Fatty acids, C14-18 and C18-unsatd., branched and linear, esters with pentaerythritol

Endpoint summary

Description of key information

Skin Irritation (OECD 439, EpiDerm): not irritating

Eye irritation (OECD 492, EpiOcular): not irritating

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:

01 - 24 Jun 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

FREIE UND HANSESTADT HAMBURG, Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: not specified

Source strain:

not specified

Justification for test system used:

This test method provides an in vitro procedure that, depending on information requirements, may allow determining the cytotoxic potency and skin irritancy of test items as a stand-alone replacement test within a testing strategy, in a weight of evidence approach. The test method is based on reconstructed human epidermis models, which in their overall design (the use of human derived epidermal keratinocytes as cell source, representative tissue and cyto-architecture) closely mimic the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. The procedure described under this test method allows the hazard identification of irritant substances in accordance with UN GHS category 1 or 2.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm (EPI-200) MatTek In Vitro Life Science Laboratories, Bratislava II, Slovak Republic
- Tissue batch number(s): 23342
- Date of initiation of testing: 01 Jun 2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C (for the first 35 min) and room temperature (for the second 25 min)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: At the end of the exposure period, the test item was carefully washed from the skin surface with Dulbecco's phosphate buffered saline (D-PBS) (not further specified)

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 h
- Spectrophotometer: Tecan Sunrise (Magellan Version 6.4)
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: The optical density (OD) of the extraction solvent alone should be sufficiently small, i.e. OD <0.1. The tissue treated with the negative control (NC) should exhibit stability in culture (provide similar viability measurements) for the duration of the test exposure period.
- Barrier function: Concurrent negative (NC) and positive controls (PC) were used, each in triplicate, to demonstrate that viability (NC), barrier function and resulting issue sensitivity (PC) of the tissues are within a defined historical acceptance range
- Contamination: The skin model was free of contamination by bacteria, viruses, mycoplasma, or fungi.
- Reproducibility: The assay meets the acceptance criterion if the standard deviation (SD) calculated from individual % tissue viabilities of the 3 identically treated replicates is ≤18%.

NUMBER OF REPLICATE TISSUES: Triplicate tissues for test item, negative and positive control each

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Freeze-killed tissues
- Procedure used to prepare the killed tissues: The frozen tissues4 were stored in the freezer (-20 ± 5°C). The test item was applied to two freeze-killed tissues. In addition, two freeze-killed tissues were left untreated. The entire assay protocol was performed on the frozen tissues in parallel to the assay performed with the live EpiDerm tissues.
- N. of replicates: Two treated and untreated freeze-killed tissues each
- Method of calculation used: True viability = (Viability of treated tissue – Interference from test chemical) = OD tvt – OD kt (where OD kt = (mean OD tkt – mean OD ukt)); tvt = treated viable tissue, kt = killed tissues, tkt = treated killed tissue, ukt = untreated killed tissue (NC treated tissue)

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced below or is equal to 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritating to skin if the mean relative tissue viability of three individual tissues exposed to the test substance is above 50% of the mean viability of the negative controls.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 30 µL
- Concentration: 100% (undiluted)

NEGATIVE CONTROL
- Amount(s) applied: 30 µL (D-PBS)
- Concentration: 100% (undiluted)

POSITIVE CONTROL
- Amount(s) applied: 30 µL SDS
- Concentration: 5%

Duration of treatment / exposure:

60 min

Duration of post-treatment incubation (if applicable):

42 h

Number of replicates:

triplicate tissues for each treatment and concurrent control groups

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1

Value:

112.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

viability 100%

Positive controls validity:

valid

Remarks:

viability 9.6%

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS
- Direct-MTT reduction: The test item was evaluated for the potential to interfere with the MTT assay reagent. Therefore, 30 μL of the test item were added to 1 mL of the MTT medium and incubated at 37°C, 5% CO2, and 95% humidity for 60 min. Untreated MTT solution was used as control. A discoloration of the test item to lilac was noted. Hence, due to interacting of the test item with the MTT measurement (reduction of MTT) an additional test with freeze-killed tissues had to be performed.
- Colour interference with MTT: Prior to the testing, the test item was evaluated for colour changes under aqueous conditions. Therefore, 30 μL of the test item was diluted in 300 μL deionized water and incubated at 37°C, 5% CO2 and 95% relative humidity (RH) for 60 min. At the end of exposure time, the mixture was evaluated for the presence and intensity of the staining. No discolorations were noted.

ACCEPTANCE OF RESULTS
- Acceptance criteria met for negative control: The mean optical density (OD) of the negative control of 3 tissues was 1.417 and was well within the acceptable range of ≥ 1.0 to ≤ 2.5.
- Acceptance criteria met for positive control: The viability of cells treated with the positive reference item, 5% SDS, was 9.6% of the negative control and fulfilled the acceptance criterion of ≤ 20%.
- Acceptance criteria met for variability between replicate measurements: The standard deviation of all triplicates determined was below the limit of acceptance of 18%.

Table 1: Summarized results of in vitro skin irritation

	Optical density (n = 3 tissues)	Standard deviation	Corrected OD (n = 3 tissues)	% Corrected OD / OD compared to the control
Negative control	1.417	0.015	-	100
Test item	1.684	0.107	1.597	112.7
Positive control	0.136	0.012	-	9.6

Interpretation of results:

GHS criteria not met

Conclusions:

CLP: not classified

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:

01 - 23 Jun 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-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:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

FREIE UND HANSESTADT HAMBURG, Behörde für Gesundheit und Verbraucherschutz, Hamburg, Germany

Species:

human

Strain:

other: three-dimensional Reconstructed human Cornea-like Epithelium (RhCE) tissue

Details on test animals or tissues and environmental conditions:

- Justification of the test method and considerations regarding applicability: EpiOcular RhCE tissue construct (Mattek) consist of relevant human-derived cells and reconstruct a cornea-like epithelium three-dimensional tissue, which is composed of progressively stratified but not cornified cells. Multiple layers of viable, non-keratinized epithelial cells are present in the reconstructed cornea-like epithelium. The RhCE tissue construct has an epithelial surface in direct contact with air so as to allow for direct topical exposure of test chemicals in a fashion similar to how the corneal epithelium is exposed in vivo. The RhCE tissue construct forms a functional barrier with sufficient robustness to resist rapid penetration of cytotoxic benchmark substances, e.g., Triton X-100. The containment properties of the RhCE tissue construct prevent the passage of test chemical around the edge of the viable tissue, which could lead to poor modelling of corneal exposure. The RhCE tissue construct was free of contamination by bacteria, viruses, mycoplasma, and fungi. The test item was applied topically to three-dimensional Reconstructed human Cornea-like Epithelium (RhCE) tissue constructs and tissue viability was measured following exposure and a post-treatment incubation period.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 50 µL
- Concentration: 100% (undiluted)

Duration of treatment / exposure:

30 min

Duration of post- treatment incubation (in vitro):

120 min

Number of animals or in vitro replicates:

duplicate tissues for each treatment

Details on study design:

- Details of the test procedure used: Two tissue replicates were used for each treatment, negative and positive control. Concurrent negative (NC) and positive controls (PC) were used to demonstrate that viability (NC), barrier function and resulting issue sensitivity (PC) of the tissues are within a defined historical acceptance range. Prior to treatment the tissues were pre-wetted with 20 μL DPBS (without Mg2+ and Ca2+) and incubated at 37°C, 5% CO2 and 95% relative humidity for 30 ± 2 minutes (pre-treatment). After the 30 ± 2 minutes pretreatment each test item, negative control and positive control was tested by applying 50 μL topically on EpiOcular tissues. The tissues were incubated at 37°C, 5% CO2 and 95% relative humidity for 30 ± 2 minutes (exposure). At the end of treatment, test items were removed by extensively rinsing the tissues with DPBS (without Mg2+ and Ca2+) at room temperature. The rinsing was performed by dipping the tissues three times in containers pre-filled with fresh 100 mL DPBS (without Mg2+ and Ca2+). For each treatment different fresh DPBS (without Mg2+ and Ca2+) solutions were used. The rinsing was repeated twice, each with fresh DPBS (without Mg2+ and Ca2+). After rinsing, the tissues were immediately transferred in 5 mL pre-warmed Assay Medium (room temperature) in a pre-labeled 12-well plate for 12 ± 2 minutes (post-soak). After the Post-Soak each insert is removed and remaining media was decanted, the inert is blotted on absorbent material and transferred to the appropriate well of the pre-labeled 6-well containing 1 mL of warm Assay Medium. The tissues were incubated at 37°C, 5% CO2 and 95% relative humidity for 120 ± 2 minutes (post-treatment). After the Post-treatment Incubation the MTT assay was performed. Therefore, tissues were transferred into wells of a 24-well plate containing 0.3 mL of MTT solution (1 mg/mL). The tissues were incubated at 37°C, 5% CO2 and 95% relative humidity for 180 ± 10 minutes. After incubation each insert was removed from the 24-well plate. The bottom of the insert blotted on absorbent material, and then the insert was transferred to a pre-labeled 24-well plate containing 2.0 mL of isopropanol in each designated well so that isopropanol will flow into the insert on the tissue surface. The plates were sealed with parafilm and were either stored overnight at 2°C to 8 °C in the dark or immediately extracted on a plate shaker for 2 to 3 hours at room temperature. At the end of the extraction period, the tissue was pierced and the liquid within each insert decanted into the well from which it was taken. The extract solution was mixed and transferred to a 96-well plate. The absorbance (Optical Density (OD) at 540 nm) was determined with a microplate reader. As the test item was supposed to reduce the MTT solution, two freeze-killed tissues per control and test item were used. All assay procedures were performed as for the viable tissue.
- RhCE tissue construct used, including batch number: OCL-200-EIT, MatTek In Vitro Life Science Laboratories, Bratislava II, Slovak Republic (Lot. No. 23715)
- Doses of test chemical and control substances used: 100% (undiluted)
- Duration and temperature of pre-treatment, exposure and post-exposure incubation periods: 30 ± 2 min (pre-treatment, exposure) and 120 ± 2 min (post-exposure) at 37 °C, 5% CO2 and 95% relative humidity (each treatment period)
- Number of tissue replicates used per test chemical and controls (positive control, negative control: duplicate tissues each
- Wavelength and band pass used for quantifying MTT formazan: 540 - 590 nm
- Description of the method used to quantify MTT formazan: MTT assay was used for quantifying tissue viability. Viable cells of the EpiOcular RhCE tissue construct reduce the vital dye MTT into a blue MTT formazan precipitate, which was then extracted from the tissue using isopropanol. The extracted MTT formazan was quantified using a standard absorbance (Optical Density (OD)) measurement procedure in the range between 540 and 590 nm
- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model: The individual % of control OD540 values was averaged to calculate the mean percent of control. The relative tissue viability was determined against the negative control. If the test item-treated tissue viability is >60% relative to negative control-treated tissue viability, the test item is predicted to be non-irritant. If the test item-treated tissue viability is ≤60% relative to negative control-treated tissue viability, the test article is predicted to be an irritant.
- Positive and negative control means and acceptance ranges based on historical data: 1.885 OD (= 100% viability for negative control), 30.65% (positive control)
- Acceptable variability between tissue replicates for positive and negative controls: 1.0 - 2.9% (negative control), 0.4 - 5.6% (positive control)
- Acceptable variability between tissue replicates for the test chemical: the difference of viability between two replicate tissues is <20%

Irritation parameter:

other: % tissue viability

Run / experiment:

1

Value:

115.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

viability 100%

Positive controls validity:

valid

Remarks:

viability 30.1

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: the negative control OD is >0.8 and <2.5
- Acceptance criteria met for positive control: the mean relative viability of the positive control is below 50% of control viability
- Acceptance criteria met for test item: the difference of viability between two replicate tissues is <20%

Table 1: Summarized results of in vitro eye irritation

	Test item	Negative control	Positive control	Test item (corrected reduction control)
Mean corrected OD (n=2 tissues)	1.663	1.434	0.431	0.007
Percent difference tissues 1 + 2 (%)	1.5	4.0	4.4	4.6
Viability compared to control (%)	116.0	100.0	30.1	0.5
Reduction corrected viability (%) of test item	115.5	-	-	-

Interpretation of results:

GHS criteria not met

Conclusions:

CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

In an in vitro skin irritation study performed according to OECD guideline 439 and in compliance with GLP human-derived epidermal keratinocytes (EpiDerm, EPI-200) were exposed to 30 µL of undiluted Fatty acids, C16-18 (even numbered) and C18-unsatd., branched and linear, tri- and tetraesters with pentaerythritol (CAS 85186-72-7) for 60 minutes followed by a post-treatment incubation period of 42 hours (Oleon, 2016a). The irritation potential of the test material was predicted from the relative mean cell viabilities compared to the mean viability of the negative control. Positive (5% SDS) and negative (D-PBS) controls were included in the study and gave the expected results. The mean corrected cell viability for the test material was calculated to be 112.7% when compared to the negative control after an incubation period of 42 hours. Therefore, the test material is considered to be non-irritating to skin under the conditions of the test.

Eye irritation

The eye irritation properties of Fatty acids, C16-18 (even numbered) and C18-unsatd., branched and linear, tri- and tetraesters with pentaerythritol (CAS 85186-72-7) have been investigated in an in vitro study performed according to OECD guideline 492 and in compliance with GLP using the EpiOcular Eye Irritation Test (Oleon, 2016b). For the assessment of the eye irritation properties 50 µL of the neat test material and the concurrent negative (sterile deionised water) or positive control (methyl acetate) were administered by topical application onto duplicate tissues of three-dimensional Reconstructed human Cornea-like Epithelium (RhCE) constructs (OCL-200-EIT, Mattek) for 30 min followed by a 12 min post-treatment immersion and a 120 min post-treatment incubation period, prior to MTT measurement. The corrected mean viability of the cells exposed to the test material was 115.5% of the mean negative control value. Hence, the test material was considered not to be irritating to eyes under the conditions of the test.

Justification for classification or non-classification

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