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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017 (report date 31st October 2017)
Reliability:
2 (reliable with restrictions)
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
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
not specified
GLP compliance:
no
Remarks:
Lab was not set up to do GLP for these specific assays. Studies were performed in accordance with the principles of GLP and relevant OECD guideline. They were monitored by QA in accordance with GLP.

Test material

Constituent 1
Chemical structure
Reference substance name:
Potassium 2,5-dihydroxybenzenesulphonate
EC Number:
244-584-7
EC Name:
Potassium 2,5-dihydroxybenzenesulphonate
Cas Number:
21799-87-1
Molecular formula:
(HO)2C6H3SO3K
IUPAC Name:
potassium 2,5-dihydroxybenzenesulphonate
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Source: Mitsuboshi Chemical Co. Ltd. Lot: YY00G7P285
- Expiration date of the lot/batch: Date not specified - "The certificate of analysis lists the test material as having an expiration date of 2 years after shipment date (Miyazaki, 2016)"
- Purity test date: 99.8%

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not specified
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: Not specified
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test material(s) were tested as neat (100%) or as provided, following the OECD 431 test guideline.
- Preliminary purification step (if any): n/a
- Final dilution of a dissolved solid, stock liquid or gel: n/a
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material) n/a

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) n/a

OTHER SPECIFICS: n/a

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: Not specified
Source strain:
not specified
Details on animal used as source of test system:
n/a
Justification for test system used:
The EpiDerm three-dimensional human skin model has been extensively characterised and is approved for identification and classification of non-corrosive and corrosive substances and mixtures in accordance with United Nations Global Harmonised System (UN GHS).
Vehicle:
unchanged (no vehicle)
Details on test system:
SKIN DISC PREPARATION
- Procedure used: Upon receipt, the EpiDerm tissues were stored at 2-8ºC until used. On the day prior to testing, EpiDerm assay medium was warmed to room temperature and an aliquot of 0.9 mL was
dispensed into each well of a 6-well plate. The EpiDerm tissues were transferred from a 24-well shipping plate to a 6-well plate containing the fresh, pre-warmed assay medium. The EpiDerm tissues were incubated at approximately 37ºC in a humidified atmosphere of approximately 5% CO2 for 60 ± 5 min. At the end of the first preincubation period, the inserts were transferred into wells containing fresh warm assay medium and were incubated overnight (18 ± 3 hrs) to acclimate the tissue.
- Quality control for skin discs: Electrical resistance obtained with two of the isolated skin discs was [complete, e.g. 10 kΩ] : Not specified

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps:
- Observable damage in the tissue due to washing:
- Modifications to validated SOP:

DYE BINDING METHOD
- Dye used in the dye-binding assay: [none / MTT / Sulforhodamine B / other:]
- Spectrophotometer:
- Wavelength:
- Filter:
- Filter bandwidth:

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. if the mean TER value is less than or equal to 5 kΩ and the skin disk is obviously damaged, or if the mean TER value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage, but the mean disc dye content is greater than or equal to the mean disc dye content of the 10M HCl positive control obtained concurrently.]
- The test substance is considered to be non-corrosive to skin if [complete, e.g. if the mean TER value obtained for the test substance is greater than 5 kΩ, or if the mean TER value is less than or equal to 5 kΩ, and the skin disc is showing no obvious damage, and the mean disc dye content is well below the mean disc dye content of the 10M HCl positive control obtained concurrently.]
- Justification for the selection of the cut-off point(s) if different than recommended in TG 430:
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): not specified

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): not specified

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): not specified
Duration of treatment / exposure:
60 minutes +/- 1 minute
Duration of post-treatment incubation (if applicable):
42 hours +/- 2 hours
Number of replicates:
3

Test animals

Species:
other: EpiDerm
Strain:
other: Human
Details on test animals or test system and environmental conditions:
n/a

Test system

Type of coverage:
not specified
Preparation of test site:
other: Administered by topical application to the surface of the EpiDerm tissues: directly dispensed atop the tissue
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 µL
- Concentration (if solution): not specified - as supplied by sponsor

VEHICLE
- Amount(s) applied (volume or weight with unit): n/a
- Concentration (if solution): n/a
- Lot/batch no. (if required): n/a
- Purity: n/a

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): not specified

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): not specified
Duration of treatment / exposure:
60 minutes +/- 1 minute
Observation period:
42 +/- 2 hours
Number of animals:
n/a
Details on study design:
TEST SITE
- Area of exposure: 0.6 cm^2
- % coverage: not specified
- Type of wrap if used: not specified

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes - tissues rinsed with sterile DPBS to remove test substance after 60 +/- 1 minute treatment period
- Time after start of exposure: 60 +/- 1 minute

OBSERVATION TIME POINTS
(indicate if minutes, hours or days) : not specified


SCORING SYSTEM:
- Method of calculation: The mean of the corrected OD570 values for the negative control was calculated using the formula:

Corrected Individual Tissue OD570 = individual tissue OD570 – mean blank OD570

For each individual tissue, % viability relative to negative control was calculated using the following formula:

% Viability= (Corrected Individual OD570 of Test Chemical (or Control) / Corrected Mean OD570 of Negative Control) X 100

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute +/- 1 minute
Value:
ca. 103
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: Not specified
- Direct-MTT reduction: Results from the assessment of direct test method reduction of MTT experiment suggested no direct reduction of MTT dye by K-Salt, as the test material did not turn the MTT solution to a blue/purple color.
- Colour interference with MTT: n/a

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes: The corrected mean OD570 value of the negative control tissues (exposed for 60 minutes) was 1.00 (i.e., ≥ 1.0 and ≤ 2.5; criteria set by the tissue manufacturer).
- Acceptance criteria met for positive control: Yes: The relative mean viability of positive control (1% TRITON™ X-100) was 16.4% (i.e., ≤ 20% compared to negative control; criteria set by the tissue manufacturer).
- Acceptance criteria met for variability between replicate measurements: Not specified
- Range of historical values if different from the ones specified in the test guideline: n/a

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, K-Salt was interpreted as a potential non-irritant to skin (UN GHS No Category) in the EpiDerm irritation assay
Executive summary:

K-Salt was evaluated for skin irritation potential in an in vitro EpiDerm skin irritation assay. In this assay, K-Salt was topically applied to the EpiDerm tissue for 60 minutes, followed by a 42-hour post-exposure recovery. Following recovery, the cell viability was measured in treated and control tissues using MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and the data reported as a percentage of the mean of negative control.

The mean relative cell viability of K-Salt and positive control-treated tissues were 103% and 16.4% (i.e. ≤ 50%), respectively. Therefore, under the conditions of this study, K-Salt was interpreted as a potential non-irritant to skin (UN GHS No Category) in the EpiDerm irritation assay.