Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine

Endpoint summary

Description of key information

Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine was non- predicted to be non-irritant to skin in an experiment employing an artificial three-dimensional model of human skin (EpiDerm™).

An IVIS of 50.035 ± 4.921 is calculated for Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine within a BCOP study according to OECD Guideline 437.

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:

2018-04-13 to 2018-04-27

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:

July 2015

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm
- Tissue batch number(s): 25899
- Certificate date: 2018-04-25


TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: The incubation conditions were 37°C, 5% CO2 and 95% relative humidity for the first 35 minutes followed by 25 minutes at room temperature under a sterile hood.
- Temperature of post-treatment incubation (if applicable): 42 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: no details

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours
- Spectrophotometer: Tecan Sunrise Magellan Version 7.2
- Wavelength: 540 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: MTT WC assay (OD 540 -570 nm) passed
- Barrier function: ET50 assy (ET50 4.77 -8.72 hrs) passed
- Morphology: passed
- Contamination: sterility an d biological contaminats tested: passed
- Reproducibility: validity criteria fullfilled

NUMBER OF REPLICATE TISSUES: 3 tissues

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
Not applicable, no possible interference with the MTT measurement

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

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
According to the EU and GHS classification (H314 or H315 / Category 1/2 or no label), an irritant is predicted if the mean relative tissue viability of three individual tissues exposed to the test substance is reduced below or equal to 50% of the mean viability of the negative controls.

mean tissue viability ≤ 50% Irritant (I), (H314 or H315 or GHS Category 1 or 2 )
mean tissue viability > 50% non-irritant (NI).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
25 mg of test item were applied to the skin model.
The test item was grounded and applied as a fine powder.
For better contact of the test item to the skin, the skin surface was moistened with 25 µL Dulbecco’s phosphate buffered saline (D-PBS)

NEGATIVE CONTROL
D-PBS was used as the negative control.
30 µL

POSITIVE CONTROL
5% aqueous sodium dodecyl sulphate (SDS)
30 µL

Duration of treatment / exposure:

The whole exposure period for the used EpiDermTM skin model was 60 minutes. The incubation conditions were 37°C, 5% CO2 and 95% relative humidity for the first 35 minutes followed by 25 minutes at room temperature under a sterile hood.

Duration of post-treatment incubation (if applicable):

Post treatment incubation period of the rinsed tissues in fresh assay medium of 42 hours

Number of replicates:

3 tissues

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test substance

Value:

79.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

The mean viability of cells exposed to the test item was 79.5% of the negative controls and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%.
The test substance was considered to be non-cytotoxic and predicted to be non-irritant to skin.

The mean optical density (OD) of 3 negative control tissues was 1.747 and was well within the acceptable range of ≥ 0.8 to ≤ 2.8.
The viability of cells treated with the positive reference item, 5% SDS, was 6.6% of the negative control and fulfilled the acceptance criterion of ≤ 20%.
The standard deviation (15.7%) determined for all triplicates w3as below the limit of acceptance of 18%.
Hence, all acceptance criteria were met.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the present test conditions, Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine tested at an exposure time of 60 minutes and a 42-hour post-treatment incubation period, was non-cytotoxic and, hence, predicted to be non-irritant to skin in an experiment employing an artificial three-dimensional model of human skin (EpiDerm™). Hence, the test item did not show irritant properties and is therefore not classified as irritant (UN GHS no category)..

Executive summary:

The EpiDerm^TM model was used to distinguishes irritants in accordance with UN GHS Category 1 or Category 2 from non-classified test substances.

Three tissues were used for each treatment and concurrent control groups. The optical density (OD) was determined by using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue) reduction assay and expressed as relative percentage of viability of the negative control-treated tissues.

Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine was applied as solid test item to the model skin surface, which was moistened with Dulbecco’s phosphate buffered saline (D-PBS). D-PBS was used as the negative control. 5% aqueous sodium dodecyl sulphate (SDS) was used as the positive reference item. An exposure time of 60 minutes was employed followed by a 42-hour post-treatment incubation period in fresh medium.

The mean viability of cells exposed to the test substance was 79.5% of the negative controls and, hence, was well above the cut-off percentage cell viability value that distinguishes irritant from non-irritant test items of > 50%.Octadecanoic acid, reaction products with acetic acid and tetraethylenepentaminewas considered to be non-cytotoxic and predicted to be non-irritant to skin.

The mean optical density (OD) of 3 negative control tissues was 1.747 and was well within the acceptable range of ≥ 0.8 to ≤ 2.8.

The viability of cells treated with the positive reference item, 5% SDS, was 6.6% of the negative control and fulfilled the acceptance criterion of ≤ 20%.

The standard deviation (15.7%) determined for all triplicates was below the limit of acceptance of 18%.

Hence, all acceptance criteria were met.

 

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:

2018-06-04 to 2018-06-20

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)

Version / remarks:

October 2017

GLP compliance:

yes (incl. QA statement)

Species:

cattle

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse.
Hubert Bahlmann GmbH & Co. Versandschlachterei Spezialmischfutterwerk KG, 49699 Lindern, Germany

Vehicle:

physiological saline

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

The solid test item was suspended in a 0.9% sodium chloride solution with a final test substance concentration of 20% as recommended in the test guideline 437 for non-surfactant solids.
750 μL were added to completely cover the cornea’s epithelium in the anterior chamber.

Duration of treatment / exposure:

240 min (recommended exposure time for non-surfactant solids)

Duration of post- treatment incubation (in vitro):

at 32 ± 1 °C for 90 ± 5 minutes

Number of animals or in vitro replicates:

3 corneas per group

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
Bovine eyes from cattle in the age range of 6 to 12 months were obtained from a slaughterhouse.
To minimise deterioration and bacterial contamination, on collection the eyes were completely submerged in Hanks’ Balanced Salt Solution (HBSS) containing penicillin at 100 IU/mL and streptomycin at 100 μg/mL.
Upon arrival at the laboratory, the eyes were examined for defects such as but not limited to increased opacity, scratches, and neovascularisation.
Only corneas from eyes free of defects were used.

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas that had opacity greater than seven opacity units or equivalent for the opacitometer and cornea holders used after an initial one hour equilibration period had to be discarded.

NUMBER OF REPLICATES : 3

NEGATIVE CONTROL USED : 0.9% sodium chloride solution
Positive control item: 20% Imidazole (CAS no. 288-32-4)6 in 0.9%
sodium chloride solution

POSITIVE CONTROL USED
20% Imidazole (CAS no. 288-32-4)6 in 0.9% sodium chloride solution

APPLICATION DOSE AND EXPOSURE TIME :
750 μL of the test or control items were added to completely cover the cornea’s epithelium in the anterior chamber.
Exposure period: 240 minutes (recommended exposure time for non-surfactant solids)

TREATMENT METHOD:The open-chamber method was used.

REMOVAL OF TEST SUBSTANCE
Subsequently, the epithelium was washed with EMEM containing phenol red at least three times.
Washing was repeated until no test item or discolouration (yellow or purple) of phenol red was visible.
The corneas were rinsed a final time with EMEM only to remove any remaining phenol red from the chamber.


METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
Corneal opacity was determined by the amount of light transmission through the cornea measured quantitatively with the aid of an opacitometer (BASF 2011-13; 67063 Ludwigshafen am Rhein, Germany)
resulting in opacity values measured on a continuous scale.
- Corneal permeability:
To determine the corneal permeability 1 mL sodium fluorescein9 solution (5 mg/mL in 0.9% sodium chloride solution) was added to the anterior chamber (epithelial surface) while the posterior chamber (endothelial surface)
was refilled with fresh EMEM. The holder was incubated in a horizontal position at 32 ± 1 °C for 90 ± 5 minutes.
The amount of sodium fluorescein that crossed from the anterior to the posterior chamber was measured quantitatively using a microplate reader (Tecan Sunrise Magellan Version 7.2).
Measurements at 490 nm were recorded as optical density (OD490).
The fluorescein permeability values were determined using OD490 values based upon a visible light spectrophotometer (Tecan Sunrise) using a standard 1 cm path length.


SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: the decision criteria as indicated in the TG was used.

Irritation parameter:

in vitro irritation score

Run / experiment:

Experiment 1

Value:

50.035

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no information reported

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The corneas treated with the negative control item 0.9% sodium chloride solution revealed a mean opacity value of 0.585 ± 1.163 and a mean permeability value of 0.023 ± 0.013.
The calculated IVIS value of 0.925 ± 1.294 was within two standard deviations of the current historical mean and well below the cut-off value of 3 (UN GHS no category).

- Acceptance criteria met for positive control:
The corneas treated with the positive control item 20% Imidazole in 0.9% NaCl solution revealed a mean opacity value of 57.702 ± 9.732 and a mean permeability value of 2.496 ± 0.384 compared to the solvent control.
The calculated IVIS value of 95.137 ± 14.345 was within two standard deviations of the current historical mean and well above the cut-off value of 55.

The acceptance criteria for the test were fulfilled.

Conclusions:

The calculated IVIS for Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine is 50.035 ± 4.921.

Executive summary:

The purpose of this study was to determine a possible potency of Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine of being 'ocular corrosive and severe irritant' employing an in vitro system. The Bovine Corneal Opacity and Permeability Assay (BCOP) test method is an organotypic model that provides short-term maintenance of normal physiological and biochemical function of the bovine cornea in vitro.

 

In this test method, possible damage by the test item was assessed by quantitative measurements of changes in corneal opacity and permeability in isolated corneas from

bovine eyes.Corneal opacity was measured quantitatively as the amount of light transmission through the cornea. Permeability was measured quantitatively as the amount of sodium fluorescein dye that passes across the full thickness of the cornea, as detected in the medium in the posterior chamber. The measurements were used to calculate an in vitro irritancy score (IVIS), which was used to assign an in vitro irritancy hazard classification category for prediction of the in vivo ocular irritation potential of the test item.

 

Three corneas were used for each treatment group (test item, solvent control and positive control).

The solid test item was suspended in a 0.9% sodium chloride solution with a final concentration of 20% test item as recommended in the test guideline 437 for non-surfactant solids.

0.9% NaCl solution was used as the solvent control and 20% Imidazole in 0.9% NaCl solution as the positive control item.

 

The test item and the controls were applied to the epithelial surface of the cornea by addition to the anterior chamber of the corneal holder. The exposure time for the test item and the

controls was 240 minutes. The optical density (OD) was measured at a wavelength of 490 nm.

The acceptance criteria of validity were fulfilled in this test.

 

Following treatment with test substance a mean opacity of 48.645 ± 4.760 and a mean permeability value of 0.093 ± 0.015 compared to the negative control were determined.

The calculated IVIS of 50.035 ± 4.921 is above the cut-off value of 3 (UN GHS no category) and below the cut-off value of 55, (identifying test substances as inducing serious eye damage UN GHS Category 1).

According to criteria of the guideline no prediction concerning irritating potential of the test item can be made.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Justification for classification or non-classification

Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine does not need to be classified for skin irritation according to the criteria of CLP, EU GHS (Regulation (EC) No 1272/2008).

The IVIS scored of 50.035 calculated for Octadecanoic acid, reaction products with acetic acid and tetraethylenepentamine within a BCOP study according to OECD Guideline 437, the score is below the cut-off value of 55, (identifying test substances as inducing serious eye damage GHS Category 1).

Therefore according to CLP, EU GHS (Regulation (EC) No 1272/2008) the substance does not need to be classified for serious eye damage Category 1.

At present there are no validated in vitro methods available for the direct identification of Category 2 Eye irritants. According to ECHA guidance R7a further in-vivo testing is not required for an Annex VII data set.

In conclusion, based on the fact that eye irritation was expected for the substance and the calculated IVIS of 50.035 which is far above the cut-off value of 3 for GHS no category, it is considered appropriate to classify the substance for Eye irritation Category 2 (CLP) without further testing.