Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-11-10 to 2017-01-26
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
Details on test material:
- Source and lot/batch No.of test material: 201601004
- Expiration date of the lot/batch: 01.01.2019
- Purity: 99.93 %
- Storage condition of test material: room temperature

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
This test uses the EPISKIN-SM™ reconstructed human epidermis model (SkinEthic) 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 skin, i.e. the epidermis.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN-SM™ (SkinEthic)
- Tissue batch number(s): 16-EKIN-048

SkinEthic Kit:
- EPISKIN-SM™ plate containing 12 reconstructed epidermis units (area: 0.38 cm²); each reconstructed epidermis is attached to the base of a tissue culture insert with an O-ring set and maintained on nutritive agar for transport (Lot No.: 16-EKIN-048):
1x 12-well assay plate
1x flask of sterile maintenance medium (basic medium for incubations, Lot No.: 16-MAIN3-079)
1x flask of sterile assay medium (basic medium for use in MTT assays, Lot No.: 16-ESSC-051)

- Validity controls as provided by the supplier (SkinEthic):
Morphology:
Histology scoring (HES stained vertical paraffin sections, n = 6):
Specification ≥ 19.5
Result: 22.3 ± 0.3, CV = 1.2%
Well-differentiated epidermis consisting of a basal layer, several spinous and granular layers and a thick stratum corneum.
Barrier function:
IC50 determination (SDS concentration, MTT test, n= 14):
Specification ≥ 1.5 mg/mL
Result: 1.7 mg/mL

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: Room temperature
- Temperature of post-treatment incubation (if applicable): 37 ± 1 °C, 5.0 % CO2

REMOVAL OF TEST MATERIAL AND CONTROLS
- washed with DPBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT solution
MTT stock solution: 3 mg/mL MTT (Sigma; Lot No.: MKBR6576V) in PBS (Gibco; Lot No.: 1722256)
MTT medium: MTT stock solution was diluted 1 + 9 with DMEM-based medium (final concentration 0.3 mg/mL)
- Acidic isopropanol
0.04 N HCl (AppliChem; Lot No.: 0000687102) in isopropanol (AppliChem; Lot No.: 0000694025)
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Wavelength: 570 nm
- Filter bandwidth: ± 30 nm

NUMBER OF REPLICATE TISSUES: 3 tissues per dose group

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant to skin if the viability after 15 minutes exposure and 42 h post-treatment incubation is greater than 50%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
yes, concurrent MTT non-specific colour control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 ± 2 mg + 5 µL aqua dest.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL DPBS (Gibco, Cat No. 14040-091, Lot No.: 1737107)

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL Sodium dodecyl sulfat (SDS; AppliChem, Art.-No. A1112,0500, CAS No.: 151-21-3, Lot No.: 40015277)
- Concentration (if solution): 5% in aqua dest.
Duration of treatment / exposure:
15 min ± 0.5 miin
Duration of post-treatment incubation (if applicable):
42 ± 1 h
Number of replicates:
3 tissues per dose group

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean of three tissues
Value:
70.3
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
For detailed results see "Any other information on results" Table 1

Any other information on results incl. tables

Results of thePre-Experiments

The mixture of 10 mg test item per 2 mL MTT medium showed reduction of MTT as compared to the solvent. The mixture turned blue/purple.

For quantitative correction of results the part of absorption due to the non-specific reduction of MTT (NSMTT) was determined by using killed tissues. Therefore, three killed tissues were treated with the test item (KT) and with the negative control DPBS (KU), respectively. NSMTT was calculated relative to the negative control of living tissues (NK) according to the following formula:

NSMTT [%] = [(ODKT- ODKU)/ODNK] * 100

Mean ODKT= 0.066

Mean ODKU= 0.032

Mean ODNK= 0.685

The calculated NSMTT was ≤ 30% (4.9%) relative to the negative control of livingepidermisand could therefore be used for determination of the true MTT metabolic conversion (TODTT) of the test item treated living tissues (TM) according to the following formula:

TODTT= ODTM- (ODKT- ODKU) (results see Table 1)

The mixtures of 10 mg of the test item per 90 µL aqua dest. and per 90 µL isopropanol showed no colouring detectable by unaided eye-assessment. Therefore, NSClivingequalled 0%. No additional controls were necessary.

Results of the main experiment

Table1:  Result of the test Item

Name Negative Control Positive Control Test Item
Tissue 1 2 3 1 2 3 1 2 3
Absolute OD570 0.606
0.643
0.671
0.728
0.737
0.726
0.096
0.102
0.076
0.084
0.096
0.093
0.540
0.546
0.558
0.565
0.372
0.382
OD570 (Blank corrected 0.563
0.600
0.628
0.685
0.694
0.683
0.053
0.059
0.033
0.041
0.053
0.050
0.497
0.503
0.515
0.522
0.329
0.342
Mean OD570 of the Duplicates (Blank corrected)  0.582 0.656 0.689 0.056 0.037 0.052 0.500 0.519 0.336
Total Mean OD570 of 3 Replicate Tissues (Blank corrected) 0.642* 0.048 0.451
SD OD570 0.055 0.010 0.101
TODIT - - 0.417
Relative Tissue Viabilities [%] 90.6 102.2 107.2 8.7 5.8 8.0 77.8 80.7 52.3
Mean Relative Tissue Viabilities [%] 100.0 7.5** 70.3
SD Tissue Viabilities [%]*** 8.5 1.5 15.7
CV [% Viability] 8.5 20.5 22.3

*Corrected mean OD570of the negative control corresponds to 100% absolute tissue viability.

**Mean relative tissue viability of the three positive control tissues is40%.

***Standard deviation (SD) obtained from the three concurrently tested tissues is 18%.

Table 2: Test Acceptance Criteria

 

Value

Cut off

pass/fail

Mean OD570 nmBlank

0.043

< 0.1

pass

Mean Absolute OD570 nmNK

0.685

0.6 ≤ NK ≤1.5

pass

Mean Relative Viability [%] PC

7.5

≤ 40%

pass

Max. SD of % Viability [%]

15.7

≤ 18%

pass

 

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”.
Executive summary:

In the present study the skin irritant potential of Propyl gallate (> 99.93% purity) was analysed according to OECD 439 using the EPISKIN-Standard Model™ (EPISKIN-SMTM), a reconstituted three-dimensional human epidermis model 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 15 min exposure and 42 h post-incubation period and compared to those of the concurrent negative controls.

In this study under the given conditions the test item showed no irritant effects (70.3% mean relative tissue viability). As the relative mean tissue viability after 15 min of exposure and 42 h post-incubation was > 50%, the test item is therefore classified as “non-irritant” in accordance with UN GHS “No Category”.