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Description of key information

The test item does not possess an irritant potential to skin (reference 7.3.1-1).


The test item does not possess an irritant potential to eyes (reference 7.3.2-1).

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:
2019-09-18 to 2019-10-18
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:
June 18, 2019
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:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic™ RHE-model RHE/S/17
- Tissue batch number: 19-RHE-156
- Expiry date: September 23, 2019
- Date of initiation of testing: September 4, 2019

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 of washing steps: 25 mL DPBS
- Observable damage in the tissue due to washing: 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, Bad Friedrichshall, Germany)
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD 1.4 (OD>0.7)
- Barrier function: 5.8 h (4.0h - Morphology: Multi-layered, highly differentiated epidermis consisting of basal, spinous and granular layers, and a multi-layered Stratum corneum.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- N. of replicates : 3
- Method of calculation used: It is necessary to evaluate the OD due to non-specific reduction and to subtract it before calculation of the cell viability.

Non specific MTT reduction = [OD(KT) - OD(KU) / OD (neg. control)] * 100

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 3

PREDICTION MODEL
- The test substance is considered as non-irritant to skin (UN GHS No Category) if the tissue viability after exposure and post-treatment incubation is > 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 16 µL ± 0.5 µL per tissue

NEGATIVE CONTROL
- Amount applied: 16 µL ± 0.5 µL per tissue

POSITIVE CONTROL
- Amount applied 16 µL ± 0.5 µL per tissue
- Concentration: 5% aqueous solution
Duration of treatment / exposure:
42 min
Duration of post-treatment incubation (if applicable):
42 h
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
96.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: Yes
- Colour interference with MTT: 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
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the test item is not 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 according to OECD TG 439. 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 test item, the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Based on results from pre-tests, the test item was shown to directly reduce MTT. To evaluate the extent of non-specific interaction, three killed tissues were treated with the test item and three killed untreated tissues were used as negative control. The treatment and the MTT assay of the killed tissues was comparable to that 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. All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5% aqueous Solution of sodium dodecyl sulfate) were met. Following treatment with the test item, the tissue viability was 96.2% and, thus, higher than 50%, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category). Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).

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:
2019-08-30 to 2019-09-27
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 Ocular Corrosives and Severe Irritants)
Version / remarks:
October 9, 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
- Characteristics of donor animals (age): 17-58 months
- Storage, temperature and transport conditions of ocular tissue: in transport medium cooled on ice
- Indication of any existing defects or lesions in ocular tissue samples: No
- Indication of any antibiotics used: No
- Quality check of the isolated corneas: Corneal diameter ca. 25-27 mm
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 750 µL
Duration of treatment / exposure:
10 min
Number of animals or in vitro replicates:
3
Details on study design:
NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: 0.9% sodium chloride Solution

POSITIVE CONTROL USED: N,N-dimethylformamide (undiluted)

APPLICATION DOSE AND EXPOSURE TIME: 750 µL for 10 min

TREATMENT METHOD: closed chamber

POST-INCUBATION PERIOD: yes, 120 min

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

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity value of each individual cornea was corrected for background opacity by subtracting the initial baseline opacity reading from the post treatment opacity reading. In addition, the opacity values of both the treatment and positive control groups were corrected for the mean negative control opacity values.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: The decision criteria as indicated in the OECD TG 437 were used.

IVIS
<3 No Category
>3<55 No prediction can be made
>55 Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
-2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes (IVIS = 3.3)
- Acceptance criteria met for positive control: yes (IVIS = 93.1)
Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the present study, the test item did not show an eye hazard potential. The test item is not requiring classification for eye irritation or serious eye damage.
Executive summary:

To determine the eye hazard potential the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item according to OECD TG 437. As negative control 0.9% sodium chloride solution and as positive control N,N-dimethylformamide 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 test item, positive or negative control were applied on the corneas and incubated for 10 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 3.3 (study acceptance criteria range: -1.4 - 5.4). Treatment with the positive control (N,N-dimethylformamide) revealed an IVIS of 93.1 (study acceptance criteria range: 78.0 -118.0). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was -2.0 and, thus, lower than 3, i.e. according to OECD 437 the test item is not requiring classification for eye irritation or serious eye damage (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

skin irritation in vitro


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 according to OECD TG 439. 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 test item, the negative control (DPBS-buffer) or the positive control (5% aqueous solution of sodium dodecyl sulfate) were applied to the tissues. Based on results from pre-tests, the test item was shown to directly reduce MTT. To evaluate the extent of non-specific interaction, three killed tissues were treated with the test item and three killed untreated tissues were used as negative control. The treatment and the MTT assay of the killed tissues was comparable to that 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. All acceptability criteria after treatment with the negative control (DPBS-buffer) and the positive control (5% aqueous solution of sodium dodecyl sulfate) were met. Following treatment with the test item, the tissue viability was 96.2% and, thus, higher than 50%, i.e. according to OECD 439 the test item is considered as non-irritant to skin (UN GHS: No Category). Under the conditions of the present study, the test item is not considered to possess an irritant potential to skin (UN GHS: No Category).


 


Eye irritation in vitro


To determine the eye hazard potential, the induced opacity and increased permeability was investigated in isolated bovine corneas after exposure to the test item according to OECD TG 437. As negative control 0.9% sodium chloride solution and as positive control N,N-dimethylformamide 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 test item, positive or negative control were applied on the corneas and incubated for 10 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 3.3 (study acceptance criteria range: -1.4 - 5.4). Treatment with the positive control (N,N-dimethylformamide) revealed an IVIS of 93.1 (study acceptance criteria range: 78.0 -118.0). Therefore, the study fulfilled the acceptance criteria. The IVIS obtained after treatment with the test item was -2.0 and, thus, lower than 3, i.e. according to OECD 437 the test item is not requiring classification for eye irritation or serious eye damage (UN GHS: No Category).

Justification for classification or non-classification

The available data for skin irritation/corrosion are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is considered to be not classified for skin irritation or corrosion under Regulation (EC) No 1272/2008.


The available data for eye irritation are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this data, the substance is considered to be not classified for causing serious eye damage or irritation under Regulation (EC) No 1272/2008.

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