Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-09-07 to 2018-01-16
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:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
July 28, 2015
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
July 06, 2012
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
recommended by guideline
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: reconstituted three-dimensional human skin model EpiDerm™ (MatTek)
- Tissue batch number(s): Lot No.: 25849, 25864
- Date of initiation of testing: 2017-10-06

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ± 1 °C
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C for 35 ± 1 min followed by room temperature for 60 ± 1min.

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, staggered again in e.g. 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.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3h ± 5 min
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: specification: MTT QC assay, 4 h, n=3, acceptance: OD (540-570 nm) [1.0-3.0], result: 1.572 ± 0.078
- Barrier function: specification: ET 50 assay 100 µL 1% Triton X-100, 4 time-points, n=3, MTT assay; acceptance: ET-50 [4.77-8.72 h]; result: 4.62 h
- Contamination:
HIV-1 virus - Oligonucleotide-directed amplification - Not detected
Hepatitis B virus - OligonucIeotide- directed amplification - Not detected
Hepatitis C virus - Oligonucleotide- directed amplification - Not detected
Bacteria, yeast, and other fungi - long term antibiotic, antimycotic free culture - Not detected
- Reproducibility: In accordance to the test guideline the assay is considered to be of sufficient reproducibility if the
- mean absolute OD 570 nm of the three negative control tissues is ≥ 0.8 and ≤ 2.8
- mean relative tissue viability of the three positive control tissues is ≤ 20%
- standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%.
The respective results and historical control data are shown in table 1.

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : Fresh and killed tissues were used for determination of non-specific MTT-reduction capability, fresh tissue was used to determine the colouring potential of the test substance and killed tissue was used if the substance acts as non-specific MTT reducer and shows non-specific colouring in order to avoid a possible double correction.
- N. of replicates : 2
- Method of calculation used: To check the non-specific MTT-reducing capability of the test 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 (TODTT ) of the test item treated living tissues TM was corrected according to the following formula:
TODTT = OD TM – (OD KT – OD KU )
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.
To check the colouring potential of the test item 25 mg of the test item were 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 MTT-staining was performed with the test item treated with tissues, which were incubated in medium without MTT. 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
If NSC living is > 30% relative to the negative control of living epidermis, the test item is considered as incompatible with the test method. 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 MTT-staining was performed with the test item treated with tissues, which was incubated in medium without MTT. 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: 1

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after exposure and post-treatment incubation is less than 50%.
- The test substance is considered to be non-irritant to skin if the viability after exposure and post-treatment incubation is greater than or equal to 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 mg of the test item + 25 µL of DPBS

VEHICLE
- Amount(s) applied (volume or weight with unit): 25 µL DPBS
- Lot/batch no. (if required): 1838067

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% solution
Duration of treatment / exposure:
95 min ± 1 min
Duration of post-treatment incubation (if applicable):
24 h ± 2 h
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
92.8
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: 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. 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 = 0.25%
NSCliving was ≤ 5% relative to the negative control of living epidermis, therefore no correction of the results was necessary.

ACCEPTANCE OF RESULTS:
Acceptance criteria: The test is is considered to be valid if
- mean absolute OD 570 nm of the three negative control tissues is ≥ 0.8 and ≤ 2.8
- mean relative tissue viability of the three positive control tissues is ≤ 20%
- standard deviation (SD) of relative tissue viability obtained from each three concurrently tested tissues is ≤ 18%.

Any other information on results incl. tables

Table 2: Result of the NSClivingcontrol

NSCliving

TVT

Negative Control

Tissue

1

2

1

2

3

absolute OD570 -values

0.046

0.050

1.911

1.656

1.728

0.048

0.047

1.911

1.672

1.722

absolute OD570 - 

Blank corrected values

0.0027

0.0064

1.8676

1.6125

1.6844

0.0045

0.0037

1.8674

1.6286

1.6789

mean OD570 

(mean of 3 aliquots)

0.004

0.005

1.868

1.621

1.682

total mean OD570

(mean of replicate tissues)

0.004

1.723*

SD OD570 

(of the 2 replicate tissues)

0.001

0.129

NSCliving[%]

0.25

-

Relative Tissue Viability [%]

-

108.4

94.0

97.6

Mean Relative Tissue Viability [%]

-

100.0

SD Tissue Viability [%]

-

7.5

CV [% Viabilities]

-

7.5

Table 3: Result of the Test Item Tetrahydrofolic acid (THFA)

Name

Negative Control

Positive Control

Test Item

Tissue

1

2

3

1

2

3

1

2

3

Absolute OD570

1.518

1.554

1.533

1.471

1.619

1.578

0.123

0.124

0.119

0.121

0.112

0.111

1.344

1.394

1.515

1.520

1.427

1.421

OD570(Blank Corrected)

1.475

1.510

1.489

1.428

1.576

1.535

0.080

0.080

0.075

0.078

0.068

0.067

1.3001

1.351

1.471

1.476

1.384

1.377

OD570(Blank Corrected) - NSClivingCorrected

 -

 -

1.300

1.351

1.471

1.476

1.384

1.377

Mean OD570of the Duplicates (Blank

Corrected)

1.493

1.459

1.555

0.080

0.076

0.068

1.326

1.474

1.381

Total Mean OD570of 3 Replicate Tissues (Blank Corrected)

1.502*

0.075

1.393

TODTT

 -

 -

1.393

SD OD570

0.049

0.006

0.075

Relative Tissue Viability [%]

99.4

97.1

103.5

5.3

5.1

4.5

88.2

98.1

91.9

Mean Relative Tissue Viability [%]

100.0

5.0**

92.8

Mean Relative Tissue Viability [%] - NSClivingCorrected

 -

 -

92.8

SD Tissue Viability [%]***

3.3

0.4

5.0

CV [% Viabilities]

3.3

8.4

5.4

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 test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.