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Administrative data

Description of key information

OECD 439, RL1: irritating to skin

OECD 431, RL1: non-corrosive to skin

OECD 437, RL1: corrosive to eye

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
05 Oct 2016 - 16 Dec 2016
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)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The reconstructed human epidermis model in vitro method is an accepted in vitro method to replace animal testing. The human skin RHE™ model closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e the epidermis, and has been validated by the ECVAM in 2008.
Vehicle:
unchanged (no vehicle)
Details on test system:
SkinEthic™ RHE-model RHE/S/17
- Batch no.: 16-RHE-114

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: rinsed with minimum 25 mL DPBS; excess DPBS removed by shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper
- Observable damage in the tissue due to washing: no data
- Modifications to validated SOP: none

DYE BINDING METHOD
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: microplate reader ELx800, BioTek Instruments GmbH
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin category 2 if the viability is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability is greater than 50%.



Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Application volume: 16 ± 2 mg per tissue

NEGATIVE CONTROL
- Application volume: 16 ± 5 µL per tissue

POSITIVE CONTROL
- Application volume: 16 ± 5 µL per tissue
Duration of treatment / exposure:
42 minutes (± 1 minute)
Duration of post-treatment incubation (if applicable):
42 hours (± 1 hour)
Number of replicates:
The test item as well as the positive and negative control were tested in batch-triplicates.
Irritation / corrosion parameter:
% tissue viability
Remarks:
replicate 1
Run / experiment:
Tissue 1
Value:
1.47
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
replicate 2
Run / experiment:
Tissue 2
Value:
1.54
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Remarks:
replicate 3
Run / experiment:
Tissue 3
Value:
1.28
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
1.43
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation
Other effects / acceptance of results:
The pre-test for direct MTT-reducing capacity of the test item not result in blue colour, i.e. the test item is a direct MTT reducer but the test item has no colorant properties.
Due to the fact that MTT was not reduced after treatment with the test item, no additional killed epidermises were tested to evaluate the extent of a non-specific interaction.

Table 1 Optical density and Tissue viability

Group

Tissue 1

Tissue 2

Tissue 3

Mean

SD

 

 

OD

viability

OD

viability

OD

viability

OD

viability

viability

Negative Control

1.925

98.52%

1.978

101.22%

1.959

100.26%

1.954

100.0%

1.37%

Positive Control

0.029

1.48%

0.028

1.45%

0.030

1.52%

0.029

1.48%

2.30%

Test item

0.029

1.47%

0.030

1.54%

0.025

1.28%

0.028

1.43%

9.28%

Interpretation of results:
Category 2 (irritant) based on GHS criteria
Conclusions:
The tissue viability after treatment with the test item was lower than 50 % (men viability: 1.43%). Therefore the test item is considered to possess an irritant potential to skin.
Executive summary:

The objective of the present study was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model.

The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin irritation potential.

Triplicates of the human skin RHE-model were treated with the test item, the negative or the positive control for 42 minutes (± 1 minute). 16 µL of either the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Before application of 16 mg of the solid test item, 10 µL of deionized water was spread to the epidermis surface to improve the contact between the test item and the epidermis.

After treatment with the negative control (DPBS-buffer) the mean OD was 1.954 (study acceptance criterion: >1.431). Treatment with the positive control (5% aqueous solution of sodium dodecyl sulfate) revealed a mean viability value of 1.48% (study acceptance criterion: <3.14%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 1.43% and, thus, lower than 50%, i.e. according to OECD 439 the test item is considered as irritant to skin (UN GHS: Category 2).

 

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
05 Dec 2016 - 03 Mar 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on animal used as source of test system:
The SkinEthic(TM) RHE model RHE/S17 was obtained form EpiSkin/SkinEthic Laboratories, Lyon, France
Justification for test system used:
The reconstructed human epidermis model is an accepted in vitro method to replace animal testing. The model 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: SkinEthic™ RHE-model RHE/S/17
- Tissue batch number: 16-RHE-130

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: ambient temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
- Washing step: using minimum volume of 20 mL DPBS, Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper
- Observable damage in the tissue due to washing: no data
- Modifications to validated SOP: none

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: ELx800, BioTek Instruments GmbH
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Application volume: 20 ± 3 mg per tissue

NEGATIVE CONTROL
- Application volume: 40 ± 3 µL per tissue

POSITIVE CONTROL
- Application volume: 40 ± 3 µL per tissue
Duration of treatment / exposure:
3 minutes or 1 hour
Number of replicates:
The test item as well as the negative control were tested with two replicate tissues per time point (3 min and 1 hour exposure). The positive control was tested with two replicate tissues only for 1 hour.
Irritation / corrosion parameter:
% tissue viability
Remarks:
replicate 1 / 3 minutes
Run / experiment:
Tissue 1
Value:
60.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
replicate 1 / 1 hour
Run / experiment:
Tissue 1
Value:
63.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
replicate 2 / 3 minutes
Run / experiment:
Tissue 2
Value:
61.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
replicate 2 / 1 hour
Run / experiment:
Tissue 2
Value:
78.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
3 minutes
Run / experiment:
Mean
Value:
61
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Remarks:
1 hour
Run / experiment:
Mean
Value:
70.9
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The pre-test for direct MTT reducing capacity did result in blue color, i.e. the test item is a direct MTT reducer, but the test item has no colorant properties.

Following treatment with the test item the tissue viability was >50 % after 3 min exposure and >15 % after 1 h exposure (Mean viability 70.9%), i.e. according to OECD 431 guideline the test item is not considered corrosive to skin.

Table 1 Optical density and Tissue viability

     Tissue 1    Tissue 2    Mean    CV
 Group  time  OD  viability  OD  viability  OD  viability  viability
 negative control  3 min  2.019  99.00 %  2.059  100.98 %  2.039  100.00 %  1.4 %
 neagtive control  1 h  1.675  99.30 %  1.699  100.70 %  1.687  100.00 %  1.00 %
 positive control  1 h  0.009  0.50 %  0.011  0.70 %  0.010  0.60 %  16.70 %
 test item  3 min  1.235  60.60 %  1.250  61.30 %  1.243  61.00 %  0.80 %
 test item  1 h  1.072  63.5 %  1.319  78.20 %  1.196  70.90 %  14.70 %
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the test item is considered not to possess a corrosive potential to skin (combination of optional sub-categories 1B and 1C).
Executive summary:

The objective of the present study was to investigate the potential of the test item to induce skin corrosion in an in vitro human skin model.

The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin corrosion potential.

Duplicates of the human skin RHE-model were treated with the test item or the negative control for 3 minutes and additional 1 hour. Duplicates with the positive control were only treated for 1 hour. 40 ± 3 µL of either the negative control (deionised water) or the positive control (potassium hydroxide, 8N) were applied to the tissues. Before application of 20 ± 3 mg of the solid test item, 20 ± 2 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis.

The test item has the ability to directly reduce MTT. To evaluate the extent of non-specific interaction, two killed tissues were treated for 3 minutes and 1 hour with the test item or the negative control, respectively. The treatment and MTT assay of the killed tissues was similar to the handling of the living tissues. The obtained OD for a non-specific reduction was subtracted from OD-values obtained after treatment of living tissues with the test item to calculate the cell viability. After treatment with the negative control (deionised water) the mean OD per tissue replicate was 2.019 and 2.059 after 3 minutes exposure and 1.675 and 1.699 after 1-hour exposure (study acceptance criterion: >= 0.8 and <= 3. 0). Treatment with the positive control (Potassium hydroxide, 8N) revealed a mean viability of 0.6% after 1 hour (study acceptance criterion: <15%). Therefore, the study fulfilled the validity criteria.

Following treatment with the test item, the tissue viability was 50% after 3 minutes exposure (mean viability: 61.0%) and 15% after 1-hour exposure (mean viability: 70.9%), i.e. according to OECD 431 the test item is considered as non-corrosive to skin.

Under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin.

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:
05 Oct 2016 - 06 Dec 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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:
2013
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Odenwaldschlachthof Brensbach, 64395 Brensbach, Germany
- Age at study initiation: 20-43 months
- Corneal diameter: 25 - 27 mm
- Storage, temperature and transport conditions of ocular tissue: cooled on ice
- Time interval prior to initiating testing: The corneas were prepared immediately after delivery of the eyes to the laboratory.
- Indication of any existing defects or lesions in ocular tissue samples: Eyes presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Volume applied: 150 mg/750 µL
- Concentration: 20% (w/v) in a 0.9% NaCl solution

VEHICLE
- Volume applied: 750 µL

Duration of treatment / exposure:
240 minutes at 32 +/- 1 °C
Number of animals or in vitro replicates:
Three corneas were used per group (negative control, positive control and test item group).
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
All eyes were carefully examined macroscopically for defects. A rim of about 2 to 3 mm of tissue (sclera) was left for stability and handling of the isolated cornea.

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas presenting defects such as vascularization, pigmentation, opacity or scratches were discarded.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9% sodium chloride solution

POSITIVE CONTROL USED: N,N-dimethylformamide

APPLICATION DOSE AND EXPOSURE TIME: 750 µL of a 20% solution were applied for 240 minutes

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes, 120 minutes

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period:at least 3 times with washing medium

- POST-EXPOSURE INCUBATION: in fresh medium

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: baseline opacity was determined with a calibrated opacitometer, the light transmission through the corneas, given as lux value, was recorded in a table and thereafter converted into an opacity value (baseline opacity values).
- Corneal permeability: corneas were incubated again in an incubator in a horizontal position at 32 ± 1°C for 90 minutes, amount of fluorescein that crossed the cornea was measured spectrophotometrically 490 nm

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)
The following formula (referring to OECD Guideline 437) was used to determine the In Vitro Irritancy Score (IVIS) of the negative control:
IVIS = mean opacity value + (15 x mean permeability OD490 value)
The following formula was used to determine the In Vitro Irritancy Score (IVIS) of the positive control and the test item:
IVIS = corrected opacity value + (15 x corrected permeability OD490 value)
- The In Vitro Irritancy Score (IVIS) was calculated for each individual treatment and positive control cornea. The mean In Vitro Irritancy Score (IVIS) value of each treated group was calculated from the individual In Vitro Irritancy Score (IVIS) values.

DECISION CRITERIA:
IVIS ≤ 3 No Category (according to GHS)
IVIS > 3; ≤ 55 No prediction can be made
IVIS > 55 Serious eye damage, Category 1 (according to GHS)

ACCEPTANCE CRITERIA:
A test is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean(IVIS positive control: 75.3 - 120.2).
The negative control responses should result in an IVIS that falls within three standard deviations of the current historical mean (IVIS negative control: -1.2 - 5.3).
A single test run with three corneas should be sufficient for a test item when the resulting classification is unequivocal. In cases of the following borderline results in the first testing run, a second test run should be considered.
- 2 of the 3 corneas give discordant predictions from the mean of all 3 corneas or
- 1 of the 3 corneas give discordant predictions from the mean of all 3 corneas, and the discordant result is >10 IVIS units from the cut-off threshold of 55.


Irritation parameter:
cornea opacity score
Remarks:
replicate 1
Run / experiment:
1
Value:
95.9
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Remarks:
replicate 2
Run / experiment:
1
Value:
99.3
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Remarks:
replicate 3
Run / experiment:
1
Value:
97.9
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Remarks:
mean
Run / experiment:
1
Value:
97.7
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: yes


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
The resulting classification of the test item in this study is unequivocal and no borderline results were obtained. Therefore, a single testing run composed of three corneas per group was considered sufficient.

Table 1 Opacity, permeability and IVIS results

 

Opacity

Permeability

IVIS

per cornea

per group
(mean value)

Standard
deviation

Negative
control

0.9% sodium
chloride solution

3.0

0.000

3.000

1.5

1.8

1.9

0.001

1.915

-0.5

-0.001

-0.515

Positive
control

20 % Imidazole in 0.9 % NaCl solution

69.6

2.422

105.930

105.4

8.6

49.1

3.168

96.620

72.9

2.720

113.700

Testitem

Test item 20 % in 0.9 % NaCl solution

53.1

2.853

95.895

97.7

1.7

44.4

3.661

99.315

54.3

2.906

97.890

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
Under the conditions of the present study the test item induces serious eye damage.
Executive summary:

The objective of the present study was to examine the potential of the test item 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 lrritancy Score (IVIS).

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.5 (study acceptance criteria range: - 1.4 - 3.4). Treatment with the positive control (20 % Imidazole) revealed an IVIS of 105.4 (study acceptance criteria range: 78.6 - 135.0). Therefore, the study fulfilled the acceptance criteria.

The IV IS obtained after treatment with the test item was 97.7 and, thus, higher than 55, i.e. according to OECD 437 the test item induces serious eye damage (UN GHS: Category 1).

 

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irritating)

Additional information

In vitro skin irritation study

The objective of the present study was to investigate the potential of the test item to induce skin irritation in an in vitro human skin model (OECD 439).

Following treatment with the test item, the tissue viability was 1.43% and, thus, lower than 50%, i.e. according to OECD 439 the test item is considered as irritant to skin.

 

In vitro skin corrosion study

The objective of the present study was to investigate the potential of the test item to induce skin corrosion in an in vitro human skin model (OECD 431).

Following treatment with the test item, the tissue viability was 50% after 3 minutes exposure (mean viability: 61.0%) and 15% after 1-hour exposure (mean viability: 70.9%), i.e. according to OECD 431 the test item is considered as non-corrosive to skin.

Based on the Weight of Evidence approach, the test item is considered to be irrating to skin.

In vitro eye corrosion study

The objective of the present study was to examine the potential of the test item 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.

After treatment with the negative control (0.9% sodium chloride solution) the calculated IVIS was 1.5 (study acceptance criteria range: - 1.4 - 3.4). Treatment with the positive control (20 % Imidazole) revealed an IVIS of 105.4 (study acceptance criteria range: 78.6 - 135.0). Therefore, the study fulfilled the acceptance criteria.

The IV IS obtained after treatment with the test item was 97.7 and, thus, higher than 55, i.e. according to OECD 437 the test item induces serious eye damage (UN GHS: Category 1).

Respiratory tract irritation

There are currently no validated tests on respiratory tract irritation, however, it is a reasonable precaution to assume that corrosive (and severely irritating) substances would also cause respiratory tract irritation. Since the substance induced serious eye damage, it is assumed as a precautionary measure that the test item cause also respiratory tract irritation.

Justification for classification or non-classification

Based on the reults, the test item is classified for skin irritation category 2 (H315), for eye corrosion category 1 (H318), and STOT SE category 3 (H335) according to Regulation (EC) No 1272/2008.