[ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids

Endpoint summary

Description of key information

Skin and eye irritation studies were conducted according to OECD guideline methods under GLP conditions.

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:

May 15 - August 7, 2018

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP compliance certification included in full study report

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm™ tissues, Lot No. 28368 Kit D, were received from MatTek Corporation (Ashland, MA) on 22 May 2018 and refrigerated at 2-8°C. Before use, tissues were incubated (37±1°C, 5±1% CO2) with assay medium (MatTek) for a one-hour equilibration. Equilibration medium was replaced with fresh medium before dosing.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

50 μl of the test article were applied to the top of each EpiDerm™ tissue. Nylon mesh was then placed on top to facilitate even distribution of the test article.

Duration of treatment / exposure:

The test article remained in contact with the EpiDerm™ tissue for 3 minutes at room temperature and 60 minutes at 37±1°C, 5±1% CO2.

Duration of post-treatment incubation (if applicable):

At the end of the exposure period, each EpiDerm™ tissue was rinsed with phosphate buffered saline (PBS) and transferred to a 24-well microplate containing 300 μl of MTT solution (1 mg/ml MTT in DMEM). The tissues were then returned to the incubator for a three-hour MTT incubation period. Following the MTT incubation period, each EpiDerm™ tissue was rinsed with PBS and then treated overnight at room temperature with 2.0 ml of extractant solution (isopropanol) per well.

Number of replicates:

Each treatment with test article or control was conducted in duplicate.

Irritation / corrosion parameter:

% tissue viability

Value:

92.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% mean tissue viability

Positive controls validity:

valid

Remarks:

2% mean tissue viability

Remarks on result:

other: no indication of corrosion

Other effects / acceptance of results:

Viability differences between the two identically treated tissues in all samples and controls at 3 minutes were 0.7% to 8.8%. Viability differences between the two identically treated tissues at 60 minutes were 0.1% to 2.3%. Inter-tissue viability differences at both time points met the acceptance criterion (≤30%).

The summarized results and corrosion classifications are as follows:

Test and Control Article Identity	Mean Tissue Viability (3 min.)	Mean Tissue Viability (3 min.)	Predicted Corrosivity
[ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids	105.5%	92.6%	Not corrosive
Tissue culture water (Negative Control)	100.0%	100.0%	Not corrosive
8.0N Potassium hydroxide solution (Positive Control)	24.0%	2.0%	Corrosive

Interpretation of results:

GHS criteria not met

Conclusions:

The in vitro skin corrosion test was conducted according to OECD 431 guideline and GLP principles. It is concluded that this test is valid and that the test substance is not corrosive in the in vitro skin corrosion test.

Executive summary:

In an in vitro skin corrosion test using a human skin model (MatTek EpiDerm™ human epidermis) performed according to OECD 431 guideline and GLP principles, the influence of the test substance on the viability of human skin was tested. The test substance was applied directly to cultured tissue (50 μL/ tissue, n=2). After 3-minute and 60-minute treatments the substance was removed and the viability of the cells was tested by reduction of MTT. The realtive mean tissue viability obtained after 3-minute and 1-hour treatments with the test substance was 105.5% and 92.6%, respectively. The positive control had a mean cell viability of 2.0% after 1 -hour exposure.

Since the mean relative tissue viability was not below 50% after the 3-minute treatment and not below 15% after the 1-hour treatment, it is concluded that the test substance is not corrosive in the in vitro skin corrosion test.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 15 - August 7, 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP compliance certification included in full study report

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Vehicle:

unchanged (no vehicle)

Details on test system:

EpiDerm™ tissues, Lot No. 28368 Kit C, were received from MatTek on 22 May 2018 and refrigerated at 2-8°C. Before use, the tissues were incubated (37±1°C, 5±1% CO2) with assay medium (MatTek) for a one-hour equilibration. The tissues were then moved to new wells with fresh medium for an additional overnight equilibrium, for 18±3 hours. Equilibration medium was replaced with fresh medium before dosing.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

30 μl of the test article were applied to the EpiDerm™ tissue.

Duration of treatment / exposure:

A nylon mesh was then placed on top to facilitate even distribution of the test article. A negative control (30 μl of PBS) and a positive control (30 μl of 5% SDS solution) were each tested concurrently, with a nylon mesh placed on top to facilitate even distribution of the material. Each treatment with test article or control was conducted in triplicate. The exposure period for the test article and controls was 60 minutes. The dosed tissues were placed in an incubator at 37±1°C, 5±1% CO2 for 35±1 minute, and then returned to the sterile hood for the remainder of the 60-minute exposure period.

Duration of post-treatment incubation (if applicable):

After dosing and incubation, the tissues were rinsed with PBS, blotted to remove the test substance and dry the tissue, and transferred to fresh medium. The rinsed EpiDerm™ tissues were returned to the incubator for 24±2 hours. Medium was changed at 24±2 hours. Tissues were returned to the incubator for an additional 18±2 hours.

Number of replicates:

Three tissue samples were treated with the test substance.

Irritation / corrosion parameter:

% tissue viability

Remarks:

Expressed as a mean of three treated tissues

Run / experiment:

The individual viabilities of three treated tissues were 4.0%, 26.6% and 46.0%.

Value:

25.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

The mean tissue viability of three treated tissues was 25.5% with a standard deviation of 21.03%. Although the standard deviation did not meet the acceptance criterion (not greater than 18%), all the individual tissue viabilities were below the classification cut-off of 50%. Therefore the material is classified as a skin Irritant.

Other effects / acceptance of results:

The three test article-treated tissue viabilities were 4.0%, 26.6%, and 46.0%, with a mean tissue viability of 25.5%, and a standard deviation of 21.03%. Although the standard deviation did not meet the acceptance criterion, all the individual tissue viabilities were below the classification cut-off of 50%. Therefore, the material was classified as a skin irritant.

Interpretation of results:

Category 2 (irritant) based on GHS criteria

Conclusions:

The in vitro skin irritation test was conducted according to OECD Guideline 439 – In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method and GLP principles. It is concluded that this test is valid and that the test substance is a Category 2 irritant.

Executive summary:

An in vitro test using a reconstructed human epidermis model was performed according to OECD Guideline 439 – In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method under GLP conditions. The test substance was applied directly to cultured tissues and covered with a nylon mesh for 60 minutes (50 μL/ tissue, n=2). After exposure, the tissues were rinsed and cells were incubated for 66 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed tissue was set at 100%, the positive control had a mean cell viability of 2.3%and the test substance showed cell viability of 25.5%.

 

Since the mean relative tissue viability after exposure to the test substance was below 50%, it is concluded that the test substance is irritating to skin according to the in vitro skin irritation test and the substance is classified as a Category 2 skin irritant according to Regulation (EC) No. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (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:

May 15 - August 7, 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)

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP cokpliance certificate included in full study report

Details on test animals or tissues and environmental conditions:

EpiOcular™ tissues, Lot No. 27430 Kit E, were received from MatTek on 22 May 2018 and refrigerated at 2-8°C. Before use, the tissues were incubated (37±1°C, 5±1% CO2) with assay medium (MatTek) for a one-hour equilibration. Equilibration medium was replaced with fresh medium for an additional overnight equilibration of 16 to 24 hours. After the overnight incubation, the tissues were moistened with 20 μl of phosphate-buffered saline (PBS) and incubated at 37±1°C, 5±1% CO2 for 30±2 minutes.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

50 μl of the test article were applied to duplicate EpiOcular™ tissues. Each treatment with test article or control was conducted in duplicate.

Duration of treatment / exposure:

The tissues were incubated at 37±1°C, 5±1% CO2 for 30±2 minutes.

Duration of post- treatment incubation (in vitro):

After dosing and incubation, the tissues were rinsed with phosphate-buffered saline (PBS) and soaked in 5 ml of room-temperature assay medium in a 12-well plate for 12 minutes. The soaked tissues were then incubated in fresh assay medium at 37±1°C, 5±1% CO2 for 120 minutes. At the end of the incubation period, each EpiOcular™ tissue was transferred to a 24-well plate containing 300 μl of MTT solution (1 mg/ml MTT in Dulbecco's Modified Eagle's Medium). The tissues were then returned to the incubator for a three-hour MTT incubation period. Following the MTT incubation period, each EpiOcular™ tissue was rinsed with PBS, and then treated with 2.0 ml of extractant solution (isopropanol) overnight at room temperature.

Irritation parameter:

other: Tissue viability

Run / experiment:

Mean tissue viability of two treated tissues.

Value:

45.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Remarks:

The test item is determined to be an irritant based on a mean tissue viability less than or equal to 60% according to the OECD Test Guideline No. 492, “Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage”.

Other effects / acceptance of results:

The mean OD570 of the negative control tissues was 2.022, which met the acceptance criteria of greater than 0.8 and less than 2.5. The mean relative viability of the positive control tissues was 35.3%, which met the acceptance criterion of less than 50%. The differences in viability between identically treated tissues were 0.21% to 8.33%, which met the acceptance criterion of less than 20%. All controls passed the acceptance criteria for a valid study.

The summarized results and irritation classifications are as follows:

Test and Control Article Identity	Mean Tissue Viability	Tissue Viability Std. Deviation	Irritancy Classification	GHS Calssification
[ω-hydroxy-C16 (saturated and unsaturated) and C16 (unsaturated)] fatty acids	45.8%	8.3%	Irritant	Eye Irritation Category 1 or 2
Tissue culture water (Negative Control)	100%	0.2%	Non-Irritant	Not classified
Methyl acetate (Positive Control)	35.3%	1.3%	Irritant	Eye Irritation Category 1 or 2

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Based on the outcome of a Reconstructed Human Cornea-like Epithelium (RhCE) Test Method performed according to OECD 492 guideline and GLP principles, it is concluded that the substance is irritanting to the eye.

Executive summary:

A Reconstructed Human Cornea-like Epithelium (RhCE) Test Method was performed with the substance according to OECD guideline 492 and GLP principles. The substance was applied undiluted (50 μL/ tissue, n=2). The mean in vitro tissue viability of the positive control was 35.3%, and the mean in vitro tissue viability of the negative control (tissue culture water) was 100%. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. The mean viability of tissue exposued to the test substance was 45.8%. Since the mean tissue viability of the treated tissues was ≤60%, the substance is considered an eye irritant and should be classified as either Category 1 or 2 according to Regulation (EC) No 1272/2008.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A literature review was conducted for irreversible effects of the major fatty acid constituents of the UVCB substance (hexadecanoic acid and related 16-carbon compounds) on eye tissues. There was no evidence of eye corrosion or irreversible eye damage caused by these compounds identified in the literature. One reference in the Journal of the Americal Oil Chemists' Society offers a general guideline that organic fatty acids having carbon chain lengths of at least 14 and greater should not be considered to be corrosive to the eyes (Kabara, J. J. (1979), Toxicological, bacteriocidal and fungicidal properties of fatty acids and some derivatives. J Am Oil Chem Soc, 56: 760A-767A).

Justification for classification or non-classification

The eye irritation test indicates that the test substance is expected to have adverse effects on the eye. However, OECD Test Guideline No. 492 is not intended to differentiate between UN GHS Category 1 and UN GHS Category 2. The registrant has classified the substance as Cateogry 2 (causing serious eye irritation) based on internal experience and an absence of reported eye damage caused by the major fatty acid constituents of the UVCB substance (hexadecanoic acid and related 16-carbon compounds) in the published literature. The registrant finds no cause to classify a mixture of weak organic fatty acids as corrosive to the eyes causing irreversible eye damage based on expert knowledge material handling experience with the substance.