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

Endpoint summary

Description of key information

In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD 442C.

The method is not applicable for the testing of substances of unknown or variable composition, complex reaction products or biological materials (UVCB substances) due to the unknown and/or variable composition of the test substance as the defined molar ratio of test chemical and peptide is needed for the assessment of the test results. Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid is a UVCB, therefore this test was not performed.

In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method (KeratinoSens^TM), OECD 442D.

A GLP compliant OECD 442D test was performed on Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid to assess its skin sensitisation potential. This study concluded the substance is not a skin sensitiser.

In Vitro Skin Sensitisation: Human Cell Line Activation Test (h-CLAT), OECD 442E.

Substances with Log Kow up to 3.5 can be tested whereas substances with Log Kow higher than 3.5 tend to produce negative results. For such substances positive results could be used to support the identification of a test chemical as sensitiser. Negative results should be considered as inconclusive.

Although Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid was determined to have a high Log Kow >6.5, a GLP compliant OECD 442E test was performed, yeilding positive results to support classification as a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08/02/2018 - 03/04/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

Remarks:

see principles of method below for full details

Principles of method if other than guideline:

Deviations from the guidelines:
- Standard states MTT musted be used for an incubation period of 4 hours, solubilised overnight in 10% SDS and read at 690 nm, the study uses MTT for a 3 hr incubation period, which is solubilised in isopropanol and read at 570 nm.
- The standard states that cinnamic aldehyde must be used at 4 - 64 μM, whereas the study uses cinnamic aldehyde 8 - 128 μM.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The test (KeratinoSens) test is a method for which validation studies have been completed followed by an independent peer review conducted by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) and is considered scientifically valid when used as part of an integrated approach to testing to determine skin sensitisers and non-sensitisers for the purposes of hazard classification and labelling.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0001230562
- Expiration date of the lot/batch: 08/06/2019
- Purity test date: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Assumed to be stable
- Stability under test conditions:Assumed to be stable
- Solubility and stability of the test substance in the solvent/vehicle: Assumed to be stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Assumed to be stable

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design: Using a test system (KeratinoSens cell line (test system) derived from the HaCaT human keratinocytes) a luciferase reporter gene transcriptionally controlled by the Anti-oxidant Response Element from a gene that is known to be up regulated in contact with sensitisers was quantitatively measured. Experiments were performed in plates where cells were exposed to in individual concentrations of the test substance or appropriate control substances. Test concentrations were determined based on the solubility of the test item in ethanol (max. 20 mg/mL in Ethanol), and for subsequent dilution in cell culture medium a top concentration of 200 μg/mL) with 3 replicates per concentration per treatment.

Positive control results:

The positive control, 'Cinnamic Aldehyde' showed that there was a dose-dependent increase of induction, which was > 1.5 - fold in at least one concentration and the CV% of blank values was < 20% so was accepted. The positive control was positive at both 64 and 129 μM.

Key result

Run / experiment:

other: Test Item

Parameter:

other: EC1.5

Value:

1.5

Vehicle controls validity:

not valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Test item

Parameter:

other: Imax

Value:

0.854

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Not specified.
- Acceptance criteria met for positive control: Not all criteria met, however considered valid based on Positive Control results.
- Acceptance criteria met for variability between replicate measurements: No specified.
- Range of historical values if different from the ones specified in the test guideline: Same.

Interpretation of results:

GHS criteria not met

Conclusions:

The Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid was classified as a Non-sensitiser to human skin as EC1.5 value value did not exceed the minimum threshold (1.5) and the Imax value was between 0.854 - 1.183.

Executive summary:

The study assesses the In vitro sensitisation potential of Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid using the test system (KeratinSens test method). The study is GLP compliant and is performed according to the OECD 442D guidelines. The experiment use the quantification of luciferase expression in the cell reporter line to determine the response to the test item, a negative control and a positive control, reporting the EC1.5 and Imax values. In all test item experiments the EC1.5 value did not exceed the minimum threshold value (1.5) and the Imax value range was 0.854 - 1.183, which lead to conclusion that the test item is not a skin sensitiser (GHS criteria determined).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23/01/2018 - 14/03/2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)).

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: THP-1 cell surface marker expression

Justification for non-LLNA method:

The test method (h-CLAT) has been evaluated in a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-lead validation study and subsequent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC) and was considered scientifically valid to be used as part of an Integrated Approach to Testing and Assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.
Note that for this method, test items with a Log Kow (octanol/water partition coefficient) of up to 3.5 have been tested successfully. However, test items with a Log Kow of greater than 3.5 can still be tested at lower soluble concentrations. In such a case, a negative result (non-sensitiser) should be considered inconclusive, whereas a positive result (sensitiser) could still be considered valid.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0001230562
- Expiration date of the lot/batch: 08/06/2019
- Purity test date: N/A

Details on the study design:

Skin sensitisation (In vitro test system)
- Details on study design: THP-1 cells were pre-cultured for either 48 or 72 hrs. Following this, the cells were dosed with the test item over an 8 dose range and incubated for 24 ±0.5hrs. The cells were then washed and stained with propidium iodide which allows for discrimination of live/dead cells by flow cytometry. The dose of test item that yields 75% cell viability (CV75) was calculated and taken forward for the next stage of testing. This dose finding assay was carried out over two independent runs.
THP-1 cells were pre-cultured again for 48hrs. Once the CV75 was determined, a narrower dilution series based around the CV75 value was produced for the test item. This dilution range was used to dose the cells again for 24 ±0.5hrs. The cells were then washed and stained with propidium iodide and also with antibodies that detect CD54 and CD86 expression as well as a negative control antibody. This allowed for discrimination of live/dead cells and also changes in CD54 and CD86 marker expression by flow cytometry.
Prior to the CV75 determination, the test item was assessed for solubility and was found to be soluble in Isopropanol at 25 mg/mL.

Positive control results:

Positive control - CV75:
Complete RPMI culture medium containing 10% Human Serum, 0.05mM 2-mercaptoethanol and 0.4% DMSO was used for reference to the positive control that was solubilised in DMSO. Positive control was accepted as both criteria where met (cell viability ≥ 75% at the lowest dose and the highest test item concentration produced cytotoxicity).

Positive Control - CD54 and CD86 Expression:
Complete RPMI culture medium containing 10% Human Serum, 0.05mM 2-mercaptoethanol and 0.4% Isopropanol was used for reference to the test item that was solubilised in Isopropanol. Positive control was accepted as all criteria where met.

Run / experiment:

other: 1

Parameter:

other: EC200

Remarks:

CD54

Value:

16

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: EC200

Remarks:

CD54

Value:

26

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 1

Parameter:

other: EC150

Remarks:

CD86

Value:

13.76

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: EC150

Remarks:

CD86

Value:

25

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid was classified as a skin sensitiser to human skin as EC200 and EC150 values for CD54 and CD86 expression were 26 and 25 µg/mL, respectively.

Executive summary:

The study assesses the in vitro sensitisation potential of Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid using the test system (h-CLAT test method). The study is GLP compliant and is performed according to the OECD Guideline 442E. The experiment uses the quantification of cytotoxic effects observed (CV75 assay) and cell surface marker expression on THP-1 cells (CD54 and CD86 assays) after 24-hour exporure to the test substance to determine the response. The relative fluorescence intensity (RFI) is used a measure of expression of CD54 and CD86, calculated from the results at test item doses in duplicate.

The expression of CD54, as measured by the RFI, crossed the threshold (RFI ≥200) in 3/8 doses in run 1 and 4/7 doses in run 2.

The expression of CD86, as measured by the RFI, crossed the threshold (RFI ≥150) in 7/8 doses in run 1 and 4/7 doses in run 2.

Final EC200 and EC150 values for CD54 and CD86 expression were 26 and 25 µg/mL, respectively.

As the CD54/CD86 expression crossed the thresholds, the test item is classified as a skin sensitiser. Cell viability did not fall below 50% at any of the test item concentrations and therefore the result is deemed to be valid.

Reference 3

Endpoint:

skin sensitisation: in chemico

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

The Direct Peptide Reactivity Assay (DPRA) is not applicable for the testing of UVCB substances due to the unknown and/or variable composition as the defined molar ratio of test chemical and peptide is needed for the assessment of the test results. Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid is a UVCB, therefore this test was not performed.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

The skin sensitisation potential of Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid was assessed experimentally by two of the three available in vitro tests (OECD 442D and 442E). OECD 442C could not be performed as the substance is a UVCB. The results of OECD 442D were negative, whilst those of 442E were positive and therefore no overall conclusion on classification can be determined based on these two results alone.