TEA-Esterquat

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 November 2020 - 11 December 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Test material form:

liquid: viscous

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm, lot 34108

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL 1 mg/mL
- Incubation time: 3 hour ± 5 minutes
- Spectrophotometer: Versamax® Molecular Devices
- Wavelength: 570 nm


FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
positive control: Mean OD 0.07, range: 0.03 – 0.11
Mean Tissue Viability [%] 3.93, range: 2.24% - 6.19 %
negative control: Mean OD 1.71, range: 1.28 - 2
(Data of 60 sets of controls performed from August 2015 until May 2020 and shared between 226 studies.)

NUMBER OF REPLICATE TISSUES:

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
1) The Color Interference – Test
Colored test items or test items which become colored after application to the tissues may interfere with the photometric MTT measurement if the colorant binds to the tissue and is extracted together with MTT. Therefore, each test item had to be checked for its colorant properties.
Therefore, 30 μL of the test item was added to 300 μl of deionised water and mixed. 330 μl of deionised water was used as control (blank). Both were incubated for 60 min under standard conditions.
In parallel, 30 μL of the test item was added to 2 mL of isopropanol and mixed. A control (2 mL of isopropanol, blank) was run concurrently. Both were incubated for 1 hour at room temperature.
Since the test item was not soluble in water the suspension was centrifuged.
After incubation the change of color was determined by the unaided eye.

2) The MTT Interference – Test
To test if a test item directly reduces MTT, 1 ml of a MTT solution (1 mg/mL) including 30 μL of the test item was incubated for 1 hour at standard conditions. Untreated MTT/DMEM solution (1 mg/mL) was used as negative control.
After incubation the change of color was determined by the unaided eye.

Since the test item/ water or test item/ isopropanol solutions changed color significantly in the first pre-experiment it was presumed to have the potential to stain the tissues. Two additional controls in duplicates run with the main experiment – additional viable tissues (colored controls = CC)
• Deionised water treated tissues (NC_CC)
• Test item treated tissues (TI_CC)
These four tissues were incubated in medium without MTT solution in the MTT assay. At the end, Data Correction Procedure I was performed.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

PREDICTION MODEL / DECISION CRITERIA
The test item is identified as requiring classification and labelling according to UN GHS/ EU CLP “Category 2” or “Category 1”, if the tissue viability after exposure and post-incubation is ≤ 50%. Since this assay covered by this OECD 439 cannot resolve between UN GHS Categories 1 and 2 further information on skin corrosion will be required to decide on its final classification. In case the test item is found to be non-corrosive the test item is considered to be irritant to skin in accordance with UN GHS Category 2.
The test item is identified as non-irritant to skin in accordance with UN GHS / EU CLP “No Category” if the tissue viability after exposure and post-treatment incubation is more 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): 30 µL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- DPBS Rinse Solution

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 30 µL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

77.3

Vehicle controls validity:

not applicable

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: yes; respective controls were included

DEMONSTRATION OF TECHNICAL PROFICIENCY: the controls gave the appropriate response

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

Any other information on results incl. tables

Treatment Group	Tissue No.	OD 570 nm			Mean OD of 3 Wells	Mean OD of 3 Wells blank corrected	Mean OD of 3 tissues	Rel. Viability [%] Tissue 1, 2 + 3	Standard Deviation	Mean Rel. Viability [%]
		Well 1	Well 2	Well 3
Blank		0.039	0.039	0.039	0.039
Negative Control	1	2.015	1.945	1.958	1.973	1.934	1.876	103.125	0.053	100.0
	2	1.950	1.870	1.877	1.899	1.860		99.188
	3	1.890	1.855	1.868	1.871	1.832		97.687
Positive Control	1	0.160	0.149	0.140	0.150	0.111	0.088	5.909	0.020	4.69
	2	0.121	0.117	0.116	0.118	0.079		4.223
	3	0.114	0.110	0.114	0.113	0.074		3.944
Test Item	1	1.707	1.678	1.681	1.689	1.650	1.457	87.983	0.183	77.30*
	2	1.480	1.465	1.476	1.473	1.435		76.490
	3	1.314	1.319	1.340	1.324	1.286		68.551
NC_CC	1	0.049	0.050	0.049	0.049	0.011	0.008	0.569	0.003	0.45
	2	0.045	0.045	0.044	0.045	0.006		0.322
TI_CC	1	0.044	0.045	0.044	0.044	0.005	0.007	0.291	0.002	0.38
	2	0.047	0.048	0.048	0.047	0.009		0.466

NC_CC     Negative Control Colored Control
TI_CC      Test Item treated Colored Control

* Corrected viability (%) = TI viability – TI_CC viability

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported, Esterification products of triglycerides, C18 (unsaturated) with triethanolamine, dimethyl sulphate-quaternized is non-irritant to skin according to UN GHS and EU CLP regulation.

Executive summary:

Thisin vitrostudy was performed to assess the irritation potential of Esterification products of triglycerides, C18 (unsaturated) with triethanolamine, dimethyl sulphate-quaternized by means of the Human Skin Model Test in accordance with OECD TG 439.

The test item did not prove to be a MTT reducer in the MTT interference pre-experiment. It changed color when mixed with deionised water. Therefore, additional tests with viable tissues (without MTT addition) had to be performed.

Three tissues each of the human skin model EpiDerm™ were treated with the test item, the negative control (DPBS) or the positive control (5% SDS) for 60 minutes.

After treatment with the negative control the absorbance values were well within the required range of the acceptability criterion of mean OD ≥ 0.8 and ≤ 2.8 for the 60 minutes treatment interval, thus assuring the quality of the tissues.

Treatment with the positive control induced a sufficient decrease well below 20% in the viability as compared to the negative control for the 60 minutes treatment interval, thus assuring the validity of the test system.

After treatment with the test item Esterification products of triglycerides, C18 (unsaturated) with triethanolamine, dimethyl sulphate-quaternized the mean relative viability value was 77.30% compared to the relative absorbance value of the negative control. This value is above the threshold for irritancy of ≤ 50%. Therefore, the test item is not considered to possess an irritant potential.

In conclusion, it can be stated that in this study and under the experimental conditions reported, Esterification products of triglycerides, C18 (unsaturated) with triethanolamine, dimethyl sulphate-quaternized is non-irritant to skin.