TEA-Esterquat

Endpoint summary

Description of key information

skin irritation: not irritating (OECD TG 439, RL1, GLP): mean tissue viability = 77.3%

eye irritation: not irritating (OECD TG 437, RL1, GLP): mean in vitro irritancy score = 1.52

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

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

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 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

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

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany
- Characteristics of donor animals (e.g. age, sex, weight): 14 month old donor cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): isolated eyes were stored in HBSS containing 1% (v/v) penicillin/streptomycin (100 units/mL penicillin and 100 μg/mL streptomycin) in the cooled slaughter-house and during transportation on the same morning to the laboratory.
- Time interval prior to initiating testing: The corneae were isolated on the same day after delivery of the eyes and used in the BCOP test on the same day.
- Indication of any existing defects or lesions in ocular tissue samples: corneas presenting defects such as vascularization, pigmentation, opacity and scratches were discarded
- Selection and preparation of corneas: Only corneae with a value of the basal opacity < 7 were used

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

0.75 mL

Duration of treatment / exposure:

ten minutes (± 30 seconds)

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

3

Details on study design:

NEGATIVE CONTROL USED: Saline (0.9% NaCl in deionised water)

POSITIVE CONTROL USED: 2-Ethoxyethanol

TREATMENT METHOD: open chamber

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: as indicated in the TG

The test is acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control.

Irritation parameter:

in vitro irritation score

Run / experiment:

mean of 3 corneas

Value:

1.52

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: no

DEMONSTRATION OF TECHNICAL PROFICIENCY: the controls gave the appropriate responses

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Results after 10 Minutes Treatment Time Test Group	Opacity value = Difference (t130-t0) of Opacity	Permeability at 490 nm (OD490)	IVIS	Mean IVIS	Standard Deviation IVIS	Proposed Category
Negative Control	0	0.066	0.99	0.95	0.07	No Category
	0	0.058	0.87
	0	0.065	0.98
Positive Control	91.00*	1.319*	110.79	114.08	5.67	Category 1
	86.00*	1.656*	110.84
	94.00*	1.775*	120.63
Test item	2.00*	0.003*	2.05*	1.52	0.45	No Category
	1.00*	0.018*	1.27*
	1.00*	0.017*	1.26*

*corrected values

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, according to the current study and under the experimental conditions reported, Esterification products of triglycerides, C18 (unsaturated) with triethanolamine, dimethyl sulphate-quaternized is not categorized (GHS).

Executive summary:

This in vitro study was performed to assess the corneal damage potential of Esterification products of triglycerides, C18 (unsaturated) with triethanolamine, dimethyl sulphate-quaternized by means of the BCOP assay using fresh bovine corneae.
After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contained incubation medium as well. Afterwards, opacity was measured a second time (t130).
After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.
With the negative control (0.9% (w/v) NaCl solution in deionised water), neither an increase of opacity nor permeability of the corneae was observed.
The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (EU CLP/EPA/GHS (Cat 1)).
Relative to the negative control, the test item Esterification products of triglycerides, C18 (unsaturated) with triethanolamine, dimethyl sulphate-quaternized did not cause a relevant increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 1.52. According to OECD TG 437 the test item is not categorized (GHS).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

This in vitro study 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.

 

Eye irritation

This in vitro study was performed to assess the corneal damage potential of Esterification products of triglycerides, C18 (unsaturated) with triethanolamine, dimethyl sulphate-quaternized by means of the BCOP assay using fresh bovine corneae in accordance with OECD TG 437. After a first opacity measurement of the fresh bovine corneae (t0), the neat test item, the positive, and the negative controls were applied to corneae fixed in an incubation chamber in horizontal position for 10 minutes at 32 ± 1 °C. The posterior chamber contained incubation medium. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae. Further, the corneae were incubated for another 120 minutes at 32 ± 1 °C in a vertical position, while the anterior chamber contained incubation medium as well. Afterwards, opacity was measured a second time (t130). After the opacity measurements, permeability of the corneae was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C. With the negative control (0.9% (w/v) NaCl solution in deionised water), neither an increase of opacity nor permeability of the corneae was observed. The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae corresponding to a classification as serious eye damaging (EU CLP/EPA/GHS (Cat 1)). Relative to the negative control, the test item Esterification products of triglycerides, C18 (unsaturated) with triethanolamine, dimethyl sulphate-quaternized did not cause a relevant increase of the corneal opacity or permeability. The calculated mean in vitro irritancy score was 1.52. According to OECD TG 437 the test item is not categorized (GHS).

 

Respiratory irritation

No data on the respiratory irritation of Esterification products of triglycerides C18 unsaturated with triethanolamine, dimethyl sulfate quaternized are available.

 

There are no data gaps for the endpoint irritation/corrosion. No human information is available for this endpoint. However, there is no reason to believe that these results would not be applicable to humans.

 

Justification for classification or non-classification

Based on the available data, Esterification products of triglycerides C18 unsaturated with triethanolamine, dimethyl sulfate quaternized does not need to be classified for skin and eye irritation according to regulation (EC) 1272/2008. Thus, no labelling is required.