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Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Feb. 14, 2018 to Feb. 16, 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Deviations:
no
Qualifier:
according to
Guideline:
other: Method B.40bis of Commission Regulation (EC) No 440/2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(UK, OECD and EC Commission Directive principles of GLP)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of the test material: Benzenesulfonic acid, mono-C10-13-alkyl derivs., compds. with ethanolamine; monoethanolamine neutralized linear alkyl benzene sulphonate
- Trade name: MEA LAS
- Common name: MEA mono-C10-13 LAS
- TSIN: GTS122043
- Physical state/Appearance: Off-white opaque viscous liquid
- Expiry Date: May 01, 2019
- Storage Conditions: Room temperature in the dark
- Lot: M989214EX-001

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: Epithelial, derived from human skin, and formed into a stratified, cornified epithelium
Details on animal used as source of test system:
No animal was used as source of test system.
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPIDERM™ Human Skin Model
- Supplier: MatTek In Vitro Life Sciences Laboratories, Bratislava, Slovakia
- EpiDermTM Tissues (0.63cm2) lot number: 25878
- Assay Medium lot number: 020818ALD
- Date received: 13 February 2018
- Date of initiation of testing: 14 February 2018

TEMPERATURE USED FOR TEST SYSTEM
- Storage prior to test: The sealed 24-well plate was stored in a refrigerator until use.
- Temperature used during treatment / exposure: Room temperature and incubator (37°C)
- Temperature of post-treatment incubation: Room temperature (overnight)

REMOVAL OF TEST MATERIAL AND CONTROLS:
- Volume and number of washing steps: After the appropriate exposure time, Dulbecco’s Phosphate Buffered Saline (DPBS) (without Ca++ Mg++) was used for rinsing to remove any residual test substance. Rinsing was achieved by filling and emptying each tissue twenty times for approximately 40 seconds using a constant soft stream of DPBS.
- Observable damage in the tissue due to washing: No

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL (prepared from a MatTek MTT-100 kit immediately prior to usage)
- MTT extractant solution: 2 mL Isopropanol
- Incubation time: 3 hours
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA: No details were provided in the source.

NUMBER OF REPLICATE TISSUES: Two. One 6-well plate was used for each exposure period. Two cells in the 6-well plate were assigned for each negative control, test substance and positive control.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: No interference of the test substance with MTT. The MTT solution containing the test substance did not turn blue/purple indicating that the test substance did not reduce MTT.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One for each exposure period

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
Amount/concentration applied:
TEST MATERIAL
- Amount applied: 50 μL
- Concentration: 20% formulation (nominal) corresponding to 19.01% formulation (measured)

VEHICLE: No

NEGATIVE CONTROL
- Amount applied: 50 μL sterile distilled water
- Concentration: Not applicable

POSITIVE CONTROL
- Amount applied: 50 μL Potassium Hydroxide
- Concentration: 8.0 N
Duration of treatment / exposure:
3 and 60 minutes
Duration of post-treatment incubation (if applicable):
3 hours
Number of replicates:
One 6-well plate was used for each exposure period. Two cells in 6-well plate were assigned for each negative control, test substance and positive control.

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 minute exposure period
Value:
107.5
Vehicle controls validity:
not examined
Negative controls validity:
valid
Remarks:
(According to OECD guideline 431, the mean OD570 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time. The mean OD570 was 1.737 for the 3-minute exposure period and 1.815 for the 60-minute exposure period.)
Positive controls validity:
valid
Remarks:
(According to OECD guideline 431, the mean viability of the tissue replicates exposed for 1 hour with the positive control (8 N KOH) should be ≤15%. The mean viability for tissues were 5.3 and 3.7% for the 3 and 60 minute exposure periods, respectively.)
Remarks on result:
other: The test substance was considered to be non-corrosive to the skin.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
60 minute exposure period
Value:
62.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Remarks:
(According to OECD guideline 431, the mean OD570 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time. The mean OD570 was 1.737 for the 3-minute exposure period and 1.815 for the 60-minute exposure period.)
Positive controls validity:
valid
Remarks:
(According to OECD guideline 431, the mean viability of the tissue replicates exposed for 1 hour with the positive control (8 N KOH) should be ≤15%. The mean viability for tissues were 5.3 and 3.7% for the 3 and 60 minute exposure periods, respectively.)
Remarks on result:
other: The test substance was considered to be non-corrosive to the skin.
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No
- Direct-MTT reduction: The MTT solution containing the test substance did not turn blue/purple, indicating the test substance did not reduce MTT.
- Color interference with MTT: The solution containing the test substance did not become colored, indicating the test substance did not have the potential to cause color interference.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes. According to OECD guideline 431, the mean OD570 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time. The mean OD570 was 1.737 for the 3-minute exposure period and 1.815 for the 60-minute exposure period.
- Acceptance criteria met for positive control: Yes. According to OECD guideline 431, the mean viability of the tissue replicates exposed for 1 hour with the positive control (8 N KOH) should be ≤15%. The mean viability for the positive control treated tissues were 5.3 and 3.7% for the 3 and 60 minute exposure periods, respectively.
- Acceptance criteria met for variability between replicate measurements: Yes. In the range 20 and 100% viability, the Coefficient of Variation (CV) between tissue replicates should be ≤ 30%. The CV between the two tissue replicates of each treatment group did not exceed 30%.

Any other information on results incl. tables

Table 1: Mean OD570 values and viabilities for the negative control, positive control and test substance (Study# 98499)

Tissue Exposure period Mean OD570 of individual tissues Mean OD570 of duplicate tissues Standard Deviation Coefficient of Variation (%) Relative Mean Viability (%)
Negative control 3 minutes 1.679 1.737 0.082 4.7 100*
1.795
60 minutes 1.831 1.815 0.023 1.2
1.799
Positive control 3 minutes 0.098 0.093 0.008 na 5.3
0.087
60 minutes 0.064 0.068 0.006 na 3.7
0.072
Test substance 3 minutes 1.944 1.867 0.109 5.8 107.5
1.790
60 minutes 1.216 1.137 0.112 9.9 62.6
1.057

OD = Optical density

* = The mean percentage viability of the negative control tissue is set at 100%

na = Not applicable

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In an in vitro test for skin corrosion using the EpiDerm™ Human Skin Model, benzenesulfonic acid, mono- C10-13-alkyl derivs., MEA salts (CAS RN 85480-55-3; 19.01% active) was considered to be non-corrosive to the skin after treatment periods of 3 and 60 minutes.
Executive summary:

An in vitro study was conducted to evaluate the corrosivity potential of benzenesulfonic acid, mono- C10-13-alkyl derivs., MEA salts (CAS RN 85480-55-3; 19.01% active) according to OECD guideline 431 and EU Method B.40bis using the EpiDerm™ Human Skin Model.

Duplicate EpiDerm™ tissues were treated with the test substance for exposure periods of 3 and 60 minutes. Negative control (sterile distilled water) and positive control (8.0 N potassium hydroxide) groups were also treated for each exposure period. At the end of the exposure period, the tissues were rinsed, before each tissue was taken for MTT-loading. After MTT loading each tissue was placed in 2 mL isopropanol for MTT extraction.

At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 μL samples were transferred to the appropriate wells of a pre-labelled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).

The acceptance criteria for negative control and positive control groups were met in the study. The mean OD570 for negative control treated tissues was 1.737 for the 3-minute exposure period and 1.815 for the 60-minute exposure period. The mean viability for the positive control treated tissues were 5.3 and 3.7% for the 3 and 60 minute exposure periods, respectively. The coefficient of variation (CV) between tissue replicates did not exceed 30%.

The relative mean viabilities for test substance were 107.5 and 62.6% for 3 and 60 minutes exposure periods, respectively. Based on the mean viabilities, the test substance was categorized as non-corrosive.

In an in vitro test for skin corrosion using the EpiDerm™ Human Skin Model, benzenesulfonic acid, mono- C10-13-alkyl derivs., MEA salts (CAS RN 85480-55-3; 19.01% active) was considered to be non-corrosive to the skin after treatment periods of 3 and 60 minutes.

This skin corrosion test is classified as acceptable and satisfies the guideline requirement for the current OECD guideline 431 (In vitro skin corrosion: Reconstructed Human Epidermis (RhE) test method).