Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid

Endpoint summary

Description of key information

The skin corrosion study investigates the potential for Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid to cause skin corrosion in vitro. The study was performed to GLP standards and the was compliant with the Guideline for the Testing of Chemicals No. 431 (In vitro skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method) and EU Method B.40 (In vitro skin corrosion: Transcutaneous Electrical Resistance Test (Ter)) with no deviations. Using a human skin model, EpiDerm tissue (the target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium) the test substance was assessed for its potential to cause in vitro skin corrosion based on a MTT colourmetric assay. The results shown that the test substance was non-corrosive over both a 3 mins and 60 mins time period as cell variability was only reduced to 98% and 84.1%, respectively. This experimentation was in the presence of a suitable negative and positive control which allowed the outcome of the test substance on cell viability to be validated (Cell viability for the negative control was 100 % (3 mins and 60 mins) and for the positive control was 4.6% (3 mins) and 3.9 % (60 mins)). The experiment also showed that the result was not influenced by a direct reduction or colour interference with MTT from the test substance.

Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid was determined not to be a skin irritant under CLP Regulation (EC) No 1272/2008. This conclusion was based on a tissue viability of 108.8 % following a 15-minute exposure period and 42-hour post-exposure incubation period.

The eye irritation/damage study investigates the potential for Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid to cause in vitro eye irritation. This study was performed using The Bovine Corneal Opacity and Permeability (BCOP) assay according to the OECD Guideline for the Testing of Chemicals No. 437 (updated 26 July 2013) “Bovine Corneal Opacity and Permeability Assay” and the EU Method B.47 of Commission Regulation (EC) No. 440/2008. The study is also GLP compliant. The BCOP assay reports results for both opacity and permeability of prepared corneas for the test item, a negative control and a positive control (3 replicated per treatment type). Using the In vitro Irritancy Score the test item achieved a value of 142.5, the positive control achieved a value of 116.7 and the negative control achieved a value of 0.5, all of which were within the acceptance criteria of the standard. In conclusion, the test substance was awarded a classification of Category 1. UN GHS or EU CLP Causes serious eye damage.

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:

31/07/2017 - 17/11/2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

Method B.40b

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: 0001230562
- Expiration date of the lot/batch: 08 June 2019
- Purity test date: 100% (UVCB)


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature in the dark
- Stability under test conditions: Not specified
- Solubility and stability of the test substance in the solvent/vehicle: Not specified
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None

Test system:

human skin model

Remarks:

Normal human epidermal keratinocytes cells (EpiDermTM Tissue)

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Source strain:

not specified

Justification for test system used:

The target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium.

This human skin model is advantageous for the study of dermal corrosivity - Corrosion is directly related to cytotoxicity in the EpiDerm™ tissue. Cytotoxicity is determined by the reduction of MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) to formazan by viable cells in the test item treated tissues relative to the corresponding negative control. The results are used to make a prediction of the corrosivity potential of the test item.

Vehicle:

unchanged (no vehicle)

Details on test system:

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C for 3 mins and 60 mins.
- Temperature of post-treatment incubation (if applicable): 37 °C for 3 hours

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: 1 washing stage, 2 blotting stages.
- Observable damage in the tissue due to washing: N/A
- Modifications to validated SOP: N/A

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Labtech LT 4500 microplate reader
- Wavelength: 570nm (OD570)
- Filter: N/A
- Filter bandwidth: N/A

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 runs per time point per type (i.e. test substance, positive control, negative control).

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 0 mg of test substance

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 mg of test substance

Duration of treatment / exposure:

3 mins or 60 mins

Duration of post-treatment incubation (if applicable):

3 hours

Number of replicates:

2 per time point (3 mins or 60 mins) per type (e.g. test substance, positive control, negative control).

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Negative control (3 mins)

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Negative control (60 mins)

Value:

100

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive Control (3 mins)

Value:

4.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Positive Control (60 mins)

Value:

3.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item (3 mins)

Value:

98

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Test Item (60 mins)

Value:

84.1

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:

- OTHER EFFECTS:
- Visible damage on test system: N/A
- Direct-MTT reduction: No
- Colour interference with MTT: No

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: No

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin based on the results (98% cell viability at 3 mins and 84.1% cell viability at 60 mins).

Executive summary:

The skin corrosion study investigates the potential for Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid to cause skin corrosion in vitro. The study was performed to GLP standards and is compliant with the Guideline for the Testing of Chemicals No. 431 (In vitro skin Corrosion: Reconstructed Human EpiDermis (RHE) Test Method) and EU Method B.40 (In vitro skin corrosion: Transcutaneous Electrical Resistance Test (Ter)) with no deviations. Using a human skin model, EpiDerm tissue (the target cells are epithelial, derived from human skin, and formed into a stratified, cornified epithelium) the test substance was assessed for its potential to cause in vitro skin corrosion based on a MTT colourmetric assay. The results shown that the test substance was non-corrosive over both 3 mins and 60 mins time period, as cell variability was only reduced to 98% and 84.1%, respectively. This experimentation was in the presence of a suitable negative and positive control which allowed the outcome of the test substance on cell viability to be validated (Cell viability for the negative control was 100 % (3 mins and 60 mins) and for the positive control was 4.6% (3 mins) and 3.9 % (60 mins)). The experiment also showed that the result was not influenced by a direct reduction or colour interference with MTT from the test substance.

Reference 2

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 1 - 6, 2017

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test system:

human skin model

Remarks:

(EpiSkin™ model)

Source species:

human

Cell type:

other: Reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute

Vehicle:

unchanged (no vehicle)

Details on test system:

SKIN DISC PREPARATION
- Procedure used: Before removal from the transport plate, each tissue was inspected for air bubbles between the agarose gel and the insert. 2 mL of maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labelled 12-well plate. Each epidermis unit was transferred into the maintenance medium filled wells (3 units per plate). A different 12-well plate was used for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.

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

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: At the end of the exposure period, each tissue was removed from the well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved by filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item.

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter: Without a reference filter

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritating to skin if relative mean tissue viability is ≤50%.
- The test substance is considered to be non-irritating to skin if relative mean tissue viability is >50%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: Approximately 10 mg (26.3 mg/cm2) of the test item was then applied to the epidermal surface.

NEGATIVE CONTROL
- Amount(s) applied: 10 µL of DPBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10 µL of SDS 5% w/v

Duration of treatment / exposure:

15-minute exposure.

Duration of post-treatment incubation (if applicable):

The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

Number of replicates:

Triplicate

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15-minute exposure / 42-hour post-exposure incubation

Value:

108.8

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:

- OTHER EFFECTS:
- Visible damage on test system: No.
- Direct-MTT reduction: The MTT solution containing the test item did not turn blue or purple which indicated that the test item did not directly reduce MTT.
- Colour interference with MTT: The solution containing the test item was a white color. This color was attributed to the intrinsic color of the test item itself. It was therefore unnecessary to run color correction tissues.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The mean OD570 for the negative control treated tissues was 0.817 and the standard deviation value of the viability was 2.2%. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control: The relative mean tissue viability for the positive control treated tissues was 12.6 % relative to the negative control treated tissues and the standard deviation value of the viability was 5.1 %. The positive control acceptance criteria were therefore satisfied.
- Acceptance criteria met for variability between replicate measurements: The standard deviation calculated from individual tissue viabilities of the three identically test item treated tissues was 4.6%. The test item acceptance criterion was therefore satisfied.

Interpretation of results:

GHS criteria not met

Conclusions:

A skin irritation test for reaction product of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid was undertaken using the EPISKIN™ Reconstructed Human Epidermis Model. At 10 mg (26.3 mg/cm2) tissue viability following a 15 minute exposure period was 108.8 %, resulting in classification as a non-irritant to skin (CLP Regulation (EC) No 1272/2008).

Executive summary:

To assess the skin irritation potential of Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid, an experiment was performed according to Good Laboratory Practise (GLP), OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method), and EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test). No deviation from the protocol was reported. 10 mg (26.3 mg/cm2) of the test item, 10 µL of Dulbecco’s Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ (negative control), and 10 µL of  5% w/v (positive control) was applied to tissue in triplicate for a 15 minute exposure period and 42-hour post-exposure incubation period. All tissues were subsequently taken for MTT-loading and, thereafter, exposed to acidified isopropanol for extraction of formazan crystals and then measured for optical density at 570 nm. The quality criteria required for acceptance of results in the test were satisfied. The relative mean viability of the tissues treated with the test substance was 108.8 %, from which it is possible to infer that Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid is not a skin irritant under the conditions of the test (CLP 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:

31/7/2017 - 13/12/2017

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- batch No.of test material: 0001230562
- Expiration date of the lot/batch: 08 June 2019
- Purity test date: 100 % UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark.
- Stability under test conditions: Assumed to be stable.
- Solubility and stability of the test substance in the solvent/vehicle: Assumed to be stable, dissolved test item is assumed to be homogeneous in solution.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not specified.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: 20% w/v solution.
- Final preparation of a solid: 20% w/v solution in sodium chloride 0.9% w/v.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : In solution.

Species:

cattle

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir. The eyes were excised after slaughter and placed in Hanks’ Balanced Salt Solution supplemented with penicillin at 100 IU/mL and streptomycin at 100 µg/mL. They were transported to the test facility on ice packs on the same day and the corneas were prepared immediately on arrival.

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75mL applied per prepared cornea.
- Concentration (if solution): 20% w/v test item solution in sodium chloride 0.9% w/v.

Duration of treatment / exposure:

240 minutes.

Duration of post- treatment incubation (in vitro):

90 minutes (for permeability assay).

Number of animals or in vitro replicates:

3 per treatment (Test item, Negative control, Positive control).

Details on study design:

SELECTION AND PREPARATION OF CORNEAS
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

QUALITY CHECK OF THE ISOLATED CORNEAS
Corneas were incubated in Eagle’s Minimum Essential Medium (EMEM) without phenol red at 32 ± 1 ºC for 70 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

NUMBER OF REPLICATES
3 per treatment type (Test item, negative control, positive control).

NEGATIVE CONTROL USED
0.9% w/v sodium chloride

POSITIVE CONTROL USED
20% w/v Imidazole solution in 0.9% w/v sodium chloride

APPLICATION DOSE AND EXPOSURE TIME
0.75 mL of 20% w/v test item solution in sodium chloride 0.9% w/v applied to cornea at 32 +- 1 ºC for 240 minutes.

TREATMENT METHOD: BCOP holder with anterior and posterior chambers. EMES in posterior chamber and test solution in anterior chamber.

POST-INCUBATION PERIOD: Yes, for permeability determination the corneas were incubated with a 5 mg/mL sodium fluorescein solution at 32 ± 1 ºC for 90 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: Three washes with fresh complete EMEM containing phenol red and then a final final rinse with complete EMEM without phenol red.

POST-EXPOSURE INCUBATION: for permeability determination the corneas were incubated with a 5 mg/mL sodium fluorescein solution at 32 ± 1 ºC for 90 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: A pre-treatment and a post-treatment opacity reading was taken for each cornea using a calibrated opacitometer and each cornea was visually observed.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a Labtech LT-4500 microplate reader (quantitative viability analysis) at 492 nm (without a reference filter).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) .

DECISION CRITERIA: Decision criteria as indicated in the testing guidelines.

Irritation parameter:

in vitro irritation score

Run / experiment:

Test item

Value:

142.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Negative Control

Value:

0.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Irritation parameter:

in vitro irritation score

Run / experiment:

Positive control

Value:

116.7

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: All eyes were macroscopically examined before and after dissection for damage and prepared corneas were visually examined for defects.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes.
- Acceptance criteria met for positive control: Yes .
- Range of historical values if different from the ones specified in the test guideline: No, values fall within study acceptance criteria.

Interpretation of results:

Category 1 (irreversible effects on the eye) based on GHS criteria

Conclusions:

Eye damage as a result from exposure to test item (In vitro irritancy score of 142.5) resulted in a classification of "Category 1. UN GHS or EU CLP Causes serious eye damage".

Executive summary:

The study investigates the potential for Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid to cause in vitro eye irritation. This study was performed using The Bovine Corneal Opacity and Permeability (BCOP) assay according to the OECD Guideline for the Testing of Chemicals No. 437 “Bovine Corneal Opacity and Permeability Assay” and the EU Method B.47 of Commission Regulation (EC) No. 440/2008. The study is GLP compliant. The BCOP assay reports results for both opacity and permeability of prepared corneas for the test item, a negative control and a positive control (3 replicated per treatment type). Using the In vitro Irritancy Score the test item achieved a value of 142.5, the positive control achieved a value of 116.7 and the negative control achieved a value of 0.5, all of which were within the acceptance criteria of the standard. In conclusion, the test item was assigned a classification of Category 1. UN GHS or EU CLP Causes serious eye damage.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)