negative control (optical density between 1.2 and 2.5; 1.75 in this itest) are met.

In vitro eye irritation study

The objective of the present study was to examine the potential of the structural analogue substance to induce serious eye damage in the BCOP assay. The BCOP assay with isolated fresh bovine corneas is an accepted in vitro model for ocular hazard assessment.

To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item as a 20% (w/v) suspension in a 0.9% sodium chloride solution. As negative control 0.9% sodium chloride solution and as positive control 20% (w/v) Imidazole was used. Three corneas were used per group (negative control, positive control or test item group).
After a first opacity measurement of the untreated bovine corneas, 750 µL of the suspended test item, positive or negative control were applied on the corneas and incubated for 240 minutes. After the incubation phase the test item, the positive, and the negative control were rinsed from the corneas and the opacity was measured again. After the opacity measurements, the permeability of the corneas was determined by application of a fluorescein solution for 90 minutes. The amount of fluorescein solution that crossed the cornea was measured spectrophotometrically. The opacity and permeability assessments were combined to determine an In Vitro Irritancy Score (IVIS).

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 0.1 (study acceptance criteria range: -1.4 – 3.4). Treatment with the positive control (20% Imidazole) revealed an IVIS of 111.2 (study acceptance criteria range: 77.4 – 136.3). Therefore, the study fulfilled the validity criteria. The IVIS obtained after treatment with the test item was 113.9 and, thus, higher than 55.

Under the conditions of the present study, the test item induces serious eye damage in this in vitro assay.

Justification for classification or non-classification

Based on the data provided, the test item is not classified for skin irritation according to Regulation (EC) No 1272/2008. However, based on the results obtained from the structural analogue substance the test item is classified for severe eye damage cat. 1 and labelled with H318 according to Regulation (EC) No 1272/2008.