Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Remarks:
Test performed according to OECD 439 & GLP.
Adequacy of study:
key study
Study period:
1 December 2017 - 27 March 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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
3,4,5-trihydroxybenzoic acid
EC Number:
205-749-9
EC Name:
3,4,5-trihydroxybenzoic acid
Cas Number:
149-91-7
Molecular formula:
C7H6O5
IUPAC Name:
3,4,5-trihydroxybenzoic acid
impurity 1
Chemical structure
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
Oxidane
impurity 2
Reference substance name:
sum of impurities (organic and inorganic) not relevant for classification
Molecular formula:
not available for mixtures
IUPAC Name:
sum of impurities (organic and inorganic) not relevant for classification
Test material form:
solid
Details on test material:
Name: gallic acid
Form: powder
Color: white
Specific details on test material used for the study:
Test Item Name: Gallic Acid
Chemical Name (IUPAC): 3,4,5-Trihydroxybenzoic acid
CAS No.: 149-91-7
Physical Appearance: White powder
Batch Produced by: Archroma
Date of Expiry: 13.07.2019
Storage Conditions: Ambient (21 to 29ºC)

In vitro test system

Test system:
human skin model
Remarks:
Reconstructed Human Epidermis (RHE) EpiDerm™ (EPI-200-SIT)
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: human derived non-transformed keratinocytes
Justification for test system used:
Test system recommended in the OECD test Guideline No. 439.
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM:
The Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SIT) was used as test system.

SOURCE OF THE TEST SYSTEM:
MatTek In Vitro Life Science Laboratories, s.r.o, MlynskéNivy 73, 821 05, Bratislava II, Slovak Republic, www.mattek.com; Phone: +421-2-3260-7401; Fax: +421-2-3260-7404.

PREPARATION OF REAGENTS AND MEDIA:
1. MTT Solution
MTT solution was prepared by first thawing the MTT concentrate (MTT-100-CON). MTT concentrate of 2 mL (5 mg/mL) was then diluted to 10 mL by adding 8 mL of MTT diluent (MTT-100-DIL) to obtain the final concentration of 1 mg/mL. As the MTT is light sensitive MTT solution was stored at 2 to 8°C in vial wrapped with aluminum foil.

2. Negative control
The sterile Dulbecco’s Phosphate Buffered Saline (DPBS) provided by MatTek was used as negative control (NC).

3. Positive control
Ready to use 5% Aqueous Sodium Dodecyl Sulfate (SDS) provided by MatTek was used as positive control (PC).
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
The tissues were exposed to 25 mg of test item.
Duration of treatment / exposure:
60 minutes (35 minutes at 37°C and 25 minutes at room temperature°C).
Duration of post-treatment incubation (if applicable):
Approximately 45 hours.
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Value:
ca. 70.3
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
TEST ACCEPTANCE:
1. The assay met the acceptance criterion as the mean OD570 of the NC tissues is 1.757, which is in the range of ≥0.8 and ≤ 2.8.
2. The assay met the acceptance criterion as the mean viability of PC tissues was 6.4% which is ≤ 20% of the negative control tissues and the SD of the three tissues replicates is 4.34.
3. The assay met the acceptance criterion as the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is <18% i.e., in the range of 0.41 to 4.34.

Any other information on results incl. tables

The mean OD of the negative control tissues is 1.757 which within the range of ≥0.8 and ≤2.8, hence the tissues were considered as viable after shipping and storing procedures and under specific conditions of use.

Percentage viability of tissues was measured by performing MTT assay after 60 minutes of treatment and post incubation of 44 hours and 47 minutes.

The mean percentage viability of test item treated tissues was 70.3, which is >50% of the negative control, hence the test item is considered as non-irritant.

The mean percentage of viability in the positive control treated tissues was 6.4, which is less than 50%, which shows the irritative potential of the PC and the suitability of the test method.

Table 1: Summary of Optical Density (OD) and Viability

Treatment

 

OD

Viability (%)

Classification

Negative Control

(DPBS)

Mean

1.757

100

NI

±SD

0.076

4.34

n

3

3

Positive Control

(5% Sodium Dodecyl Sulphate)

Mean

0.112

6.4

I

±SD

0.007

0.41

n

3

3

Test Item

(Gallic Acid)

Mean

1.235

70.3

NI

±SD

0.037

2.08

n

3

3

NI: Non-irritant; I= irritant; n= No. of tissues; SD: Standard Deviation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results obtained under the laboratory testing conditions, the test item Gallic Acid has been categorized as non-irritant to Reconstructed Human Epidermis (RhE) in accordance with UN GHS, as the mean percentage of tissue viability was greater than 50% of the negative control.
Executive summary:

The objective of this study was to evaluate the skin irritation potential of Gallic Acid using Reconstructed Human Epidermal Model - EpiDerm™ (EPI-200-SIT) as per the OECD Guideline for the testing of chemicals No. 439, “In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method”, adopted on 28 July 2015 and Commission Regulation (EU) No 640/2012 of 06 July 2012 amending, for the purpose of its adaptation to technical progress, Regulation (EC) No 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH), B.46. In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method.

The test item did not develop any colour when dissolved in distilled water and was considered as non-reducer of MTT as no purple colour was developed when mixed and incubated with MTT solution.

After receipt of the tissues, visual inspection was done to verify the defects. As there were no tissue defects, air bubble or excess moisture observed, all the tissue inserts were used for the study. Tissue inserts were transferred to the upper row of 6-well plates prefilled with 0.9 mL of assay medium and incubated in CO2 incubator for 16 hours. After the incubation period, the tissues were topically exposed to 30 µL of DPBS (negative control: NC), 30 µL of 5% aq. SDS solution (positive control: PC) or 25 mg of test item (Gallic Acid). All the treatments were maintained in triplicates. After 60 minutes of exposure with negative control, positive control or test item, the tissues were washed using DPBS. Later, the tissue inserts were blotted and transferred to fresh medium and incubated in CO2 incubator for 23 hours and 50 minutes. After the incubation period (Day 1), tissue inserts were shifted from upper wells to lower wells of 6-well plates prefilled with 0.9 mL of assay medium. After the medium change, tissues were incubated for an additional 20 hours and 57 minutes in CO2 incubator. After the post-incubation period, the bottom of the tissue inserts was blotted and transferred into an MTT solution and incubated for 2 hours and 55 minutes. The resultant purple-blue formazan salt, formed mainly by mitochondrial metabolism, was extracted for 2 hours and 16 minutes using an extraction solvent (MTT-EXT-100). The optical density of the extracted formazan was measured in a 96-well plate spectrophotometer at 570 nm. Viability of tissues was calculated by entering OD values in the spreadsheet provided by MatTek.

Percentage viability of negative control, positive control and test item was 100±4.34, 6.4±0.41 and 70.3±2.08 respectively. As the percentage viability of the test item was greater than 50% of the negative control, the test item is considered as non-irritant. The percentage of viability in the positive control (PC) was less than 50%, which shows the irritative potential of the PC and the suitability of the test method

Conclusion

Based on the results obtained under the laboratory testing conditions, the test item Gallic Acid has been categorized as non-irritant to Reconstructed Human Epidermis (RhE) in accordance with UN GHS no Category, as the mean percentage tissue viability was greater than 50% of the negative control.