Oleic acid, compound with (Z)-octadec-9-enylamine (1:1)

Endpoint summary

Description of key information

No alert for skin sensitization was obtained in silico (OECD Toolbox). A weak positive, close to negative result in the DPRA (in chemico testing) and a negative result in vitro (KeratinoSens negative) confirmed this assessment. The positive result obtained in the mouse LLNA is considered to be not reliable, due to false-positive reactivity of the fatty acid moiety oleic acid in the LLNA. Furthermore, the physico-chemical properties of Oleylamine oleat will limit skin penetration. In summary, the performed tests and assessments do not indicate a substantial skin sensitizing activity of Oleylamine oleate. The substance is thus considered to be not classified for skin sensitization.

Due to the skin irritating properties seen in vitro and the classification with Skin Irrit 2 (H315) skin protection is needed in occupational settings that will markedly decrease the risk of dermal contact.

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:

weight of evidence

Study period:

04 - 14 Oct 2017

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)

Version / remarks:

adopted Feb 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the study design:

TEST METHOD
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test substance by addressing the second molecular key event of the Adverse Outcome Pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement compared with the respective solvent controls is used to support the discrimination between skin sensitiser and non-sensitisers.

TEST CELL LINE
- Type: KeratinoSens™
- Source: Givaudan, Switzerland
- Passage number: batch number 40, passage 15 for Experiment 1 and batch number 43, passage 12 for Experiment 2

CELL CULTURE CONDITIONS
- Type and identity of media:
Maintenance medium: Dulbecco’s Modified Eagle Medium (DMEM) containing serum and geneticin
Treatment medium: Dulbecco’s Modified Eagle Medium (DMEM) containing serum but without geneticin
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0

TEST CONCENTRATIONS
Experiment 1: 0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 61.5, 125, 250, 500, 1000 and 2000 μM
Experiment 2: 0.076, 0.015, 0.031, 0.061, 0.12, 0.24, 0.49, 1.0, 2.0, 3.9, 7.8 and 15.6 µM

CONTROLS
Vehicle control:
- Substance: DMSO
- Final concentration: 1%
Positive control:
- Substance: cinnamic aldehyde
- Final concentration: 4 - 64 μM
Untreated control:
- Substance: Isopropanol
- Final concentration: 1%

EXPOSURE CONDITIONS
- Exposure duration: 48 ± 1 h
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0

NUMBER OF REPLICATIONS: triplicates in two independent experiments

DETERMINATION OF CELL VIABILITY
- Method: MTT assay
- Incubation time: 4 h
- Device: SpectraMax M2e
- Wavelength: 600 nm
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0

DETERMINATION OF LUMINESCENCE
- Incubation time: 20 minutes
- Temperature (°C): 25 ± 2
- Device: luminescence plate reader

Key result

Run / experiment:

other: Experiment 1 at the lowest concentration (0.98 µM)

Parameter:

other: I(max)

Value:

1.72

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment 2 at 0.12 µM

Parameter:

other: I(max)

Value:

1.38

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment I at the lowest concentration (0.98 µM)

Parameter:

other: Cell viability (%)

Value:

45.64

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: The average coefficient of variation of the luminescence reading for the vehicle control was < 20% (12.63% in and 11.48% in Experiment 1 and 2, respectively).
- Acceptance criteria met for positive control: Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 16 to 64 μM in Experiments 1 and 2. The EC1.5 values for the positive control were 8.59 and 10.16 μM in Experiments 1 and 2, respectively. The average induction in the two replicates for the positive control at 64 μM was 5.49 and 27.65 in Experiments 1 and 2, respectively. The assay was considered valid as there was a clear dose response with the positive control.

The overall maximal average fold increase (Imax) was 1.72 and 1.38 for Experiments 1 and 2, respectively. In Experiment 1 only two concentrations (0.98 and 1.95 μM) were analysable due to strong cytotoxicity at all higher concentrations (3.91 to 2000 μM). It was thus not possible to calculate an EC1.5 value for Experiment 1 as the Imax value was >1.5 at the two analysable concentrations. Due to the cytotoxicity in Experiment 1 the test item concentrations for Experiment 2 were chosen with 0.01 to 15.63 μM. It was not possible to calculate an EC1.5 value for Experiment 2 as there were no statistically significant increases in induction. In Experiments 1 and 2, there was no apparent overall dose response for luciferase and the dose response curves were not biphasic. Cell viability at the lowest concentration with an Imax >1.5 in Experiment 1 was 45.6%. The cell viability measurement was not applicable for Experiment 2 as there was no EC1.5 determining concentration.

Interpretation of results:

other: no skin sensitising potential based on the key event “inflammatory response in keratinocytes”

Conclusions:

Under the conditions of the test, the test substance did not have a keratinocyte activating potential. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

04 - 24 Jul 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted Feb 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Integration und Soziales des Landes Nordrhein-Westfalen, Germany

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

TEST METHOD
The Direct Peptide Reactivity Assay (DPRA) is an in chemico procedure proposed to address the molecular initiating event leading to skin sensitization, namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine. Cysteine and lysine percent peptide depletion values are then calculated and used in a prediction model to categorize a substance in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers.

TEST SYSTEM
- Supplier of synthetic peptides: JPT Peptide Technologies GmbH
- Peptide stock solution preparation: Stock solutions of cysteine and lysine peptides were freshly prepared.
Cysteine-containing peptide:
- Concentration: 0.666 mM
Lysine-containing peptide:
- Concentration: 0.667 mM

VEHICLE CONTROL
- Substance: acetone
- Justification for selecting vehicle: the test substance was soluble in the vehicle at the test concentration of 100 mmol/L.

POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Preparation: The positive control was prepared as 100 mM/L solution in acetonitrile.

CO-ELUTION CONTROL
- Co-elution Control was made with the solution of the test item in acetone and phosphate buffer respectively ammonium acetate buffer. It was included in the assay to detect possible co-elution of the test item with either the lysine or the cysteine peptide.

REFERENCE CONTROLS
- Acetonitrile was used to verify the accuracy of the calibration curve for peptide quantification (Reference contol A) and to verify the stability of the respective peptide over the analysis time (Reference control B)
- Acetone and acetonitrile were used to verify that the solvents do not impact the percent peptide depletion (Reference controls C)

TEST SUBSTANCE PREPARATION
The test substance was prepared as a 100 mM solution in acetonitrile.

INCUBATION CONDITIONS
- Peptide ratios: Cysteine-containing peptide: 1:10; Lysine-containing peptide: 1:50
- Temperature used during treatment / exposure: 25 ± 2.5 °C
- Duration of treatment / exposure: 24 h

NUMBER OF REPLICATES
for each peptide triplicates were prepared for treatment substance and controls

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Wavelength: 220 nm for quantitation and 258 nm as indicator for co-elution

Positive control results:

The positive control revealed a depletion of cysteine and lysine peptides (66.1%). According to the cysteine 1:10/lysine 1:50 prediction model “high reactivity” was derived for the positive control, leading to a DPRA prediction of “positive“.

Key result

Run / experiment:

other: Cysteine/Lysine

Parameter:

other: Mean peptide depletion (%)

Value:

8.59

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Cysteine run

Parameter:

other: Mean peptide depletion (%)

Value:

11.53

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Run / experiment:

other: Lysine run

Parameter:

other: Mean peptide depletion (%)

Value:

5.64

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

The cysteine 1:10/lysine 1:50 prediction model was applied to the test item. A depletion of cysteine and lysine peptides became obvious in the DPRA (8.59%). According to the cysteine 1:10/lysine 1:50 prediction model “low reactivity” was derived for the test item in acetone, leading to a DPRA prediction of “positive“.

Table 1: Summary of results

	Mean % Cysteine peptide depletion	Mean % Lysine peptide depletion	Mean % Cysteine/Lysine peptide depletion	Reactivity Class (CYS + LYS)	Reactivity Class (CYS)	DPRA Prediction
Positive Control	71.49	61.72	66.61	High	n.a.	Positive
SD	0.16	1.54	-	-	-	-
CV	0.2%	2.5%	-	-	-	-
Test Item	11.53	5.64	8.59	Low	n.a.	Positive

SD: Standard deviation

CV: Coefficient of variation

Interpretation of results:

other: skin sensitising potential based on the key event “protein reactivity”

Conclusions:

Under the conditions of the test, the test substance showed reactivity towards selected proteins. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.

Reference 3

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Guideline adopted 22 July 2010; Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorised in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). Information on validation of IMDS and scientific justification is given in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).

Deviations:

yes

Remarks:

Measurement of cell counts instead of radioactive labelling. In addition, ear swelling and ear weights are determined to discriminate the irritating potential from the sensitizing potential of the test substance.

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

July 2012

Principles of method if other than guideline:

The study was performed according to OECD 429. However, an alternative method was used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes. In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.
Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the respective vehicle treated ones.
Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round; Toxicology 212, 60-68 (2005a);
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round; Toxicology 212, 69-79 (2005b).

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 9 weeks
- Weight at study initiation: 28 - 35 g
- Housing: The animals were kept singly in MAKROLON cages (type II). Animals were not group-housed to prevent contact of the application sites.
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period: at least 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3°C
- Humidity (%): 55% +/- 10%
- Photoperiod (hrs dark / hrs light): 12hrs dark / 12 hrs light
- IN-LIFE DATES: From: 30 May 2018 To:13 July 2018

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Three concentrations of Oleylamine oleate (5%, 10%, and 25% (w/w)), suspended in acetone/olive oil (4:1, v/v) were tested in six female NMRI mice per group and compared to a vehicle control group. A 25% (w/w) concentration was the highest feasible concentration of the test item in acetone/olive oil (4:1, v/v), see Table 1.

No. of animals per dose:

6

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: suspension/solution in acetone/olive oil (A/O, 4:1, v/v), see Table 1
- Irritation: no effects
- Systemic toxicity: no effects
- Ear thickness measurements: no effects
- Erythema scores: 0
A preliminary experiment was carried out in 3 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations out of the technically feasible concentrations (see Table below). Three concentrations of 5%, 10%, and 25% (w/w) suspended in acetone / olive oil (4:1, v/v) were examined. Doses were selected based on the OECD guideline from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5% (w/w) etc.
The preliminary experiment was conducted under conditions identical to the main LLNA study, except there was no assessment of lymph node proliferation and only 1 animal per concentration was used. Both ears of each mouse were observed for erythema. The test item suspension was administered to the dorsum of both animal's ears at an application volume of 25 µL/ear.
No clear irritating properties were observed in this preliminary experiment at concentrations of 5%, 10% or 25% (w/w), and no differences to the historical control data were noted for the ear thickness, but a trend for an increase in ear weight. A 25% (w/w) concentration was the highest feasible concentration of Oleylamine Oleate in acetone/olive oil (4:1, v/v).


MAIN STUDY
The study was performed according to OECD 429, however not employing the use of radioactive labelling to measure cell proliferation, as the radioactive method proposed by the OECD guideline led to problems in various EU laboratories: such as (i) practical difficulties/complexity of the test, in particular the radiochemical steps, which sometimes resulted in loss of specimen/activity; this in turn led to variability in the results and to a poor reproducibility and (ii) radiation protection issues. However, the OECD guideline allows other endpoints for assessment of proliferation in form of lymph node cell counts and lymph node weights if justification and appropriate scientific support exist showing the validity of this method.
The alternative method used for the study employing the lymph node weight and lymph node cell count to assess proliferation has been established by an European inter-laboratory validation exercise, as described in the two publications by Ehling et al. 2005a and 2005b. This method has the advantage of (i) more simplistic experimental work, (ii) less variability, (iii) better reproducibility, (iv) faster results, (v) reduced costs.
In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item.
By additionally measuring simple inflammatory parameters such as ear thickness or ear weight, it is possible to reliably determine the degree of response that is attributable to irritation (Vohr and Ahr, 2005 ).
Hence, in addition, the acute inflammatory skin reaction (irritating potential) was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control. The stimulation indices were calculated by dividing the average ear weight and average ear thickness on test day 4 per group to the test item treated animals by the vehicles treated ones. The cut-off threshold value for ear weight was set at 1.1.
To describe the relation between the activation of the local skin-draining lymph nodes and the skin inflammation, the Differentiation Index (DI) was calculated according to Homey et al.. The DI is the relation of '% maximal LN cell count index increase' divided by the '% of maximal ear swelling'.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: modified LLNA
- Criteria used to consider a positive response:
The so-called stimulation (or LLN-) indices to determine the sensitising potential (this value was fixed empirically during the interlaboratory validation of this method, for details see Ehling et al. 2005a and 2005b, page 12), were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated animals by the vehicle treated ones.
Thus, in case of no stimulating effect the index for the lymph node cell count is always below 1.4 (cut-off value). An index above 1.4 is considered positive.

TREATMENT PREPARATION AND ADMINISTRATION:
The experimental schedule of the assay was as follows:
- Day 1:
The weight of each animal was individually identified. The weights and any clinical signs were recorded. In addition, ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer.
Open application of 25 µL of the appropriate dilution of the test item, the vehicle alone or the positive control (as appropriate) were administered to the dorsum of each ear.
- Days 2 and 3:
The application procedure carried out on day 1 was repeated.
- Day 4 (24 hours after the last application):
Ear swelling measurements (immediately before sacrificing the mice) were carried out at the helical edge of both ears using an Oditest micrometer.
The animals were euthanized by carbon dioxide (CO2) inhalation and laparotomised.
Punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance.
Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. The lymph nodes were then stored on ice in PBS /0.5% BSA and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.

Test formulation analysis was carried out non-GLP in 2 steps:
1) The analytical method was validated by LPT before in vivo testing. The following parameters were determined:
- Linearity, Accuracy, Precision, Sensitivity, Specificity and Stability (6 hours at room temperature)
2) 3 Samples of approximately 5 mL were taken during the in-life phase from the prepared formulations of 5%, 10%, and 25% (w/w). The samples were stored at ≤ 20°C until analysis for concentration.

Observations of the animals daily for clinical signs, local irritation or systemic toxicity and body weight (day 1 and day 4) were included in the study.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

The acute inflammatory skin reaction (irritating potential) was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control.

Positive control results:

The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid.(see Table 2)

Key result

Parameter:

SI

Value:

2.279

Test group / Remarks:

5% test item in A/O: the stimulation index (SI) for lymph node cell count is above the threshold level of 1.4

Remarks on result:

other: indication of skin sensitization but also of skin irritation

Key result

Parameter:

SI

Value:

2.99

Test group / Remarks:

10% test item in A/O: the stimulation index (SI) for lymph node cell count is above the threshold level of 1.4

Remarks on result:

other: indication of skin sensitization but also of skin irritation

Key result

Parameter:

SI

Value:

3.51

Test group / Remarks:

25% test item in A/O: the stimulation index (SI) for lymph node cell count is above the threshold level of 1.4

Remarks on result:

other:

Remarks:

indication of skin sensitization but also of skin irritation

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
In the main study treatment with Oleylamine oleate at concentrations of 5%, 10% and 25% (w/w) did reveal statistical significantly increased values for the lymph node cell count and lymph node weight. The stimulation index for the lymph node cell count exceeded the threshold level of 1.4. (see Table 2)

As the stimulation indices of ear weight exceeded the threshold level of 1.1 markedly at all concentrations, the test item was considered to have irritating properties in this concentration range in this test system. (see Table 2)

The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid. (see Tabel 2)

DETAILS ON STIMULATION INDEX CALCULATION
The study was performed according to OECD 429. However, an alternative method was used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes. In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.
Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the respective vehicle treated ones.
Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).

EC 1.4 CALCULATION
The so-called stimulation (or LLN-) indices to determine the sensitising potential (this value was fixed empirically during the interlaboratory validation of this method, for details see Ehling et al. 2005a and 2005b, page 12), were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated animals by the vehicle treated ones. No values were above the threshold of 1.4 (lymph node cell count) or 1.1 (ear weight). Thus, under the present test conditions, the test item at concentrations of 5%, 10% and 25% (w/w) in acetone/olive oil (4:1, v/v) did reveal skin irritating and sensitising properties in the local lymph node assay. A 25% (w/w) concentration of the test item in acetone/olive oil (4:1, v/v) was the maximum feasible concentration.

DIFFERENTIATION INDICES (DI)
Based on the DI values (DI > 1), the test item Oleylamine Oleate is classified to be sensitizing. (see Table 2)

CLINICAL OBSERVATIONS and BODY WEIGHTS
No signs of local or systemic intolerance were recorded. The mean animal body weight of the test item-dosed animals was decreased in a dose-related way.

In a preliminary experiment, concentrations of 5%, 10% and 25% (w/w) of Oleylamine oleate, employing 1 animal per concentration, were examined. No clear irritating properties were observed in this preliminary experiment at concentrations of 5%, 10% and 25% (w/w), but there was a trend for an increase in ear weight. A 25% (w/w) concentration was the highest feasible concentration of the test item in acetone/olive oil (4:1, v/v).

Table 2: Stimulation indices (SI) in the main experiment (mean of 6 animals per group):

Parameter	negative control (A/O)	5% (w/w) test item in A/O	10% (w/w) test item in A/O	25% (w/w) test item in A/O	positive control (20% alpha-hexyl cinnamic aldehyde (v/v) in A/O)
Lymph node cell count	1.000	2.279*	2.990*	3.510*	1.907*
Lymph node weight	1.000	1.935*	2.717*	2.870*	1.891*
Ear weight	1.000	1.101*	1.286*	1.321*	1.107*
Ear thickness	1.000	1.012	1.131	1.193	1.119
Lymph node cell count index#		32	50	63
Ear swelling (% of maximum)		9	27	29
Differentiation index (DI)		4	2	2

* significantly different from control at p ≤ 0.01

# according to Homey et al. (1989) An integrated model for the differentiation of chemical-induced allergic and irritant skin reactions; Toxicology and Applied Pharmacology 153: 83-94

The analysis of the test item vehicle suspensions of the 5%, 10% and 25% (w/w) concentrations for the actual test item levels was carried out (non-GLP) under conditions employing a validated analytical method. The analysis resulted in actual levels of 101.9% (5% (w/w) concentration), 99.5% (10% (w/w) concentration) and 101.9% (25% w/w) concentration) of the nominal concentration.

Interpretation of results:

GHS criteria not met

Executive summary:

The purpose of this study was to determine the skin sensitising potential of Oleylamine oleate in the modified local lymph node assay in mice. The study was performed according to OECD 429. However, an alternative method was used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes. In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.

Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the respective vehicle treated ones.

Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).

Thee concentrations of Oleylamine oleate (5%, 10% and 25% (w/w)), suspended in acetone/olive oil (4:1, v/v) were tested in six female NMRI mice per group and compared to a vehicle control group. Acetone/olive oil (A/O, 4:1, v/v) was selected as it provided a suitable suspension of the test item both for administration and adherence to the mouse ear. A 25% (w/w) concentration was the highest feasible concentration of the test item in A/O. N,N-dimethylformamide, propylene glycol and dimethyl sulfoxide, other recommended vehicles, did not provide higher concentrated suitable suspensions. Methyl ethyl ketone was not applicable for analytics.

In the main study treatment at concentrations of 5%, 10% and 25% (w/w) did reveal statistical significantly increased values for the lymph node cell count and lymph node weight. The stimulation index for the lymph node cell count exceeded the threshold level of 1.4. The threshold level for the ear weight of 1.1 was also exceeded and an increase of ear thickness was observed, i.e. skin irritating properties were noted. The calculated Differentiation Index, used to discriminate sensitizing from irritating properties, was above 1 at all doses, thus, the test item is shown to be positive in this LLNA. The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid. No signs of local or systemic intolerance were recorded. The mean animal body weight of the test item-dosed animals was decreased in a dose-related manner.

In conclusion, under the present test conditions, Oleylamine oleate at concentrations of 5%, 10% and 25% (w/w) in acetone/olive oil (4:1, v/v) showed sensitising and irritating properties in this test system as evident from the increased lymph node cell count and ear weight and was judged to be positive in the LLNA. A 25% (w/w) concentration of the test item in acetone/olive oil (4:1, v/v) was the maximum feasible concentration.

Endpoint conclusion

Additional information:

No human data are available for the substance to assess the endpoint skin sensitization.

An assessment of potential skin sensitization properties based on defined approaches and individual sources was conducted for Oleylamine oleate within an IATA. The outcome of this assessment is given below:

Oleylamine oleate is a brown liquid with a molecular weight of 269 g/mole for the oleylamine moiety and about 281 g/mole for the carboxylates (mixture of about 70% oleic acid and other fatty acids). The substance has a low water solubility of 0.6. mg/L and the log Kow (octanol/water) was calculated to be above + 6.5. Based on these physico-chemical properties dermal uptake of the substance can be predicted to be low (ECHA Guidance on Information Requirements, Chapter R7c, 2017).

Oleylamine oleate was investigated in silico for structural activity relationship (QSAR), in chemico for peptide reactivity, and in vitro for keratinocyte activation (KeratinoSens). Due to positive results in vitro in vivo testing was performed in the LLNA as last option to assess the endpoint skin sensitization.

OECD Toolbox 4.0:

The structure activity relationship of the substance was investigated by using the OECD Toolbox 4.0 (released 2017). No protein binding alerts for skin sensitization were identified in silico by the OECD QSAR Toolbox (Schlecker, 2017). No activity in the Direct Peptide Reactivity Assay (DPRA) with regard to cysteine and lysine peptide depletion and no h-CLAT activity were predicted by the Toolbox. With regard to keratinocyte gene expression the substance is out of mechanistic domain.

DPRA; OECD TG 442C:

The Direct Peptide Reactivity Assay (DPRA; OECD 442C) is an in chemico procedure proposed to address the molecular initiating event leading to skin sensitization, namely protein reactivity, by quantifying the reactivity of test chemicals towards model synthetic peptides containing either lysine or cysteine. Cysteine and lysine percent peptide depletion values are then calculated and used in a prediction model to categorize a substance in one of four classes of reactivity for supporting the discrimination between skin sensitizers and non-sensitizers.

Oleylamine oleate showed a low depletion of peptides in the DPRA with a cysteine/lysine depletion of 8.59% (Schaub M, 2018). The obtained value counts for a DPRA prediction of ‘low reactivity – positive’. However, the observed low depletion value is close to a negative result (≤ 6.38%) and thus does not indicate a relevant effect taking also into account the above mentioned QSAR Toolbox profiler for DPRA Cys or Lys binding potency (“not reactive”; within applicability domain).

KeratinoSens™; OECD TG 442D:

The objective of the study was to investigate the potential of the test item to induce genes that are regulated by the antioxidant response element (ARE). The ARE-Nrf2 Luciferase Test Method (KeratinoSens) was performed acording to OECD 442D.

The test article was dissolved in isopropanol to the final concentration of the stock solution (200 mM) with further dilution in isopropanol. The assay acceptance criteria for the positive control cinnamic aldehyde and the negative control isopropanol were met in this test.

None of the four criteria specified in the prediction model for a positive prediction (Imax > 1.5-fold, cell viability > 70%, EC1.5 value < 1000 μM, dose response) were met in Experiment 1 and 2. The test item Oleylamine oleate was thus considered to be negative in the Luciferase Test ARE-Nrf2 cells derived from HaCaT human keratinocytes (Dreher D, 2018).

h-CLAT; OECD TG 442E:

The human Cell Line Activation Test (h-CLAT) is an in vitro skin sensitization test following OECD TG 442E (In Vitro Skin Sensitization: human Cell Line Activation Test (h-CLAT), adopted July 2016).  This test addresses the third key event of skin sensitization, the activation of dendritic cells in culture.

According to OECD TG 442E test chemicals with a Log Kow greater than 3.5 tend to produce false negative results and negative results with test chemicals with such high Log Kow should not be considered. Based on the results of the DPRA (low, close to no reactivity) and the KeratinoSens (negative) described above for oleylamine oleate a negative result can be expected for the substance also in the h-CLAT. Since a Log Kow of > 6.5 was determined for Oleylamine oleate the h-CLAT method is not applicable to this substance and the test was thus not performed.

In vivo testing in the mouse LLNA; OECD TG 429:

Based on inconsistent results in chemico (DPRA weak positive) and in vitro (KeratinoSens negative) in vivo testing was initiated to gain information on potential in vivo skin sensitization activities. No information on skin sensitization potency can be derived from skin sensitization hazard reported in the in vitro studies.

Oleylamine oleate was tested in the modified local lymph node assay (LLNA) in mice. The study was performed according to OECD 429. An alternative method was used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes. In addition, the acute inflammatory skin reaction is measured by ear weight and ear thickness measurements to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.

Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the respective vehicle treated ones. Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).

Three concentrations of Oleylamine oleate (5%, 10% and 25% (w/w)), dissolved in acetone/olive (A/O) oil (4:1, v/v) were tested in six female NMRI mice per group and compared to a vehicle control group. A/O was selected as it provided a suitable suspension of the test item both for administration and adherence to the mouse ear. A 25% (w/w) concentration was the highest feasible concentration in A/O.

The study revealed a dose-dependent and statistically significant increase in lymph node cell count and lymph node weight. The stimulation index for the lymph node cell count exceeded the threshold level of 1.4. The threshold level for the ear weight of 1.1 was also exceeded and an increase of ear thickness was observed, i.e. skin irritating properties were noted. The calculated Differentiation Index, used to discriminate sensitizing from irritating properties, was above 1 at all doses, thus, the test item is shown to be positive in this LLNA. No signs of local or systemic intolerance were recorded. However, the mean animal body weight of the test item-dosed animals was decreased in a dose-related manner.

In conclusion, under the present test conditions, Oleylamine oleate showed sensitising and irritating properties in this test system as evident from the increased lymph node cell count and ear weight and was judged to be positive in the LLNA.

Weight of Evidence Assessment

Oleylamine oleate is used as additive for technical lubricants. It consists of two moieties, the oleylamine and the carboxylate (mixture of about 70% oleic acid and other fatty acids). No human data are available for the substance to assess the endpoint skin sensitization. Based on the physico-chemical properties of the substance (very low water solubility, high log Kow) dermal absorption can be expected to be low. However, a skin irritating potential was recorded in an in vitro test on reconstructed human skin, leading to a classification with Skin Irrit 2 (H315) that could theoretically enhance skin penetration.

No alert for protein binding was obtained in silico (OECD Toolbox). A weak positive, close to negative, result in the DPRA (in chemico testing) and a negative result in vitro (KeratinoSens negative) was obtained. Thus, in vivo testing (mouse LLNA) was initiated to gain information on potential in vivo skin sensitization activities.

Under the test conditions of the LLNA, Oleylamine oleate at concentrations of 5%, 10% and 25% in acetone/olive oil (A/O) showed sensitising and irritating properties, as evident from a dose-dependent and statistically significant increase in lymph node cell counts and ear weights. The calculated Differentiation Index, used to discrimiate sensitizing from irritating properties, was above 1 at all doses. The test substance was thus judged to be a skin sensitizer in the LLNA.

Assessment of the fatty acid moiety:

The reliability and relevance of this positive LLNA result is, however, questionable for the following reasons. The carboxylate moiety of Oleylamine oleate mainly consists of the unsaturated oleic acid (CAS-No. 112-80-1). Oleic acid is the most widely distributed and abundant fatty acid in nature. It is used commercially in the preparation of cosmetics and as pharmaceutical solvent without any indication of skin sensitizing properties. For oleic acid no skin sensitization potential was found when tested in a total of about 600 subjects in several human repeat insult patch tests using cosmetic formulations containing between 2% and 6% oleic acid (CIR Cosmetic Ingredient Review Panel; Journal of the American College of Toxicology 6, 1987). It was shown by Kreiling et al. (Food and Chemical Toxicology 46, 1896-1904, 2008) that oleic acid gives a false-positive result in the LLNA. At doses of 25 and 50% oleic acid in A/O stimulation indices of 14.9 and 6.9 over control level were recorded, respectively. Thus, the threshold of 3 in this LLNA was clearly exceeded. In the Guinea Pig Maximization Test (GPMT) oleic acid was shown, however, to be no skin sensitizer (Kreiling et al., 2008). Testing of saturated versus unsaturated fatty acids (e.g. stearic versus oleic acid) revealed different findings in the LLNA indicating that carbon-carbon double-bonds as ‘structural/functional element’ may have an influence on the LLNA response. Several other chemicals having double-bonds in their structures showed also conflicting results. The authors postulate that diverse cell proliferative and inflammatory processes, e.g. modulation of cell-mediated immune reactions, morphological alteration of keratinocytes and epidermal Langerhans cells and enhancement of cytokine production, may trigger the positive outcome of the LLNA.

Assessment of the oleylamine moiety:

Oleylamine (CAS-No. 112-90-3; (Z)-octadec-9-enylamine) is classified as corrosive to skin. No reliable information is available with respect to skin sensitization for oleylamine or other primary alkyl amines. In a not fully reliable GPMT performed with coco alkyl amine borderline effects were observed.

In summary, the performed tests and assessments do not indicate a substantial skin sensitizing activity of Oleylamine oleate. The substance is thus considered to be not classified for skin sensitization.

Respiratory sensitisation

Endpoint conclusion

Additional information:

no study available

Justification for classification or non-classification

No alert for skin sensitization was obtained in silico (OECD Toolbox). A weak positive, close to negative result in the DPRA (in chemico testing) and a negative result in vitro (KeratinoSens negative) confirmed this assessment. The positive result obtained in the mouse LLNA is considered to be not reliable, due to false-positive reactivity of the fatty acid moiety oleic acid in the LLNA. Furthermore, the physico-chemical properties of Oleylamine oleat will limit skin penetration. In summary, the performed tests and assessments do not indicate a substantial skin sensitizing activity of Oleylamine oleate. The substance is thus considered to be not classified for skin sensitization.