Linseed oil, ester with pentaerythritol

Endpoint summary

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:

From April 24, 2017 to April 28, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted July 23, 2009

Deviations:

no

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

Cell source:

foreskin from a single donor

Source strain:

not specified

Vehicle:

unchanged (no vehicle)

Details on test system:

The test system is a commercially available EpiDermTM-Kit (EPI-212-SIT, batch no: 25809, 2017), procured by MatTek. The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on specially prepared cell culture inserts.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

- One plate (3 tissues) was used as negative control in which each tissue was treated with 30 μL DPBS buffer.
- One plate was used as positive control in which each tissue was treated with 30 μL 5% SDS-solution.
- One plate was used for treatment with the test substance in which 30 μL test substance were applied.
A nylon meshes were added to all plates in order to ensure sufficient contact with the tissue surface.

Duration of treatment / exposure:

Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2. 1 h after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals. After rinsing, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). Then, the tissues were set in the incubator for 23 h and 30 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.

Duration of post-treatment incubation (if applicable):

Post-incubation, the tissues were removed from the incubator and shaken for 5 minutes (120 rpm). 0.9 mL assay medium were filled in the lower row of the 6-well-plate. Then the inserts were transferred into the lower row of the 6-well-plate and set into the incubator for 19 h for post-incubation at 37 ± 1°C and 5.0 ± 0.5% CO2.

Number of replicates:

From each well, two replicates with 200 μL solution (each) were pipetted into a 96-well-plate which was read in a plate spectrophotometer at 570 nm.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Tissue viability is calculated as % photometric absorbance compared with the mean of the negative controls

Value:

ca. 96.9 - ca. 103.5

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The relative absorbance value was reduced to 99.9 % after the treatment. This value is above the threshold for skin irritation (50%). Therefore, the test item is considered as non-irritant to skin. Values for negative control and for positive control were within the range of historical data of the test facility. Therefore, the experiment is considered valid.

Comparison of Tissue Viability

For the test item and the positive control, the following percentage values of tissue viability (in the form of formazan production) were calculated in comparison to the negative control:

 

Designation	linseed oil, ester with pentaerythritol	Positive Control
% Tissue viability (tissue 1)	99.5%	4.1%
% Tissue viability (tissue 2)	103.5%	3.6%
% Tissue viability (tissue 3)	96.9%	3.9%
% Tissue viability (mean)	99.9%	3.8%
± SD of mean tissue viability (%)	3.3%	0.2%

 

The relative absorbance value was reduced to 99.9 % after the treatment. This value is above the threshold for skin irritation (50%). Therefore, the test substance is considered as non-irritant to skin.

 

Validity and Acceptability

Validity criteria and results are stated in the following table:

 

Criterion	Demanded	Found
OD of negative control	≥ 0.8 and ≤ 2.8	1.7
% tissue viability of positive control SDS	≤ 20% of negative control	3.8%
SD of mean viability of the tissue replicates (%)	≤ 18%	1.0% (negative control) 0.2% (positive control) 3.3% (test item)

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance was considered as non-irritating to skin in the RhE test method.

Executive summary:

A study was conducted to determine skin irritation potential of the test substance in the Reconstructed Human Epidermis (RhE) according to EU Method B.46 and OECD Guideline 439, in compliance with GLP. Three tissues of the human skin model EpiDermTM were treated for 60 minutes. The test substance was applied directly to each tissue and spread to match the tissue size (0.63 cm2). DPBS-buffer was used as negative control, 5% SDS solution was used as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.7. The positive control showed clear irritating effects. Relative absorbance was reduced to 3.8% (required: ≤20%). Variation within the tissue replicates was acceptable (required: ≤18%). After the treatment with the test item, the relative absorbance value was reduced to 99.9%. This value is above the threshold for skin irritation potential (50%). Therefore, the test substance was considered as non-irritating to skin in the RhE test method (Andres, 2017).

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:

From April 24, 2017 to April 27, 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:

adopted July 26, 2015.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

human

Strain:

not specified

Details on test animals or tissues and environmental conditions:

The EpiOcularTM tissue consists of normal, human-derived 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. These cells are not transformed or transfected with genes to induce an extended life span. The EpiOcularTM tissues are cul-tured in specially prepared cell culture inserts with a porous membrane through which nutrients can pass to the cells. The tissue surface is 0.6 cm2.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 µL unchanged

Duration of treatment / exposure:

28 minutes

Duration of post- treatment incubation (in vitro):

2 h

Number of animals or in vitro replicates:

Duplicate

Details on study design:

Pre-tests:

Assessment of direct reduction of MTT by the test substance: The test substance was tested for the ability of direct formazan reduction. To test for this ability, 50 µL of the liquid test substance were added to 1 mL of MTT reagent in a 6-well plate and the mixture was incubated in the dark at 37±1°C, 5.0±1% CO2 and 80–100% relative humidity for 3 h. 1 mL of MTT reagent plus 50 µL of deminerelised water was used as negative control. The MTT reagent did not change its colour; therefore, direct MTT reduction had not taken place, and no data correction was necessary.

Assessment of coloured or staining test substances: It was tested whether the test substance develops a colour without MTT addition. 50 µL of the test substance was added to 2 mL isopropanol, incubated in 6-well plates on an or-bital shaker for 2 h at room temperature. Then, two 200 µL aliquots of the resulting solu-tion and two 200 µL aliquots of neat isopropanol were transferred into a 96-well plate and measured with a plate reader at 570 nm. After subtraction of OD for isopropanol, the OD of the test substance solution was -0.002 (≤ 0.08). Therefore, the main test was performed without colourant controls.

Main test:

Preparations: On the day of the start of the experiment, the MTT concentrate was thawed. The concentrate was diluted with the MTT solvent directly before use. The assay medium was warmed in the water bath to 37±1°C. 6-well-plates were labelled with test substance, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. All 24 inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37±1°C, 5±1% CO2 and 80–100% relative humidity for 1 h. After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37±1°C, 5±1% CO2 and 80–100% relative humidity for 16 h and 30 minutes.

Exposition and post-treatment: After overnight incubation, the tissues were pre-wetted with 20 µL DPBS buffer and then incubated at 37±1°C, 5±1% CO2 and 80–100% relative humidity for 30 minutes. After that, 50 µL of the controls and the test substance were applied in duplicate in 1- minute-intervals. This was done in such a manner that the upper surface of the tissue was covered. At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 28 minutes at 37±1°C, 5±1% CO2 and 80–100% relative humidity. At the end of the exposure time, the inserts were removed from the plates in 1 minute in-tervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred to and immersed in 5 mL of pre-warmed assay medium in a pre-labelled 12-well plate for 12 minutes post soak at room temperature. After that, each insert was removed from the medium, the medium was decanted off the tissue and the insert was blotted on absorbent material and transferred into the respective well of a pre-labelled 6-well plate containing 1 mL assay medium. For post-treatment in-cubation, the tissues were incubated for 120 minutes at 37±1°C, 5±1% CO2 and 80–100 % relative humidity. After the post-treatment incubation, the MTT Assay was performed.

MTT assay and extraction: A 24-well-plate was prepared with 300 µL freshly prepared MTT-reagent in each well. The tissue inserts were blotted on absorbent material and then transferred into the MTT solu-tion. The plate was incubated for 180 minutes at 37±1°C, 5±1% CO2 and 80 – 100% relative humidity. At last, each insert was thoroughly dried and set into a pre-labelled 6-well-plate, containing 2 mL isopropanol, taking care that no isopropanol is flowing into the tissue insert. The plate was firmly sealed to avoid evaporation of the solvent and then stored in the refrigerator overnight. On the next day the plate was shaken on an orbital shaker for 2 h at room temperature, protected from light.

Measurement: After 2 h, the inserts were pierced with an injection needle, taking care that all colour was extracted. The inserts were then discarded and the content of each well was thoroughly mixed in order to achieve homogenisation. From each well, two replicates with 200 µL solution (each) were pipetted into a 96-well-plate. Eight wells with 200 µL isopropanol were also pipetted, serving as a blank. The plate was read in a plate spectrophotometer at 570 nm.

Irritation parameter:

other: % tissue viability

Run / experiment:

Tissue viability is calculated as % photometric absorbance compared with the mean of the negative controls

Value:

ca. 105.4

Vehicle controls validity:

not examined

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

All validity criteria were met. The criterion for optical density of the negative control was fulfilled: The OD value was 1.7 (> 0.8 and < 2.5). The positive control induced a decrease in the relative absorbance as compared to the negative control to 38.4%. Variation within the replicates was acceptable (< 20%). The related infromation on results were mentioend in table in below section 'any other information on results incl. tables'.

Absorbance values blank isopropanol (OD at 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.038	0.038	0.037	0.038	0.038	0.039	0.038	0.039	0.038

Absorbance values negative control, positive control and test Item (OD at 570 nm) :

Designation	Measurement	Negative Control	Positive Control	linseed oil, ester with pentaerythritol
Tissue 1	1	1.746	0.711	1.819
	2	1.698	0.694	1.811
Tissue 2	1	1.729	0.677	1.829
	2	1.707	0.655	1.783

Mean absorbance negative control, positive control and test Item:

Designation	Negative Control	Positive Control	linseed oil, ester with pentaerythritol
Mean – blank (Tissue 1)	1.684	0.665	1.777
Mean – blank (Tissue 2)	1.680	0.628	1.768

% Viability positive control and test Item :

Designation	Positive Control	linseed oil, ester with pentaerythritol
% Viability (Tissue 1)	39.5%	105.6%
% Viability (Tissue 2)	37.3%	105.1%
% Viability Mean	38.4%	105.4%

Interpretation of results:

GHS criteria not met

Conclusions:

Under the study conditions, the test substance was identified as non-irritating to eyes.

Executive summary:

A study was conducted to determine eye irritation potential of the test substance in the reconstructed human Cornea-like Epithelium according to OECD Guideline 492, in compliance with GLP. Before conducting the main study, the substance was tested for ability of direct formazan reduction The MTT reagent did not change its colour, therefore direct MTT reduction had not taken place. Duplicate tissues of the reconstructed human Cornea-like Epithelium were treated with 50µL of test substance for 28 minutes. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control, methyl acetate was used as positive control. The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD >0.8 and <2.5, OD was 1.7. The positive control showed clear eye irritating effects, the relative absorbance value was reduced to 38.4% (< 50%). Variation within tissue replicates was acceptable (< 20%). After treatment with the test substance, the mean value of tissue viability was 105.4%. This value is well above the threshold for eye irritation (≤60%). Under the study conditions, the test substance was identified as non-irritating to eyes (Andres, 2017).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation

A study was conducted to determine skin irritation potential of the test substance in the Reconstructed Human Epidermis (RhE) according to EU Method B.46 and OECD Guideline 439, in compliance with GLP. Three tissues of the human skin model EpiDermTM were treated for 60 minutes. The test substance was applied directly to each tissue and spread to match the tissue size (0.63 cm2). DPBS-buffer was used as negative control, 5% SDS solution was used as positive control. After treatment with the negative control, the absorbance values were within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.7. The positive control showed clear irritating effects. Relative absorbance was reduced to 3.8% (required: ≤20%). Variation within the tissue replicates was acceptable (required: ≤18%). After the treatment with the test item, the relative absorbance value was reduced to 99.9%. This value is above the threshold for skin irritation potential (50%). Therefore, the test substance was considered as non-irritating to skin in the RhE test method (Andres, 2017).

Eye irritation

A study was conducted to determine eye irritation potential of the test substance in the reconstructed human Cornea-like Epithelium according to OECD Guideline 492, in compliance with GLP. Before conducting the main study, the substance was tested for ability of direct formazan reduction The MTT reagent did not change its colour, therefore direct MTT reduction had not taken place. Duplicate tissues of the reconstructed human Cornea-like Epithelium were treated with 50µL of test substance for 28 minutes. After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution. Demineralised water was used as negative control, methyl acetate was used as positive control. The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD >0.8 and <2.5, OD was 1.7. The positive control showed clear eye irritating effects, the relative absorbance value was reduced to 38.4% (< 50%). Variation within tissue replicates was acceptable (< 20%). After treatment with the test substance, the mean value of tissue viability was 105.4%. This value is well above the threshold for eye irritation (≤60%). Under the study conditions, the test substance was identified as non-irritating to eyes (Andres, 2017).

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Justification for classification or non-classification

Based on the results of in vitro skin and eye irritation/corrosion studies, the test substance does not meet the requirements for classification according to EU CLP (Regulation EC 1272/2008) criteria.