Fatty acids, tall-oil, esters with ethylene glycol

Endpoint summary

Description of key information

In vitro skin irritation: not irritating

In vitro eye irritation: not irritating

The hazard assessment is based on the data currently available. New studies with the registered substance and/or other member substances of the glycol esters category will be conducted in the future. The finalised studies will be included in the technical dossier as soon as they become available and the hazard assessment will be re-evaluated accordingly.

For further details, please refer to the category concept document attached to the category object (linked under IUCLID section 0.2) showing an overview of the strategy for all substances within the glycol esters category.

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:

04 - 11 Jun 2018

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 Jun 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

not specified

GLP compliance:

yes

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: EPISKIN Small Model™

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small Model™
- Tissue batch number(s): 18-EKIN-023, 18-EKIN-015 and 18-EKIN-022

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: at room temperature
- Temperature of post-treatment incubation: at 37 °C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were washed with phosphate buffered saline to remove residual test substance.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/mL
- Incubation time: 3 h
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Barrier function: According to the Supplier`s Data Sheet, the IC50 value was determined to be 2.1 and 2.2 mg/mL for the tissues with the batch numbers of 18-EKIN-023 and 18-EKIN-015 (acceptance criteria: 1.5 ≤ IC50 ≤ 3.0), respectively. The result of the tissue with the batch number 18-EKIN-022 was invalid.

NUMBER OF REPLICATE TISSUES: 3 tissues were used per treatment, negative and positive control, respectively.

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Fresh tissues / killed tissues : Since the test item reacted with the MTT medium, in addition three killed tissues treated with test item and three killed untreated tissues were used for the cytotoxicity evaluation with MTT (Batch numbers: 18-EKIN-015 and 18-EKIN-022).
- Procedure used to prepare the killed tissues: Living epidermis was transferred to 12 well plates and incubated with 2 mL Milli-Q for 48 ± 1 h. After incubation, killed epidermis was stored at ≤ -15°C. Killed tissues were thawed by placing them for 1 h at room temperature in 12 well plates on 2 mL maintenance medium. Further use of killed tissues was similar to living tissues.
- N. of replicates: 3
- Method of calculation used:
Nonspecific MTT reduction (NSMTT) was calculated. NSMTT is the difference between the mean OD of the untreated killed tissues (ODkt_u+MTT) and test item treated killed tissues (ODkt_t+MTT) expressed as percentage of the mean of the negative control tissues (ODlt_u+MTT).
%NSMTT = [(ODkt_t+MTT – ODkt_u+MTT)/ mean ODlt_u+MTT] * 100

True tissue viability is calculated as the difference between the living test item treated tissues incubated with MTT medium (ODlt_t+MTT) and the difference between ODkt_t+MTT and ODkt_u+MTT.
OD= ODlt_t+MTT – (ODkt_t+MTT-ODkt_u+MTT)
%Viability = [OD/ mean ODlt_u+MTT] * 100

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: Duplicate measurements each of 3 independent tissues

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant or corrosive (category 1 or 2) to skin if after 15 min exposure and 42 h of post incubation is less than or equal to 50%.
- The test substance is considered to be non-irritant (no category) to skin if after 15 min exposure and 42 h of post incubation is greater than 50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 µL

NEGATIVE CONTROL
- Amount(s) applied: 25 µL

POSITIVE CONTROL
- Amount(s) applied: 25 µL
- Concentration: 5% (aq) sodium dodecyl sulfate in phosphate buffered saline

Duration of treatment / exposure:

15 ± 0.5 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

Duplicate measurements each of 3 independent tissues

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15 ± 0.5 min exposure with the test item

Value:

69

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- OTHER EFFECTS:
- Direct-MTT reduction and colour interference with MTT: The test item was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because a color change was observed by adding MTT-medium it was concluded that the test item did interact with the MTT endpoint. In addition to the normal procedure, three killed tissues treated with test item and three killed non-treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test item was 0.64% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test item treated viable tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the OD values of the three independent tissues for the negative control were 1.3171, 1.2761, 1.0732, 1.0738, 1.0012 and 0.9437, and thus within the range of the istorical control data (0.422 - 1.547).
- Acceptance criteria met for positive control: Yes, the positive control reduced the cell viability at 3.6 % and fulfilled the acceptance criteria.
- Acceptance criteria met for variability between replicate measurements: Yes, differences of three tissue cell viabilities in the negative control substance, the positive control substance and the test substance groups were 15%, 14% and 0.8%, respectively, and thus ≤ 18%.

Table 1: Individual absorption values

	Tissue A	Tissue B	Tissue C
	(OD570)	(OD570)	(OD570)
Negative control measurement 1	13.171	10.732	10.012
Negative control measurement 2	12.761	10.738	0.9437
Test item on viable tissue OD570measurement 1	0.8058	0.9558	0.6488
Test item on viable tissue OD570measurement 2	0.7956	0.9267	0.6155
Test item on killed tissue OD570measurement 1	0.1190	0.1076	0.1505
Test item on killed tissue OD570measurement 2	0.1203	0.1197	0.1391
Non treated killed tissue OD570measurement 1	0.0993	0.1442	0.1296
Non treated killed tissue OD570measurement 2	0.0939	0.1317	0.1166
Positive control OD570measurement 1	0.0807	0.0874	0.0726
Positive control OD570measurement 2	0.0826	0.0924	0.0716

OD: Optical density

SD: Standard Deviation

1) The test item values were corrected fot the non-specific MTT reaction (0.64%)

Table 2: Mean tissue viability (%)

	Mean tissue viability (% of control)	Standard deviation (%)
Negative control	100	15
Test item	69	14
Positive control	3.6	0.8

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

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:

30 Apr - 04 May 2018

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:

adopted Jun 2018

Deviations:

no

GLP compliance:

yes

Species:

human

Strain:

other: Reconstructed Human EpiOcular™ Model

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL

NEGATIVE CONTROL
- Amount(s) applied: 50 µL

POSITIVE CONTROL
- Amount(s) applied: 50 µL

Duration of treatment / exposure:

30 ± 2 min at 37.0 ± 1.0°C

Duration of post- treatment incubation (in vitro):

12 ± 2 min at room temperature (post-exposure immersion) and 120 ± 15 min at 37 °C (post-incubation)

Number of animals or in vitro replicates:

Duplicate measurements each of 2 independent tissues for treatment, negative and positive control group, respectively

Details on study design:

- RhCE tissue construct used, including batch number : EpiOcular™ (OCL-200-EIT MatTek Corporation, Lot: 27427 Kit A)

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: The ability of the test substance to directly reduce MTT and to change the color of the MTT medium was assessed in a pre-experiment. Because no color changes were observed it was concluded that the test item did not interact with the MTT endpoint and did not induce color interference.

- Description of the method used to quantify MTT formazan : The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Eye hazard potential of the test item was classified according to remaining cell viability following exposure of the test item.

- Description of evaluation criteria used: The test substance is considered to be not irritating to eye if the tissue viability is > 60%. The test substance is considered to be irritating to eye if the tissue viability is ≤ 60%.

- Positive and negative control means and acceptance ranges: OD in the negative control substance group is > 0.8 and < 2.5. The cell viability in the positive control substance group is < 50%.
- Acceptable variability between tissue replicates for positive and negative controls and for the test substance: Differences of two tissue cell viabilities in each treatment group are < 20%

Irritation parameter:

other: % tissue viability mean value of 2 tissues

Run / experiment:

30 ± 2 min

Value:

102

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system:

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, the OD of the negative control was 1.9660, 1.9044, 1.6966 and 1.6817, and thus within the acceptibility range (OD > 0.8 and < 2.5).
- Acceptance criteria met for positive control: Yes, the positive control reduced the cell viability at 2.9% and fulfilled the acceptance criteria.
- Acceptance criteria for variabilties: Differences of two tissue cell viabilities in the negative control substance, the positive control substance and the test substance groups were 14%, 4.2% and 1.3%%, respectively, and thus < 20%.

Table 1: Individual absorption values

	Tissue A	Tissue B
	(OD570)	(OD570)
Negative control measurement 1	19.660	16.966
Negative control measurement 2	19.044	16.817
Test item measurement 1	19.030	18.244
Test item measurement 2	18.664	17.970
Positive control measurement 1	0.1035	0.0807
Positive control measurement 2	0.1027	0.0801

OD: Optical density

Table 2: Mean tissue viability

	Mean tissue viability (% of control)	Difference between two tissues  (%)
Negative control	100	14
Test item	102	4.2
Positive control	2.9	1.3

Interpretation of results:

other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The hazard assessment is based on the data currently available. New studies with the registered substance and/or other member substances of the glycol esters category will be conducted in the future. The finalised studies will be included in the technical dossier as soon as they become available and the hazard assessment will be re-evaluated accordingly.

For further details, please refer to the category concept document attached to the category object (linked under IUCLID section 0.2) showing an overview of the strategy for all substances within the glycol esters category.

Skin

The skin irritating properties of the test substance Fatty acids, tall-oil, esters with ethylene glycol (CAS 68187-85-9) were assessed in an in vitro skin irritation study according to OECD 439 and GLP using a human three dimensional epidermal model (EPISKIN-SM™) (Charles River, 2018). 25 µL of the undiluted test material was applied directly on top of the skin tissue for 15 ± 0.5 min. After a 42 h post-incubation period, determination of the cytotoxic (irritancy) effect was performed. The test material did interact with MTT. Therefore in addition to the normal procedure, three killed tissues treated with test material and three killed untreated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by the test material was 0.64% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test material treated viable tissues. Skin irritation is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 15 ± 0.5 min treatment with the test material compared to the negative control tissues was 69%. Since the mean relative tissue viability for the test material was above 50% after 15 ± 0.5 min treatment the test material is considered to be non-irritant. The positive control had a mean cell viability of 3.6% after 15 ± 0.5 min exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was ≤ 15%, indicating that the test system functioned properly.

In conclusion, Fatty acids, tall-oil, esters with ethylene glycol is non-irritant in the EPISKIN-SM™ test under the experimental conditions described in this report.

 

Eye

The eye irritating properties of the test substance Fatty acids, tall-oil, esters with ethylene glycol (CAS 68187-85-9) were assessed in an in vitro eye irritation study according to OECD 492 and GLP using the Reconstructed Human EpiOcular™ Model (Charles River, 2018). The test material was applied undiluted (50 µL) directly on top of the tissue for 30 ± 2 min. After exposure the cornea epithelial construct was thoroughly rinsed to remove the test material and transferred to fresh medium for an immersion incubation. Afterwards, the tissues were transferred to fresh medium and incubated for 2 h at standard culture conditions, prior to determination of the cytotoxic (irritancy) effect. The positive control had a mean cell viability of 2.9% after 30 ± 2 min exposure. The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was 1.771 and within the laboratory historical control data range. The difference between the percentage of viability of two tissues treated identically was less than 14%, indicating that the test system functioned properly. Eye hazard potential is expressed as the remaining cell viability after exposure to the test material. The relative mean tissue viability obtained after 30 ± 2 min treatment with the test material compared to the negative control tissues was 102%. Since the mean relative tissue viability for the test material was above 60% after 30 ± 2 min treatment the test material is considered to be non-irritant.

In conclusion, Fatty acids, tall-oil, esters with ethylene glycol is non-irritant in the EpiOcular™ test under the experimental conditions described in this report.

Justification for classification or non-classification

The available data on skin and eye irritation do not meet the classification criteria according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.