Registration Dossier

Toxicological information

Skin irritation / corrosion

Currently viewing:

Administrative data

Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 April 2018 - 06 April 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 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
adopted 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized
EC Number:
291-707-5
EC Name:
Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized
Cas Number:
90459-62-4
Molecular formula:
C24H55N3O6S
IUPAC Name:
bis(2-aminoethyl)amine octadecanoic acid dimethyl sulfate
Test material form:
solid
Remarks:
paste

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Justification for test system used:
The Reconstructed Human Epidermal Model EpiDerm™ (EPI-200-SCT) was selected as test system to assess the skin corrosion potential of the test item as it represents a recommended in vitro test system according to OECD Guideline No. 431.
Vehicle:
unchanged (no vehicle)
Remarks:
wetted with water
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm
- Tissue batch number(s): 25892

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37±1 °C, 5±1 % CO2 (1 h exposure), room temperature (3 min exposure)
- Temperature of post-treatment incubation (if applicable): 3 h

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: tissues were rinsed with sterile PBS (fill and empty insert 20 times in a constant soft stream of 1xPBS)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Wavelength: 540 nm

NUMBER OF REPLICATE TISSUES: 2

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 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
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg + 25 µL of distilled water

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL sterile distilled water

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µL glacial acetic acid
Duration of treatment / exposure:
3 min, 1 h
Duration of post-treatment incubation (if applicable):
3 h
Number of replicates:
2

Results and discussion

In vitro

Resultsopen allclose all
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 h exposure
Value:
73.8
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
3 min exposure
Value:
67.8
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline:
positive control (glacial acetic acid): 3 min exposure 5.6±0.2, 1 h exposure: 4.3 ±0.1
negative control: 3 min exposure mean OD 2.081, 1 h exposure mean OD 1.445

Any other information on results incl. tables

SUMMARY OF Optical Density (OD) AND VIABILITY (%)

 

1 Hour Exposure                                                                                  

Treatment

 

OD

Viability (%)

Classification

Negative Control

(Sterile distilled water)

Mean

1.423

100.0

NC

±SD

0.007

0.7

n

2

2

Positive Control

(Glacial acetic acid)

Mean

0.070

4.9

C (Category 1A)

±SD

0.005

0.5

n

2

2

Test Item

Mean

1.049

73.8

NC

±SD

0.029

2.9

n

2

2

                                           

 

3 Minutes Exposure

Treatment

 

OD

Viability (%)

Classification

Negative Control

(Sterile distilled water)

Mean

1.454

100.00

NC

±SD

0.029

2.8

n

2

2

Positive Control

(Glacial acetic acid)

Mean

0.086

5.8

C (Category 1A)

±SD

0.006

0.6

n

2

2

Test Item

Mean

0.985

67.8

NC

±SD

0.008

0.7

n

2

2

NC = Non Corrosive; C = Corrosive; n = No. of tissues; SD = Standard Deviation.

Applicant's summary and conclusion

Interpretation of results:
other: non-corrosive
Conclusions:
Based on the results obtained under the laboratory testing conditions, the test item is categorized as non-corrosive to Reconstructed Human Epidermis (RhE) in accordance with UN GHS No category, as the mean percentage tissue viability was greater than 50% after 3 minutes exposure and greater than 15% after 1 hour exposure of the negative control.
Executive summary:

The objective of study was to evaluate the in vitro skin corrosion potential of Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized by measurement of tissue viability on the Epidermal Model - Epiderm™ (EPI-200-SCT) as per the OECD Guideline 431, adopted on 29th July 2016.

The test item did not develop any colour when dissolved in distilled water/isopropanol and 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 and excess moisture observed all the tissue inserts were used for the study. Tissue inserts were transferred to upper row of 6 well plates prefilled with 0.9 mL of assay medium and incubated in CO2 incubator for 60 minutes.

Test items were exposed for 1 hour and 3 minutes separately. All the treatments were maintained in duplicates. For 3 minutes treatment, quantity of 50 µL of sterile distilled water (NC) was dispensed into the first insert atop the tissue. After 60 seconds the procedure was repeated with second tissue and continued for other tissues. Similar procedure was followed in the same manner until all the tissues were treated. Tissues were treated with 25 mg test item + 25 µL distilled waterand 50 µL of positive control (glacial acetic acid).

For 1 hour treatment, 25 mg test item + 25µL distilled water, 50 µL of negative control and 50 µL of positive control were dispensed directly atop Epiderm™ tissues at 1 minute intervals to facilitate rinsing after exposure. The tissues were incubated at standard culture conditions for 1 hour.

For the 3 minutes exposure, percentage viability of negative control, positive control and test item was 100±2.8, 5.9±0.6 and 67.8±0.7, respectively. As the percentage viability of test item was greater than 50% of negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 50% of negative control clearly represents the irritation potential of positive control.

For the 1 hour exposure, percentage viability of negative control, positive control and test item was 100±0.7, 4.9±0.5 and 73.8±2.9, respectively. As the percentage viability of test item was greater than 15% of negative control, the test item is considered as non-corrosive, whereas the percentage viability of positive control (PC) is less than 50% of negative control clearly represents the irritation potential of positive control.

 

Conclusion

Based on the results obtained under the laboratory testing conditions, Octadecanoic acid, reaction products with diethylenetriamine, di-Me sulfate-quaternized is categorized as non-corrosive as the mean percentage tissue viability was greater than 50% after 3 minutes exposure and greater than 15% after 1 hour exposure of the negative control.