Fatty acids, C18 unsatd., mono- and diesters with triethanolamine, di-Me sulfate quaternized

Endpoint summary

Description of key information

Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternizedwas proven to be not irritating to skin in two in-vitro studies according to OECD guideline 431 and 439.

The substance has to be classified for eye irritation based on two in-vitro studies according to OECD guideline 437 and 492.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-09-25 to 2017-11-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Version / remarks:

adopted July 29, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended by the OECD testing guideline 431

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ tissues
- Tissue batch number(s): Lot: 25853
- QA test date of tissue: 2017-11-01

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
3 min exposure at room temperature; 60 min exposure at 37 ± 1.5 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 20 times with exess of DPBS
- Observable damage in the tissue due to washing: not reported


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL (1 mg/mL)
- Incubation time: 3 hours
- Spectrophotometer: Versamax®, Molecular Devices, SoftMax Pro Enterprise (version 4.7.1)) at 570 nm (OD570)
- Wavelength: at 570 nm (OD570)
- Filter: no
- Linear OD range of spectrophotometer: no information

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: valid
- Barrier function: valid
- Morphology: valid
- Contamination: valid
- Reproducibility: valid

NUMBER OF REPLICATE TISSUES: duplicate

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Optical evaluation of the MTT-reducing capacity of the test item after 1 hour incubation with MTT-reagent did not show blue/purple colour.

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. 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 [complete, e.g. 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 ± 2mg /tissue

VEHICLE: No vihicle

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50µL Potassium Hydroxide
- Concentration (if solution): 8.0 N

Duration of treatment / exposure:

Test Item: 3 ± 0.5 minutes, 60 ± 5 minutes
Negative Control: 3 ± 0.5 minutes, 60 ± 5 minutes
Positive Control: 3 ± 0.5 minutes, 60 ± 5 minutes

Duration of post-treatment incubation (if applicable):

no

Number of replicates:

Duplicate EpiDermTM tissues

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes exposure

Value:

96.3

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non corrosive to skin

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour exposure

Value:

86

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non corrosive to skin

The test item is considered to be non-corrosive to skin:

·        since the viability after 3 minutes exposure is greater than 50% and

·        the viability after 1 hour exposure is greater than 15%.

Interpretation of results:

GHS criteria not met

Remarks:

non-corrosive to skin

Conclusions:

Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized is non corrosive to skin according to EU CLP.

Executive summary:

In an in-vitro skin irritation study performed in accordance with OECD Guideline 431 (In Vitro Skin Corrosion, RHE) (adopted July 29, 2016), Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized (100 % a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 3 minutes and 1 hour in duplicate.

Approximately 25 mg of the test item were applied to the surface of tissues, wetted with 25 µL of deionised water prior to application in order to improve contact between the solid and the tissue.

Each 50 µL of negative and positive control were applied to sets of duplicate tissues, respectively.

After exposure period of 3 minutes (room temperature) and 1 hour (37 °C) the tissues were rinsed off, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. After rinsing, the formazan salt was extracted for about 20 hours at room temperature.

The test item did not reduce MTT, therefore additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were not necessary.

After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability < 15% of the negative control. The Coefficient of Variation (CV) in the range 20 – 100% viability between the tissue replicates is ≤ 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed.

After exposure of the tissues to the test item the mean relative absorbance value decreased to 96.3% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 86.0%. Both values did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-03-05 to 2018-03-23

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)

Version / remarks:

adopted July 28, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

Recommended by the OECD testing guideline 439

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™
- Tissue batch number(s): Lot: 25888
- Shipping date: 2018-03-20
- Experimental compleation date: 2018-03-23

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
total 60 min (35 min at 37 °C and rest of time at room temperature)
- Temperature of post-treatment incubation (if applicable): 37 ± 1.5 °C (41.5 hours)

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: at least 15 times in order to remove any residual test material.
- Observable damage in the tissue due to washing: no information


MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 µL/ tissue (1mg/mL MTT)
- Incubation time: 3 hours
- Spectrophotometer: Versamax® Molecular Devices, Softmax Pro Enterprise, version 4.7.1
- Wavelength: 570 nm


FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: valid
- Barrier function: valid
- Morphology: valid
- Contamination: valid
- Reproducibility: valid

NUMBER OF REPLICATE TISSUES: triplicates

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
The test item was not considered to reduce MTT and an additional test with freeze-killed tissues did not have to be performed.


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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
Cut-off point(s) as recommended in TG 439

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

Test item: approx. 25 mg/ tissue (wetted with 25 µL DPBS) spread to match the surface of the tissue
Negative Control: 30 µL/tissue
Positive Control: 30 µL/tissue

Duration of treatment / exposure:

60 minutes (35 min at 37 °C and rest of time at room temperature)

Duration of post-treatment incubation (if applicable):

41.5 hours (37 ± 1.5 °C, 5 ± 0.5 % CO2)

Number of replicates:

triplicates

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1

Value:

125

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The test item did not reduce MTT (test for direct MTT reduction), and it did not change colour when mixed with deionised water (test for colour interference).

Results after treatment with test item:

Treatment Group	Tissue No.	OD 570 nm Well 1	OD 570 nm Well 2	OD 570 nm Well 3	Mean OD of 3 Wells	Mean OD of 3 Wells blank corrected	Mean OD of 3 tissues blank corrected	Rel. Viability [%] Tissue 1, 2 + 3*	Standard Deviation [%]	Mean Rel. Viability [%]**
Blank		0.039	0.038	0.038	0.038
Negative Control	1	1.947	1.888	1.908	1.914	1.876	1.718	109.2	8.5	100.0
	2	1.668	1.598	1.614	1.627	1.589		92.5
	3	1.750	1.717	1.714	1.727	1.689		98.3
Positive Control	1	0.089	0.088	0.088	0.089	0.050	0.051	2.9	0.1	2.9
	2	0.091	0.088	0.093	0.091	0.053		3.1
	3	0.088	0.086	0.087	0.087	0.049		2.8
Test Item	1	2.363	2.283	2.249	2.298	2.260	2.148	131.6	5.7	125.0
	2	2.206	2.106	2.105	2.139	2.101		122.3
	3	2.156	2.090	2.119	2.122	2.083		121.3

*Relative viability [rounded values]

** Mean relative viability [rounded values]

Historical Data

Positive Control; OD at 570 nm after exposition to 5 % SDS solution in deionised water (MatTek)		Negative Control OD at 570 nm DPBS (MatTek)
MeanViability	4.28%	Mean Absorption	1.66
Standard Deviation	1.00 p.p	Standard Deviation	0.20
Rel. Standard Deviation	23.44%	Rel. Standard Deviation	11.96%
Range of Viabilities	2.24%—6.19%	Range of Absorbance*	1.28—2.00
Mean Absorption	0.07	* should be 0.8—2.8 (OECD 439) or 1.0—2.5 (MatTek)
Standard Deviation	0.02
Rel. Standard Deviation	25.96%
Range of Absorbance	0.03—0.11
Data of 36 sets of controls shared between 147 studies performed from January 2017 until January 2018. (p.p.—percentage points)

 

Interpretation of results:

GHS criteria not met

Conclusions:

Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized is not irritating to skin according to EU CLP criteria.

Executive summary:

In an in-vitro skin irritation study performed in accordance with OECD Guideline 437 (In Vitro Skin Irritation, RHE) (adopted July 29, 2016), Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized (100 % a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 60 minutes in triplicate.

Approximately 25 mg of the test item were applied to the surface of tissues, wetted with 25 µL of DPBS prior to application in order to improve contact between the solid and the tissue.

Each 30 µL of negative and positive control were applied to sets of triplicate tissues, respectively.

After exposure period of 60 minutes (35 min at 37 °C and rest of time at room temperature) the tissues were rinsed off. Following a further incubation for 41.5 hours the tissues were treated with the MTT solution for 3 hours. After rinsing, the formazan salt was extracted for about 2 hours at room temperature while shaking.

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 in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

After treatment with the test item the mean relative viability value increased to 125.0% 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.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-01-12

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)

Version / remarks:

July, 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
Test System:
Freshly isolated bovine cornea (at least 9 month old donor cattle)
Rationale: OECD 437
Source: AB Schlachthof GmbH & Co. KG, 63739 Aschaffenburg, Germany

Freshly isolated bovine eyes of at least 9 month old donor cattle were collected from the abattoir. Excess tissue was removed from the excised eyes. The 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 until transportation on the same morning to the laboratory using a Styrofoam box. The corneae were isolated on the same day after delivery of the eyes.

All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea.

Each isolated cornea was mounted in a specially designed cornea holder according to the description given in OECD guideline 437, which consists of anterior and posterior compartments, which interface with the epithelial and the endothelial sides of the cornea, respectively. The endothelial side of the cornea was positioned against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the O-ring but stretching was avoided. The anterior part of the holder was positioned on top of the cornea and fixed in place with screws. Both compartments of the holder were filled with incubation medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure that no air bubbles were present within the compartments.
For equilibration, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath.

Vehicle:

physiological saline

Controls:

yes, concurrent vehicle

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL
- Concentration: Since the test item is a surfactant substance, it was tested at a final concentration of 10% (a.i) as a solution (w/v) in saline as stated in the OECD guideline 437.
Since the test item was not liquid but had a creamy consistency, prior to the test it was warmed for 15 min at 37 °C to make it more easy to handle.
Then 0.5 mL were sampled using a syringe and were diluted to 4.5 mL saline. However, the test item did not completely dissolve.
Prior to sampling of the application volume of 0.75 mL, the suspension was vortexed in order to treat the three corneae with equivalent amounts of test item in saline.

Duration of treatment / exposure:

The corneae were incubated in a horizontal position at 32 ± 1 °C in the water-bath. The incubation time lasted ten minutes.

Duration of post- treatment incubation (in vitro):

After exposure the test item or control items, respectively, were rinsed off from the application side with saline, fresh cMEM was added into the anterior compartment.
Then the corneae were incubated at 32 ± 1 °C for further two hours in a vertical position.

Number of animals or in vitro replicates:

Sets of three corneae were used for treatment with the test item and for the negative and positive controls.

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): rinsed off from the application side with saline, fresh cMEM
- Time after start of exposure: 10 min.

SCORING SYSTEM:
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)

The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – corrected opacity value mean negative control) + (15 x corrected OD490 value)

TOOL USED TO ASSESS SCORE:
Opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France)
Permeability was assessed using fluorescein
The optical density was measured with a microplate reader (Versamax® Molecular Devices) at 490 nm (OD490).

Irritation parameter:

in vitro irritation score

Run / experiment:

Experiment 1

Value:

9.18

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: No prediction can be made

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.19).

- Acceptance criteria met for positive control:
The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 89.70) corresponding to a classification as serious eye damaging (Cat 1 according to CLP).

Results after 10 Minutes Treatment Time

Test Group	Opacity value = Difference (t130-t0) of Opacity		Permeability at 490 nm (OD490)		IVIS	Mean IVIS	Proposed in vitro Irritancy Score
		Mean		Mean
Negative Control	0	-0.67	0.048	0.057	0.72	0.19	No Category
	-1		0.071		0.06
	-1		0.052		-0.22
Positive Control	75.67*		1.009*		90.80	89.70	Category 1
	77.67*		0.879*		90.85
	76.67*		0.718*		87.44

Test item	10.67*		0.095*		12.09	9.18	No prediction can be made
	6.67*		0.064*		7.63
	6.67*		0.076*		7.81

*corrected values

The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t₁₃₀– t₀).

The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.

Interpretation of results:

GHS criteria not met

Remarks:

Classification Cat 1 according CLP “H318 cause serious eye damaging” is not required.

Conclusions:

The calculated mean in vitro irritation score for Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized was 9.18.
Since the mean in vitro irritancy score of the test substance was <55, the substance is considered to not be severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted July, 2013. Since the test item is a surfactant substance, it was tested as 10% solution (w/v) in saline as recommended by OECD guideline 437.

After a first opacity measurement of the fresh bovine corneae (t0), the 10% suspension of the test item, the positive, and the negative controls were applied to the different corneae and incubated for 10 minutes at 32 ± 1 °C. After the incubation phase, the test item as well as 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 incubation medium, and opacity was measured for a second time (t130).

The test was performed in triplicates. Opacity and permeability were determined. The in vitro irritancy score (IVIS) were calculated as mean opacity value + (15 x mean OD490 value). Substance that induces an IVIS > 55 Cat. 1 for serious eye damage has to be assigned. For substances that induce an IVIS < 3, no classification is required.

 

The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 89.70) corresponding to a classification as serious eye damaging (Cat 1 according CLP/EPA/GHS). With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.19).

Relative to the negative control, the test item Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized caused an increase of the corneal opacity and permeability. The calculated mean IVIS was 9.18. Since the mean in vitro irritancy score of the test substance was <55, the substance is considered to be not severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018-03-05 to 2018-03-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)

Version / remarks:

July, 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability
The EpiOcular ™ Eye Irritation Test (EIT) was validated by the European Union Reference laboratory for Alternatives to Animal Testing (EURL ECVAM) and cosmetics Europe between 2008 and 2013.

- Description of the cell system used, incl. certificate of authenticity and the mycoplasma status of the cell live
EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts.
Certificate of analysis from MatTek: valid

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg of test item
Each 50 µL of the negative control and of the positive control

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

18 hours

Number of animals or in vitro replicates:

duplicate

Details on study design:

Cell Culture
EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia). The EpiOcular™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a stratified squamous epithelium similar to that found in the human cornea. It consists of highly organized basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal in vivo corneal epithelium. The EpiOcular™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts.
Batch: 27027

Assessment of Direct MTT Reduction by the Test Item
Test items may have the ability to directly reduce MTT and to form a blue/purple reaction product which could have an impact on the quantitative MTT measurement. Therefore, it was necessary to assess this ability for the test item prior to conducting any assays with viable tissues. For this purpose approximately 50 mg of the test item were added to a 1 mL of a 1.0 mg/mL MTT solution (in DMEM) in a glass tube and the mixture was incubated in the dark at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 in air for three hours. A control (50 µL of deionised water in 1 mL of 1.0 mg/mL MTT solution) was run concurrently. If the MTT solution colour turned blue/purple, the test item was presumed to have reduced the MTT.
Since the MTT solution colour did not turn blue/purple, the test item was not presumed to be a MTT reducer, and an additional test with freeze-killed tissues was not necessary.

Irritation parameter:

other: mean relative absorption value %

Run / experiment:

Experiment 1

Value:

57.8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

Additional tests with viable or freeze-killed tissues were not performed, since the test item was not coloured intensively, did not dye water or isopropanol, and did not prove to be a MTT reducer.

Results after treatment for 6 hours

Treatment Group	Tissue No.	OD 570 nm Well 1	OD 570 nm Well 2	Mean OD of 2 Wells	Mean OD of 2 Wells blank corrected	Mean OD of Treatment Group blank corrected	Rel. Viability [%] Tissue 1, 2 *	Absolute Value of the Difference of Rel. Viability  Tissue 1,2[%]	Mean Rel. Viability [%]**
Blank		0.038	0.036	0.037
Negative Control	1	1.760	1.714	1.737	1.700	1.711	99.4	1.3	100.0
	2	1.781	1.736	1.759	1.722		100.6
Positive Control	1	0.632	0.609	0.621	0.584	0.530	34.1	6.4	30.9
	2	0.525	0.499	0.512	0.475		27.8
Test Item	1	1.081	1.059	1.070	1.033	0.989	60.4	5.2	57.8
	2	0.995	0.969	0.982	0.945		55.2

*  Relative viability [rounded values]

** Mean relative viability [rounded values]

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized has to be considered as an eye irritant Cat. 2 according to the criteria of CLP.

Executive summary:

This in vitro study was performed to assess the eye irritation potential of Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized in the Human Cornea Model Test. according to OECD guideline 492, adopted July, 2015. The test item did not prove to be an MTT reducer in the MTT pre-test. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) did not have to be performed. About 50 mg of the test item and each 50 µL of the controls, respectively, were applied to either one of duplicate EpiOcular issue for 6 hours.

 

After treatment with the negative control the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system.The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

 

Irritating effects were observed following incubation with the test item. Compared with the value of the negative control, the relative mean absorption value corresponding to the viability of the tissues decreased to 57.8 %. This is below the threshold of 60%, but was considered borderline (60±5%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item possesses an eye irritating potential.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

In an in-vitro skin irritation study performed in accordance with OECD Guideline 431 (In Vitro Skin Corrosion, RHE) (adopted July 29, 2016), Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized (100 % a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 3 minutes and 1 hour in duplicate.

Approximately 25 mg of the test item were applied to the surface of tissues, wetted with 25 µL of deionised water prior to application in order to improve contact between the solid and the tissue.

Each 50 µL of negative and positive control were applied to sets of duplicate tissues, respectively.

After exposure period of 3 minutes (room temperature) and 1 hour (37 °C) the tissues were rinsed off, and a 3 hour incubation period (37 ± 1 °C, 5 ± 0.5 % CO2) with MTT solution followed. After rinsing, the formazan salt was extracted for about 20 hours at room temperature.

The test item did not reduce MTT, therefore additional tests with freeze-killed or viable tissues to determine correction factors for calculating the true viability in the main experiment were not necessary.

After exposure to the negative control the absorbance values met the required acceptability criterion of a mean OD570 ≥ 0.8 and ≤ 2.8 for both treatment intervals thereby confirming the acceptable quality of the tissues. Exposure to the positive control induced a decrease in the relative absorbance as compared to the negative control, both for the 3 minutes exposure period and for the 1 hour exposure period. The 1 hour exposure caused a decrease of the cell viability < 15% of the negative control. The Coefficient of Variation (CV) in the range 20 – 100% viability between the tissue replicates is ≤ 30%, thus the validity of the test system and the specific batch of the tissue models is confirmed.

After exposure of the tissues to the test item the mean relative absorbance value decreased to 96.3% after 3 minutes exposure. After 1 hour exposure the relative absorbance value was reduced to 86.0%. Both values did not exceed the threshold for corrosivity which is defined to be 50% after the 3 minutes exposure and 15% after the 1 hour exposure. Therefore, the test item is not considered to be corrosive.

 

In an in-vitro skin irritation study performed in accordance with OECD Guideline 437 (In Vitro Skin Irritation, RHE) (adopted July 29, 2016), Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized (100 % a.i.) was applied to the three-dimensional human epidermis model tissue for an exposure period of 60 minutes in triplicate.

Approximately 25 mg of the test item were applied to the surface of tissues, wetted with 25 µL of DPBS prior to application in order to improve contact between the solid and the tissue.

Each 30 µL of negative and positive control were applied to sets of triplicate tissues, respectively.

After exposure period of 60 minutes (35 min at 37 °C and rest of time at room temperature) the tissues were rinsed off. Following a further incubation for 41.5 hours the tissues were treated with the MTT solution for 3 hours. After rinsing, the formazan salt was extracted for about 2 hours at room temperature while shaking.

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 in the relative absorbance as compared to the negative control for the 60 minutes treatment interval, and thus assuring the validity of the test system.

After treatment with the test item the mean relative viability value increased to 125.0% 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, of Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized does not need to be classified for skin irritation according to CLP, EU GHS (Regulation (EC) No 1272/2008).

 

 

Eye irritation

This in vitro study was performed to assess the corneal irritation and damage potential of Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized by means of the BCOP assay using fresh bovine corneae according to OECD guideline 437, adopted July, 2013. Since the test item is a surfactant substance, it was tested as 10% solution (w/v) in saline as recommended by OECD guideline 437.

After a first opacity measurement of the fresh bovine corneae (t0), the 10% suspension of the test item, the positive, and the negative controls were applied to the different corneae and incubated for 10 minutes at 32 ± 1 °C. After the incubation phase, the test item as well as 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 incubation medium, and opacity was measured for a second time (t130).

The test was performed in triplicates. Opacity and permeability were determined. The in vitro irritancy score (IVIS) were calculated as mean opacity value + (15 x mean OD490 value). Substance that induces an IVIS > 55 Cat. 1 for serious eye damage has to be assigned. For substances that induce an IVIS < 3, no classification is required.

The positive control (2-Ethoxyethanol) was tested undiluted and showed clear opacity and distinctive permeability of the corneae (mean IVIS = 89.70) corresponding to a classification as serious eye damaging (Cat 1 according CLP/EPA/GHS). With the negative control (saline) neither an increase of opacity nor permeability of the corneae could be observed (mean IVIS = 0.19).

Relative to the negative control, the test item Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized caused an increase of the corneal opacity and permeability. The calculated mean IVIS was 9.18. Since the mean in vitro irritancy score of the test substance was <55, the substance is considered to be not severely irritating/ corrosive in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

 

This in vitro study was performed to assess the eye irritation potential of Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized in the Human Cornea Model Test. according to OECD guideline 492, adopted July, 2015. The test item did not prove to be an MTT reducer in the MTT pre-test. Therefore, additional tests with freeze-killed tissues or viable tissues (without MTT addition) did not have to be performed. About 50 mg of the test item and each 50 µL of the controls, respectively, were applied to either one of duplicate EpiOcular issue for 6 hours.

After treatment with the negative control the absorbance values were well within the required acceptability criterion of OD > 0.8 and < 2.5, thus showing the quality of the tissues. Treatment with the positive control induced a decrease below 50% viability compared with the negative control value in the relative absorbance, thus ensuring the validity of the test system.The difference of viability between the two relating tissues was < 20% in the same run (for test item tissues, positive and negative control tissues).

Irritating effects were observed following incubation with the test item. Compared with the value of the negative control, the relative mean absorption value corresponding to the viability of the tissues decreased to 57.8 %. This is below the threshold of 60%, but was considered borderline (60±5%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item possesses an eye irritating potential.

 

The studies reflect that the substance is not corrosive, but clearly indicate an eye irritating potential of Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized. However, according to the guidelines, data from the in vitro studies are not sufficient for classification purpose as standalone information.

Within a weight of evidence approach it can be shown, that oleic acid based TEA-Esterquat has a potential for eye irritation. As the used source of fatty acid is identical and this is known to be the trigger for irritating effects a conclusive pattern for eye irritating potency can be drawn from the available data.

 

In vivo studies in detail

In a primary eye irritation study according to OECD Guideline 405, 2002 0.1 mL of the oleic acid-based TEA-Esterquat was instilled into the conjunctival sac of three New Zealand White rabbits. Animals then were observed for 28 days. Irritation was scored by the method stipulated by the OECD Guideline 405.

All 3 animals showed moderate to strong ocular reaction 1 h after application of the test substance. 24 h after application slight to moderate ocular reactions were still observed in all three animals. Slight cornea opacity was observed in all animals 24 h after application. The cornea damage was not further observed 48 h after treatment. Slight irritation of the conjunctivae and the iris persisted until day 14 in two animals and chemosis only until day 21 in one animal.. At day 21 effects were fully reversible with exception of a chemosis score 1 in one animal, which was fully reversible within 28 days.

Classification Cat.2 for eye irritation is justified according to CLP, EU GHS (Regulation (EC) No 1272/2008).

 

In conclusion, based on a weight of evidence approach including information from in vitro studies with the target substance and taking into account data from in vivo studies with the structurally closely related source substance oleic acid based TEA-Esterquat a robust conclusion including classification for the eye irritating properties can be drawn from available data.

Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized has to be classified “Cat.2 for eye irritation” according to CLP, EU GHS (Regulation (EC) No 1272/2008).

Accordingly, no further in vivo testingfor eye irritation on vertebrate animalswith Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternized is considered appropriate.

Justification for classification or non-classification

Based on data from in-vitro studies according to OECD Guideline 431 and 439, Fatty acids, C18 unsatd., mono and diester with triethanolamine, di-Me sulfate-quaternized does not need to be classified as irritating to skin according the criteria of CLP, EU GHS (Regulation (EC) No 1272/2008) and labelling is not required, accordingly.

Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternizedhas to be regarded as eye irritating based on a weight of evidence approach including data from an in-vitro study on the substance and in vivo studies with a structural similar substance.

Taken together,Fatty acids, C18 unsatd., mono and diester with triethanolamine , di-Me sulfate-quaternizedhas to be classified for eye irritation Cat. 2, based on CLP criteria, EU GHS (Regulation (EC) No 1272/2008) and labelled with H319 “Causes serious eye irritation” respectively.