Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-05-03 to 2017-10-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Reconstructed human epidermis tissue containing normal human keratinocytes
Justification for test system used:
This test uses the EpiDerm™ reconstructed human epidermis model (MatTek) which consists of normal human epidermal keratinocytes (NHEK) and therefore represents in vitro the target organ of the species of interest and closely mimics the biochemical and physiological properties of the upper parts of the human, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ reconstructed human epidermis model
- Tissue batch number(s): Lot No.: 25813, 25829, 25838
- Date of initiation of testing: 2017-05-17

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

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: the inserts were washed at least 15 times with DPBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Wavelength: 570 nm
- Filter bandwidth: a filter band pass of maximum ± 30 nm without reference wavelength

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: MTT QC assay, 4 h, n = 3; Acceptance criteria: OD (540-570 nm) [1.0 - 3.0], Result (batch: 25813): 1.563 ± 0.022, (batch: 25829) 1.857 ± 0.107, (batch: 25838) 2.403 ± 0.238
- Barrier function: ET-50 assay, 100µL 1%Triton X-100, 4 time-points, n = 3, MTT assay; Acceptance criteria: ET-50 [4.77 - 8.72]; Result (batch: 25813): 5.38 h, (batch: 25829) 5.4 h, (batch: 25838) 5.36 h
- Contamination:HIV-1, Hepatitis B, Hepatitis C, Bacteria, yeast, and other fungi: not detected

NUMBER OF REPLICATE TISSUES: triplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues:
To check the non-specific MTT-reducing capability of the test item 25 mg of the test item was mixed per 1 mL MTT medium and incubated for 60 min at 37 ± 1 °C in the incubator. If the mixture turns blue/purple, the test item is presumed to have reduced MTT and 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 (NK) according to the following formula:
NSMTT [%] = [(OD KT - OD KU )/OD NK ] * 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 (TOD TT ) of the test item treated living tissues TM was corrected according to the following formula:
TOD TT = OD TM – (OD KT – OD KU )
To check the colouring potential of the test item 25 mg of the test item was mixed per 300 µL aqua dest. and per 300 µL isopropanol each in a transparent recipient and incubated at 37 ± 1°C for 60 min. If the test item is classified as non-irritant and colouring is detected by unaided eye-assessment, and the chemical in water and/or isopropanol absorbs light in the range of 570 ± 30 nm, the test item was checked for its tissue-colouring potential. For quantitative correction of results, the test was performed using two additional living tissues treated with 25 mg of the test item (TVT). The non-specific colour
of additional viable tissues (NSC living ) was then calculated according to the following formula:
NSC living [%] = [OD TVT /OD NK ]*100
If NSC living is ≤ 5% relative to the negative control of living epidermis, no correction of the results is necessary.
If NSC living is > 5% and ≤ 30% relative to the negative control of living epidermis, the true MTT metabolic conversion (TOD TT ) was corrected according to the following formula:
TOD TT = OD TM – OD TVT
For test items which are classified as non-irritant and which act as non-specific MTT-reducers and show non-specific colouring of living tissues, a third control for non-specific colour in killed tissues (NSC killed ) was performed to avoid a possible double-correction for colour interference. Therefore, two killed tissues were treated with 25 mg of the test item (TKT). The non-specific colour of additional viable tissues (NSC killed ) was then calculated according to the following formula:
NSC killed [%] = [OD TKT /OD NK ]*100
The true tissue viability was then calculated as the percent tissue viability obtained with living tissues minus NSMTT minus NSC living plus NSC killed .
If the coloured test material or MTT reducing substance is classified as “irritant” i.e. mean tissue viability is < 50%, the correction procedures are not necessary.


NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 4

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after exposure and post-incubation is less than or equal 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 µL of sterile DPBS was 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 was 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 gently shaking the inserts or by using a bulb-headed Pasteur pipette.

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL 5% SDS
- Concentration (if solution): 5%
Duration of treatment / exposure:
35 ± 1 min
Duration of post-treatment incubation (if applicable):
24 ± 2 h
Number of replicates:
triplicates

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
test item, experiment I
Value:
50.1
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
4.7% tissue viability
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
test item, experiment II
Value:
96.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
3.9% tissue viability
Remarks on result:
no indication of irritation
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
test item, experiment III
Value:
48.7
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
3.9% tissue viability
Remarks on result:
other: Standard deviation of Viabilities was not below 18 %
Irritation / corrosion parameter:
other: absolute tissue viability
Run / experiment:
test item, experiment IV
Value:
88.8
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Remarks:
4.4% tissue viability
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: The mixture of 25 mg test item per 1 mL MTT medium showed no reduction of MTT compared to the solvent. The mixture did not turn blue/purple. Therefore, NSMTT equalled 0%.
- Colour interference with MTT: The mixture of 25 mg of the test item per 300 µL aqua dest. and/or per 300 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSC equalled 0%.

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, except for Experiment III
- Range of historical values if different from the ones specified in the test guideline:
Mean Relative viability [%] Positive Control: 3.9 ± 1.9 (n = 27), Mean OD 570 ± 30 nm NK = 1.831 ± 0.357 (n = 27)
Acceptance criteria: Mean OD 570 nm Negative Control: ≥ 0.8 and ≤ 2.8, Mean Relative viability [%] Positive Control ≤ 20, Standard deviation of viability of replicate tissues of all dose groups ≤ 18% (except Experiment III)

Any other information on results incl. tables

Table 1: Mean Absorbance of the test material, Relative Viability [%] Positive Control and SD Viability [%]

Group

Exp. I

Exp. II

Exp. III

Exp. IV

Mean Absorbance 570 nm

0.999

1.451

0.730

1.556

Relative Viability [%] Positive Control

4.7

3.9

3.9

4.4

SD Viability [%]

0.2-14.4

0.7-6.4

0.4-38.3

0.1-6.8

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In the present study conducted according to OECD guideline 439 the test material m-Xylylenebismaleimide was applied to the reconstituted three-dimensional human skin model EpiDerm™(MatTek) for 60 min followed by a 42 h post-incubation period and immediate determination of cytotoxic effects via MTT reduction assay. There were no indications for skin irritation and all acceptance criteris were fulfiled, thus, the substance does not need to be classified according to Regulation (EC) 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).