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Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
3rd to 25th May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Unknown 6
IUPAC Name:
Unknown 6
Constituent 2
Reference substance name:
Unknown 7
IUPAC Name:
Unknown 7
Constituent 3
Reference substance name:
Unknown 5
IUPAC Name:
Unknown 5
Constituent 4
Reference substance name:
Unknown 4
IUPAC Name:
Unknown 4
Constituent 5
Reference substance name:
Unknown 3
IUPAC Name:
Unknown 3
Constituent 6
Reference substance name:
Unknown 2
IUPAC Name:
Unknown 2
Constituent 7
Reference substance name:
Unknown 1
IUPAC Name:
Unknown 1
Test material form:
solid
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 4898318
- Expiration date of the lot/batch: March 2019
- Purity test date: Not specified
- Storage condition of test material: ≤ - 20°C

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: three-dimensional reconstructed human epidermis model (EPISKINTM model)
Cell source:
other: normal human epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis
Source strain:
other: Commercial test kit
Details on animal used as source of test system:
Not applicable
Justification for test system used:
This test method is able to detect chemicals that cause skin irritation, i.e. produce reversible damage to the skin and allows for hazard identification in accordance with CLP skin irritation category 2; it can also be used to identify non-classified chemicals. The test is used as a replacement for the in vivo Draize Skin Irritation Test (OECD TG 404). According to REACH Annex VII, an in vitro study must be conducted.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm (MatTek)
- Tissue batch number(s): Lot No.: 28615 for main experiment and viable tissue controls, 25883 for killed tissue controls
- Production date: Not specified
- Shipping date: Not specified
- Delivery date: Not specified
- Date of initiation of testing: 3rd May 2018

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: The 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, this process also occurred sequentially, e.g. in one-minute intervals. Subsequently, the inserts were completely submerged three times in 150 mL DPBS and shaken to remove rests of the test item. Finally, the inserts were rinsed once from the inside and the outside with sterile DPBS. Excess DPBS was removed by blotting the bottom with blotting paper.
- Observable damage in the tissue due to washing: Not specified
- Modifications to validated SOP: Not specified

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h ± 5 min
- Spectrophotometer: plate spectrophotometer
- Wavelength: 570 nm
- Filter: Yes - type of filter not specified
- Filter bandwidth: maximum ± 30 nm
- Linear OD range of spectrophotometer: Not specified

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: Mean relative viability: 3.8% (SD viability: 4.3%)
- Barrier function: Not specified
- Morphology: Not specified
- Contamination: Not specified
- Reproducibility: Not specified

NUMBER OF REPLICATE TISSUES: Three

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : The part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues. For quantitative correction of results, two killed tissues were treated with 25 mg of the test item (KT) and two killed tissues were left untreated as a control (KU), respectively. NSMTT was then calculated relative to the negative control of living tissues.
- Procedure used to prepare the killed tissues (if applicable): Not specified
- N. of replicates : Two for the test item and the killed tissues. Three for the negative control of living epidermis.
- Method of calculation used: According to the following formula: NSMTT [%] = [(ODKT - ODKU)/ODNK] * 100 If the test item is classified as non-irritant and if non-specific MTT reduction is ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues TM was corrected according to the following formula: TODTT = ODTM – (ODKT – ODKU) If non-specific MTT reduction is > 30% relative to the negative control of living epidermis, the test item is considered as incompatible with the test method.

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin in accordance with regulation EC 1272/2008 if the tissue viability after exposure and post-incubation is less or equal to 50%.
- The test substance may be considered as non-irritant to skin in accordance with regulation EC 1272/2008 if the tissue viability after exposure and post-treatment incubation is more than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: Firstly, 25 µL of sterile Dulbecco’s phosphate buffered saline (DPBS) were applied to the epidermal surface in order to improve the contact between the powder and the epidermis. Afterwards, 25 mg (39 mg/cm2) of the test item were applied directly atop the EpiDerm tissue using an application spoon avoiding compression of the test item. The test item was spread to match size of the tissue by using a bulb-headed Pasteur pipette.
- Concentration (if solution): Not applicable

NEGATIVE CONTROL
- Amount(s) applied: 30 µL DPBS
- Concentration (if solution): Not applicable

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL 5% SDS solution
- Concentration (if solution): Not applicable
Duration of treatment / exposure:
60 min
After dosing of all tissues, all plates were transferred to the incubator for 35 ± 1 min. Afterwards all plates were removed from the incubator and placed under the sterile flow for the remaining time until the 60 ± 1 min incubation time of the first dosed tissue was over.
Duration of post-treatment incubation (if applicable):
42 hours
The plates were post-incubated at 37 ± 1 °C, 5.0% CO2, humidified to 95%, for 24 ± 2 h. Following this incubation the tissues were transferred to new wells containing 0.9 mL fresh assay medium and incubated for additional 18 ± 2 h.
Number of replicates:
Three

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1
Value:
80.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
2
Value:
66.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3
Value:
79.3
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
75.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean (NSMTT Corrected)
Value:
75.3
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:
- OTHER EFFECTS:
- Visible damage on test system: None specified
- Direct-MTT reduction: Yes. The mixture of 25 mg test item per 1 mL MTT medium showed reduction of MTT compared to the solvent. The mixture turned blue/purple.
- Colour interference with MTT: The mixture of 25 mg of the test item per 300 µl aqua dest. showed colouring detectable by unaided eye-assessment. Therefore, the absorption of the chemical in water was measured in the range of 570 ± 30 nm

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The controls confirmed the validity of the study.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. The mean absolute OD570 of the three negative control tissues was ≥ 0.8 and ≤ 2.8 (1.821).
- Acceptance criteria met for positive control: Yes. The mean relative tissue viability (% negative control) of the positive control was ≤ 20% (3.4%).
- Acceptance criteria met for variability between replicate measurements: Yes. Standard deviation of viability of replicate tissues of all dose groups was ≤ 18% (0.1% – 7.7%).

Any other information on results incl. tables

Please refer to document attached below (OECD 439 Study 179040 Tables of Results) .

Applicant's summary and conclusion

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%.. The test item is therefore classified as “non-irritant” in accordance with the CLP regulation EC 1272/2008.
Executive summary:

In the present study the skin irritant potential of Escherichia coli dehalogenase catalyst was analysed. The EpiDerm™-Standard Model (EPI-200™), a reconstituted three-dimensional human epidermis model, was used as a replacement for the Draize Skin Irritation Test (OECD TG 404), to distinguish between UN GHS “Category 2” skin irritating test substances and not categorized test substances (“No Category”) which may be considered as non-irritant. Hereby, the test item was applied topically. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT after a 60 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls.

The mixture of 25 mg test item per 1 mL MTT medium showed reduction of MTT compared to the solvent. The mixture turned blue/purple.

For quantitative correction of results, two killed tissues were treated with 25 mg of the test item (KT) and two killed tissues were left untreated as a control (KU), respectively. NSMTT was calculated relative to the negative control of living tissues (NK) according to the following formula: NSMTT [%] = [(ODKT - ODKU)/ODNK] * 100 = 0.3%

Mean ODKT = 0.057

Mean ODKU = 0.052

Mean ODNK = 1.777

NSMTT was ≤ 30% (0.3%) relative to the negative control of living epidermis, the true MTT metabolic conversion (TODTT) of the test item treated living tissues TM was therefore corrected according to the following formula: TODTT = ODTM – (ODKT – ODKU) = 75.6% - 0.3% = 75.3%

The mixture of 25 mg of the test item per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment.

The mixture of 25 mg of the test item per 300 µl aqua dest. showed colouring detectable by unaided eye-assessment. Therefore, the absorption of the chemical in water was measured in the range of 570 ± 30 nm. The test item in water absorbed light in the relevant range.

For quantitative correction of results, the non-specific colour of additional viable tissues (NSCliving) was determined by using additional viable tissues without MTT-staining and calculated according to the following formula:

NSCliving [%] = [ODTVT/ODNK]*100 = 0.3%

Mean ODTVT = 0.005

NSCliving was ≤ 5% relative to the negative control of living epidermis, therefore no correction of the results was necessary.

As correction with the NSCliving control was not necessary, correction with the NSCkilled control was also not required.

The test item showed no irritant effects. The mean relative tissue viability (% negative control) was > 50% (75.3%, NSMTT-corrected) after 60 min treatment and 42 h post-incubation.