Fatty acids, C18-36, esters with ethylene glycol

Endpoint summary

Description of key information

The substance was tested in the EpiDerm™ Human Skin Model using duplicate tissues during 3 and 60 minutes. The relative mean viability of the test substance treated tissues was 97.1 % for the 3 minutes exposure and 102.4% for the 60 minutes exposure. Therefore it can be concluded that the substance is not corrosive to the skin.

The substance was tested in the EPISKIN™ Reconstructed Human Epidermis Model using triplicate tissues during 15 minutes. The relative mean viability of the test substance treated tissues was 113.3 %. Therefore it can be concluded that the substance is not irritant to the skin.

In a BCOP assay bovine corneas were exposed to the substance and assessed for opacity and permeability. No effects of the substance on opacity readings and permeability was observed. The calculation of the In Vitro Irritancy Score (1.8) showed that the substance is not severely damaging to the eyes.

The eye irritation potential of the substance was assessed by means of the Human Cornea Model Test. The substance interfered with MTT reduction, but not with color.

Each 50 µL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissues for 6 hours. Irritating effects were not observed following incubation with the substance. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues did not decrease below 60% (92.2%). In conclusion, it can be stated that in this study and under the experimental conditions reported, the substance does not possess any eye irritating potential.

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:

11 October 2017 to 13 October 2017

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:

2016

Qualifier:

according to guideline

Guideline:

other: Method B.40bis of Commission Regulation (EC) No 440/2008, of 30 May 2008,

Version / remarks:

laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

the test substance was ground into a powder before application

Test system:

human skin model

Source species:

human

Cell type:

other: EpiDerm™ Human Skin Mode

Cell source:

other: EpiDerm™ Human Skin Mode

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm™ Human Skin Mode (MatTek)
- Tissue batch number(s): 25848
- Delivery date: 10 Octobert 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ˚C, 5% CO2
- Temperature of post-treatment incubation (if applicable): 37 ˚C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS: rinsed under a constant soft stream of DPBS and blotted dry with a tissue

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL MTT
- Incubation time: 3 h
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nM

NUMBER OF REPLICATE TISSUES: 2/treatment

TISSUES:
- Fresh tissues: for treatment, negative control and positive control

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

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% (H314 1A), 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% (H314 1B or 1C)
- 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% (not classified as corrosive)
:

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 as such (tissues wetted with 25 µL sterile water)

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

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

Duration of treatment / exposure:

3 min and 60 min

Duration of post-treatment incubation (if applicable):

The plates was incubated (37 ˚C, 5% CO2) for 3 hours in presence of MTT. Thereafter tissues were placed in isopropanol for MTT extraction, which was measured in triplicate samples as optical density at 570 nm.

Number of replicates:

2/treatment

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

A 3 minutes exposure

Value:

97.1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

B 60 minutes exposure

Value:

102.4

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: non-corrosive

Other effects / acceptance of results:

The results were in agreement with the quality criteria:

Negative Control
The absolute OD570 of the negative control treated tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. The mean OD 570 of the two negative control tissues should be ≥ 0.8 and ≤ 2.8 for each exposure time, which ensures that the tissue viability meets the acceptance criteria.
Positive Control
Potassium Hydroxide 8.0N solution is used as a positive control. An assay meets the acceptance criterion if mean relative tissue viability of the 60 minute positive control is < 15%.
Coefficient of Variation
In the range 20 and 100% viability, the Coefficient of Variation between tissue replicates should be ≤ 30%.

 

	Exposure time	Mean OD570	Relative mean viability
Negative control	3 min	1.408	100%
	60 min	1.382	100%
Test substance	3 min	1.367	97.1%
	60 min	1.415	102.4%
Positive control	3 min	0.044	3.1%
	60 min	0.045	3.3%

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is considered non-corrosive

Executive summary:

The substance was tested in the EpiDerm™ Human Skin Model using duplicate tissues during 3 and 60 minutes. The relative mean viability of the test substance treated tissues was 97.1 % for the 3 minutes exposure and 102.4% for the 60 minutes exposure. Therefore it can be concluded that the substance is not corrosive to the skin.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 October 2017 to 30 October 2017

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)

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

amending, for the purpose of its adaption to technical progress, Regulation (EC) No 440/2008 as described in Commission Regulation (EC) No. 761/2009, of 23 July 2009, laying down test methods pursuant to Regulation (EC) No. 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

other: EPISKIN™ Reconstructed Human Epidermis Model Kit

Cell source:

other: EPISKIN™ Reconstructed Human Epidermis Model Kit

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN™ Reconstructed Human Epidermis Model Kit
- Tissue batch number(s):17-EKIN-043 (0.38cm2)
- Delivery date: 24 October 2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 ˚C, 5% CO2
- Temperature of post-treatment incubation (if applicable): 37 ˚C, 5% CO2

REMOVAL OF TEST MATERIAL AND CONTROLS:rinsed using a wash bottle containing DPBS

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL MTT
- Incubation time: 3 h
- Spectrophotometer: Anthos 2001 microplate reader
- Wavelength: 562 nM

NUMBER OF REPLICATE TISSUES: 3/treatment

TISSUES:
- Fresh tissues: for treatment, negative control and positive control

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- Relative mean tissue viability is ≤50% --> irritant (H314 or H315)
- Relative mean tissue viability is >50% --> non-irritant (not classified)

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg as such ( (26.3 mg/cm2)

NEGATIVE CONTROL:
- Amount(s) applied (volume or weight with unit): 50 µL DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL SDS (5%)

Duration of treatment / exposure:

15 min at room temperature (thereafter rinsed with DPBS)

Duration of post-treatment incubation (if applicable):

The plates were incubated (37 ˚C, 5% CO2) for 42 hours thereafter shaken to homogenize. Thereafter incubated for 3 hours in presence of MTT. The epidermis and the collagen matrix were separated and immersed in acidified isopropanol. The tissues were stored in a refrigirator for 6 days at 1-10 ˚C for MTT extraction, which was measured in triplicate samples as optical density at 570 nm.

Number of replicates:

3/treatment

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 minutes exposure

Value:

113.3

Negative controls validity:

valid

Remarks:

OD 6.83, SD 2%

Positive controls validity:

valid

Remarks:

viability 17%, SD 17.2%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The results were in agreement with the quality criteria:

Positive Control:
The assay establishes the acceptance criterion for an acceptable test if the relative mean tissue viability for the positive control treated tissues is ≤40% relative to the negative control treated tissues, and the standard deviation (SD) value of the percentage viability is ≤18%.
Negative Control:
The assay establishes the acceptance criterion for an acceptable test if the mean OD562 for the negative control treated tissues is ≥0.6 and ≤1.5, and the SD value of the percentage viability is ≤18%.
Test Item:
The assay establishes the acceptance criterion for an acceptable test if the standard deviation calculated from individual percentage tissue viabilities of the three identically treated tissues is ≤18%.

   

Item	OD570of tissues	Mean OD570of triplicate tissues	±SDof OD570	Relative individual tissue viability (%)	Relative mean viability (%)	± SD of Relative mean viability (%)
Negative Control Item	0.680	0.683	0.014	99.6	100*	2.0
	0.698			102.2
	0.671			98.2
Positive Control Item	0.049	0.116	0.117	7.2	17.0	17.2
	0.252			36.9
	0.048			7.0
Test Item	0.801	0.774	0.027	117.3	113.3	3.9
	0.772			113.0
	0.748			109.5

OD=Optical Density

SD= Standard deviation

*The mean viability of the negative control tissues is set at 100%

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is considered non-irrirant

Executive summary:

The substance was tested in the EPISKIN™ Reconstructed Human Epidermis Model using triplicate tissues during 15 minutes. The relative mean viability of the test substance treated tissues was 113.3 %. Therefore it can be concluded that the substance is not irritant to the skin.

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:

key study

Study period:

27 September 2017 to 09 November 2017

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:

MatTek Corporation Protocol: EpiOcular™ Eye Irritation Test (OCL-200-EIT) for the prediction of acute ocular irritation of chemicals; for use with MatTek Corporation’s Reconstructed Human EpiOcular™ Model; 29 June 2015.

GLP compliance:

yes (incl. QA statement)

Species:

other: human keratinocytes

Details on test animals or tissues and environmental conditions:

EpiOcular™ kits and MTT-100 kits are purchased from MatTek Corporation (82105 Bratislava, Slovakia).

Lot No.: 27012
Sealed 24-well plate Contains 12/24 inserts with EpiOcular™ tissues on agarose
Serum-free test medium DMEM-Medium
Positive control Methyl Acetate (CAS#79-20-9)
12-well plate Holding plate
24-well plates For MTT viability assay
6-well plates For storing inserts, or for topically applying test agents
Ca++Mg++-Free D-PBS Dulbecco´s Phosphate Buffered Saline

MTT-100 Assay Kit Components
1 vial, 2 mL MTT concentrate
1 vial, 8 mL MTT diluent (supplemented DMEM) For diluting MTT concentrate prior to use in the MTT assay
1 bottle, 60 mL Extractant solution (Isopropanol) For extraction of formazan crystals

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL MTT
- Incubation time: 3 h
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1)

NUMBER OF REPLICATE TISSUES: 2/treatment

TISSUES:
- Fresh tissues: for treatment, negative control and positive control

METHOD OF CALCULATION
viability [%] = 100* (ODti or pc/meanODnc)

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

PREDICTION MODEL / DECISION CRITERIA
If the test item-treated tissue viability is > 60% relative to the negative control-treated tissue viability, the test item is labeled non-irritant.
If the test item-treated tissue viability is ≤ 60% relative to negative control-treated tissue viability, the test item is labeled irritant.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 mg test substance, 50 uL controls

Duration of treatment / exposure:

6 hours

Duration of post- treatment incubation (in vitro):

25 minutes in Assay Medium (to remove any test item absorbed into the tissue) and 18 hours in Assay Medium at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).

Number of animals or in vitro replicates:

2 replicates per treatment (1 for blank)

Details on study design:

At the end of the 6 hours treatment time, the test item was removed by extensively rinsing the tissues (3 times) with Ca++Mg++-free DPBS (brought to room temperature).
After rinsing, the tissues were immediately transferred to and immersed in 5 mL of previously warmed Assay Medium (room temperature) in a pre-labelled 12-well plate for 25 minute immersion incubation (post-soak) at room temperature. This incubation in Assay Medium was intended to remove any test item absorbed into the tissue.
At the end of the post-soak immersion, each insert was removed from the Assay Medium, the medium was decanted off the tissue, and the inserts were blotted on absorbent material, and transferred to the appropriate well of the pre-labelled glass tube containing 1 mL of warm Assay Medium. The tissues are incubated for 18 hours at 37 ± 1.5 °C in a humidified atmosphere of 5 ± 0.5% CO2 (post-treatment incubation).
At the end of the post-treatment incubation, each insert was removed and gently blotted on absorbent material. The tissues were placed into the 24-well plate containing 0.3 mL of MTT solution. Once all the tissues are placed into the 24-well plate, the plate was incubated for 180 minutes at standard culture conditions.
Each insert was removed from the 24-well plate after 180 minutes, the bottom of the insert was blotted on absorbent material, and then transferred to a pre-labelled 24-well plate containing 2.0 mL of isopropanol in each designated well so that no isopropanol was flowing into the insert on the tissue surface. The plates were sealed with a standard plate sealer, and were stored overnight at 2-8 °C in the dark and then shaken for 2-3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert was decanted into the well from which it was taken.
The extract solution was mixed and two 200 µL aliquots were transferred to the appropriate wells of a pre-labelled 96-well plate.
The absorbance at 570 nm (OD570) of each well was measured with a plate reader (Versamax® Molecular Devices, 85737 Ismaning, Germany, Software Softmax Pro Enterprise, version 4.7.1). No reference wavelength measurement was used.

Irritation parameter:

other: viability %

Value:

92.2

Negative controls validity:

valid

Remarks:

OD 1.484 and 1.530

Positive controls validity:

valid

Remarks:

mean rel viability 28.6%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

No MTT interefernce was observed, no color interference was found

Dose Group	OD570 Well 1	OD570 Well 2	Mean OD (Tissue 1/2)	Mean OD Tissue 1 and 2 corrected	Mean OD of 2 Tissues Blank corrected	Rel. Viability [%] Tissue 1 and 2*	Absolute Value of the Difference of the Rel. Absorbances [%] Tissue 1 and 2	Mean Rel. Viability [%]**
Blank	0.035	0.034	0.035
Negative Control	1.543	1.494	1.518	1.484	1.507	98.5	3.0	100.0
	1.569	1.560	1.564	1.530		101.5
Positive Control	0.493	0.479	0.486	0.451	0.431	30.0	2.7	28.6
	0.446	0.446	0.446	0.411		27.3
Test Item	1.425	1.429	1.427	1.392	1.507	92.4	0.4	92.2
	1.412	1.429	1.420	1.385		92.0

* Relative viability [rounded values]: 100*(absorbance/absorbance neg contr)

** Mean relative viability [rounded values]: 100 * (mean absorbace)/mean absorbance negative control

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not irritating to the eyes

Executive summary:

The eye irritation potential of the substance was assessed by means of the Human Cornea Model Test. The substance interfered with MTT reduction, but not with color.

Each 50 µL of the test item, the negative control (deionised water) or the positive control (methyl acetate) were applied to each of duplicate tissues for 6 hours.

Irritating effects were not observed following incubation with the substance. Compared with the value of the negative control the relative mean absorption value corresponding to the viability of the tissues did not decrease below 60% (92.2%).

In conclusion, it can be stated that in this study and under the experimental conditions reported, the substance does not possess any eye irritating potential.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 October 2017

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)

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: local abattoir
- Number of animals: not indicated
-- Storage, temperature and transport conditions of ocular tissue: placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL), transported over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
- Time interval prior to initiating testing: not indicated
- indication of any existing defects or lesions in ocular tissue samples: checked and only undamaged tissue used

Vehicle:

unchanged (no vehicle)

Remarks:

applied neat

Controls:

yes

yes, concurrent positive control

Amount / concentration applied:

TEST MATERIAL: 0.6192 g as such (controls 0.75 mL)

Duration of treatment / exposure:

240 minutes at 32 ± 1 ºC

Duration of post- treatment incubation (in vitro):

post treatment opacity measurement immediately
permeality measurement after exposure to fluorescein for 90 min at 32 ± 1 ºC

Details on study design:

QUALITY CHECK OF THE ISOLATED CORNEAS:based on visual examination and pre-treatment opacity check

NUMBER OF REPLICATES: 3/treatment

TREATMENT METHOD:closed chamber

REMOVAL OF TEST SUBSTANCE: 3 rinses with EMEM containing phenol red followed by one rinse with EMEM only

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity and Corneal permeability

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
IVIS Classification
≤ 3 No category. Not requiring classification to UN GHS or EU CLP
> 3; ≤55 No prediction of eye irritation can be made
> 55 Category 1. UN GHS or EU CLP Causes serious eye damage

Irritation parameter:

in vitro irritation score

Value:

1.8

Negative controls validity:

valid

Remarks:

IVIS 102.4

Positive controls validity:

valid

Remarks:

OD 0.3

Remarks on result:

no indication of irritation

Treatment	Cornea Number	Opacity		Permeability (OD)		In VitroIrritancy Score
		Post-Treatment-Pre‑Treatment	Corrected Value		Corrected Value
Negative Control	1	1		0.000
	2	0		0.002
	3	0		0.014
		0.3		0.005		0.4
Positive Control	4	84	83.7	1.520	1.515
	5	78	77.7	1.635	1.630
	6	83	82.7	1.073	1.068
			81.3		1.404	102.4
+Test Item	7	2	1.7	0.076	0.071
	8	0	0.0	0.013	0.008
	9	3	2.7	0.007	0.002
			1.4		0.027	1.8

OD= Optical density          

        

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of the test the substance is considered not severely damaging to the eyes

Executive summary:

In a BCOP assay bovine corneas were exposed to the substance and assessed for opacity and permeability. No effects of the substance on opacity readings and permeability was observed. The calculation of the In Vitro Irritancy Score (1.8) showed that the substance is not severely damaging to the eyes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Justification for classification or non-classification

Based on the information available the substance does not need to be classified for skin- and eye-irritation according to Regulation (EC) No 1272/2008 (CLP).