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Description of key information

LuSens:

The test item, 4-ethyl-2-(8-heptadecenyl)- 2-oxazoline-4-methanol, was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential).

DPRA test:

Determination of the skin sensitisation potential of 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol using the direct peptide reactivity assay according to OECD 442C showed a “negative” DPRA prediction with minimal reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model. As the lysine peptide samples were turbid in all experiments, the negative result is to be treated with caution according to the guideline. This is considered reasonable, because the evaluation using the Cysteine 1:10 prediction model alone also results in the prediction “negative”.

h-CLAT:

study technically not feasible

LLNA Assay:

4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol tested at the maximum feasible (non-toxic, non-irritant) concentration of 10% (w/v) and also at concentrations of 5 %, 2.5 % or 1 % (w/v) was shown to have skin sensitization potential in the Local Lymph Node Assay in mice.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
21. Dec. 2017-17. Jul. 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
This in vitro test method was conducted to investigate skin sensitisation potential of the test item. The information needed for the classification or risk assessment of a substance has to be obtained through non-animal methods as a first step.
Non-animal methods are the default requirement.
Details on study design:
A Cytotoxicity Range Finder Test (CRFT) was performed in order to determine the concentration range applicable for the main experiments. In the CRFT cytotoxicity was determined by measuring the cell viability with MTT. A reduction of the viability below 70 % is defined as a cytotoxic effect.
In the case of a cytotoxic result, the concentrations for main experiments (I and II) should be determined so that at least one of them is in the cytotoxic range. Experiment II serves only to confirm the results of experiment I.

Determination of solubility in DMSO in non-GLP pretest was sufficiently.
Negative Control: DL-Lactic acid, Final concentration: 5000 μM
Positive Control: EGDMA (Ethylene glycol dimethylacrylate), Final concentration: 120 μM
Solvent Control: DMSO, Final concentration: 1 %

In the CRFT the following 12 nominal concentrations of the test item were tested:
0.98 μM, 1.95 μM, 3.91 μM, 7.81 μM, 15.63 μM, 31.25 μM, 62.5 μM, 125 μM, 250 μM, 500
μM, 1000 μM, 2000 μM
-exposure time: 48 h

Since a cytotoxic reaction was observed in the CRFT the following 12 nominal concentrations
were chosen for experiment I and II:
4.21 μM, 5.05 μM, 6.06 μM, 7.27 μM, 8.72 μM, 10.47 μM, 12.56 μM, 15.07 μM, 18.08 μM,
21.70 μM, 26.04 μM, 31.25 μM

At the time of seeding the cells were 80 % confluent. The cells were washed twice with PBS (without Ca2+/Mg2+) containing 0.05% EDTA. Afterwards the cells were trypsinized until the cells detached. To stop this reaction, medium no. 2 was added. After centrifugation (5 min at 380 * g), the supernatant was discarded and the cells were resuspended in medium no. 2. After quantification, the cell suspension was adjusted to 83 000 (±10 %) cells per mL. 120 μL of the cell suspension (≙ 10 000 cells) were seeded in two clear flat bottom 96 well plates (one for viability and one for luciferase induction measurement).
Both plates were incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere for 23 h and 45 min in Experiment I and 25 h and 30 min Experiment II.

The treatment procedure was performed on both 96 well plates identically:
After the incubation time the medium was removed from the cells and 150 μL medium no. 3 were added to each well. Afterwards 50 μL of each single test item concentration and the controls were added to the cells in triplicates (test item concentrations). 24 wells were used for solvent control, 12 wells were used for growth control (cells + medium no. 3), 6 wells were used for negative control, 5 wells for positive control and 1 well for blank. The plates were sealed with breathable tape to avoid evaporation of volatile compounds and to avoid cross contamination between wells. Afterwards the plates were incubated for 48 h at 37 ± 1 °C in a humidified atmosphere containing 5.0 ± 0.5 % CO2.

For the evaluation of the viability, one of the plates was used:
The MTT working solution was prepared by mixing 9 parts of medium no. 3 with 1 part of MTT solution. All solutions were removed from the wells of the 96 well plate and 200 μL MTT working solution were added to each well. The plate was incubated for 2 h at 37 ±1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere. Afterwards the solution was removed and 100 μL of lysis buffer were added to each well. The plate was agitated for 5 min before it was measured at 570 nm and at 690 nm (reference) at the photometer. The cell viability is measured by the reduction of the tetrazolium dye MTT (3-(4,5-Dimethyl thiazole 2-yl)-2,5-diphenyltetrazolium-bromide) (yellow colour) to its insoluble formazan (purple colour) in living cells and therefore indicates the amount of living cells. After the measurement of the color change, the values were transferred in a validated spreadsheet for the calculation of the viability.

For the evaluation of the Luciferase induction, the second plate was used:
For the evaluation of the Luciferase expression all solutions were removed from the wells and the cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Afterwards 100 μL per well of a Lysis buffer were added to the cells and incubated for 5 min at room temperature. During this process, the plate was slightly moved. Afterwards 100 μL Steady-Glo® Reagent were added to each well and the plate was shaken again slowly for 5 min at room temperature. Then, 160 μL per well were transferred to a white flat bottom 96 well plate and the luminescence was measured for 2 seconds using a luminometer. The luciferase induction and the relative viability were calculated.
Positive control results:
The positive control induced a clear effect with an luciferase induction value of 7.1 fold (in experiment I) and 6.0 fold (in experiment II) in comparison to the solvent control.
Key result
Parameter:
other: Luciferase induction
Run / experiment:
Experiment I and II
Value:
>= 1.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid

In experiment I cytotoxic effects were observed at the concentrations 10.47 μM to 31.25 μM. In experiment II, cytotoxic effects were observed at the concentrations 8.72 μM to 31.25 μM. Those concentrations were excluded from the evaluation of the luciferase induction.

Finally the following test item concentrations showed a viability ≥ 70 % and could therefore be evaluated for luciferase induction:

Experiment I: 4.21 μM, 5.05 μM, 6.06 μM, 7.27 μM, 8.72 μM

Experiment II: 4.21 μM, 5.05 μM, 6.06 μM, 7.27 μM

Interpretation of results:
other: Positive result in the LuSens assay. The test item is therefore considered to have the potential to activate the Nrf2 transcription factor.
Conclusions:
Under the experimental conditions of this study, the test item, 4-ethyl-2-(8-heptadecenyl)- 2-oxazoline-4-methanol, was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential).
Executive summary:

This in vitro study evaluates the potential of the test item 4-ethyl-2-(8-heptadecenyl)-2- oxazoline-4-methanol to activate the Nrf2 transcription factor (sensitizing potential) by using the LuSens cell line. Since July 2017 a reviewed version of the OECD 442D is available, which includes the LuSens test. Therefore this study is performed in accordance to this draft OECD 442D with the title “In Vitro Skin Sensitisation assays addressing the AOP Key Event on: Keratinocyte activation”.

The assay included a cytotoxicity range finder test (CRFT) and two independent experiments (experiment I and II) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the two experiments were determined.

In the experiments, the highest nominal applied concentration (31.25 μM) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.2) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the experiments.

DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as growth control. Lactic acid (5000 μM) was used as negative control and EGDMA (120 μM) as positive control.

A statistically significant and reproducible dose dependent increase in luciferase induction ≥ 1.5 fold in more than two non-cytotoxic consecutive test item concentrations was observed in both experiments.

Under the experimental conditions of this study, the test item, 4-ethyl-2-(8-heptadecenyl)- 2-oxazoline-4-methanol, was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential).

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2018- November 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
An in vivo study was conducted because an experimental study according to OECD 442E was not applicable, and the results obtained from OECD 442D and OECD 442C studies were not conclusive for classification.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/Ca Ola Hsd mice
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90. Hungary
- Females: yes, nulliparous and non-pregnant
- Hygienic level at arrival: SPF
- Age at study initiation: 12 weeks old (at start of both Dose Range Finding tests-DRFs)
- Weight at study initiation: 19.4 – 21.5 g
- Diet (e.g. ad libitum): ad libitum, ssniff® Rat/Souris-Elevage E complete diet for rats and mice
- Water (e.g. ad libitum): ad libitum
- Acclimation period (Main Test): 7 days
- Acclimation period (DRF): 28 days prior to the first DRF, 21 days prior to the second DRF
- Indication of any skin lesions:
- Animal health: Only healthy animals (and not showing any sign of skin lesion) were used.

ENVIRONMENTAL CONDITIONS

Housing during acclimatization period: Grouped caging in small groups
Housing during the test: Grouped caging (4 animals/cage)
Cage type: Type II. polypropylene/polycarbonate
Bedding: Laboratory bedding
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 – 70 %
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
The test item (formulated in AOO) was tested at four test concentrations (10 %, 5 %, 2.5 % and 1 % w/v).
No. of animals per dose:
4 animals per dose (in total 24 animals*)
* Including control animals shared with the concurrent LLNAs. Number of shared animals: 8.
Details on study design:
The pooled treatment group approach was used in this test.
The maximum dose selection was performed according to the relevant guidelines and based on results of a formulation evaluation and two consecutive Dose Range Finding tests (DRFs).
The liquid test item was adequately miscible in Acetone : Olive oil 4:1 (v/v) mixture (AOO) or N,N-Dimethylformamide (DMF) (standard vehicles which are most preferred in the LLNA). Formulations prepared with both vehicles were evaluated in the DRFs to find the most adequate vehicle and the highest applicable concentration. Symptoms of adverse effects were observed in both vehicles at high test concentrations (irritation in AOO, irritation and systemic toxicity in DMF) in the DRFs. Based on the results AOO was more adequate vehicle than DMF and the highest applicable non-toxic, non-irritant concentration was 10 % (w/v) in AOO. According to this the test item was examined in the main test as 10 %, 5 %, 2.5 % and 1 % (w/v) formulations in AOO.
Appropriate positive control (α-Hexylcinnamaldehyde, HCA) and a negative control group dosed with the vehicle of both the positive control and the test item (AOO) were employed.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Significance of the dose-response was evaluated by linear regression using the calculated SI values. All calculations were made by Microsoft Excel Software.
Positive control results:
- Visually larger lymph nodes compared to the vehicle control (AOO)
- No mortality, cutaneous reactions or signs of toxicity
- calculated SI value: 20.9.
Key result
Parameter:
SI
Value:
> 3
Test group / Remarks:
1%
Key result
Parameter:
SI
Value:
> 3
Test group / Remarks:
2.5%
Key result
Parameter:
SI
Value:
> 3
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
> 3
Test group / Remarks:
10%
Cellular proliferation data / Observations:
Visually larger lymph nodes compared to the vehicle control (AOO) were observed in the positive control group and in the 10 % (w/v) dose group. Appearance of the lymph nodes was normal in the negative control group (AOO) and in the other test item treated groups (5 %, 2.5 % and 1 %, w/v). Significant lymphoproliferative response (SI ≥ 3) was observed for the test item at all tested concentrations.
The observed stimulation index values were 20.4, 12.0, 4.6 and 4.1 at test item concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. Significance of the dose-response was evaluated by linear regression using the SI values. Significant dose-response correlation was observed (p = 0.01, r = 0.99).
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol tested at the maximum feasible (non-toxic, non-irritant) concentration of 10% (w/v) and also at concentrations of 5 %, 2.5 % or 1 % (w/v) as formulations in a suitable vehicle (Acetone : Olive oil 4:1 (v/v) mixture, AOO) was shown to have skin sensitization potential in the Local Lymph Node Assay in mice.
Executive summary:

The aim of this study was to determine the skin sensitization potential of 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol following dermal exposure in the Local Lymph Node Assay in mice. The pooled treatment group approach was used in this test.

The maximum dose selection was performed according to the relevant guidelines and based on results of a formulation evaluation and two consecutive Dose Range Finding tests (DRFs).

The liquid test item was adequately miscible in Acetone : Olive oil 4:1 (v/v) mixture (AOO) or N,N-Dimethylformamide (DMF). Formulations prepared with both vehicles were evaluated in the DRFs to find the most adequate vehicle and the highest applicable concentration. Symptoms of adverse effects were observed in both vehicles at high test concentrations (irritation in AOO, irritation and systemic toxicity in DMF) in the DRFs. Based on the results AOO was more adequate vehicle than DMF and the highest applicable non-toxic, non-irritant concentration was 10 % (w/v) in AOO. According to this the test item was examined in the main test as 10 %, 5 %, 2.5 % and 1 % (w/v) formulations in AOO.

Appropriate positive control (α-Hexylcinnamaldehyde, HCA) and a negative control group dosed with the vehicle of both the positive control and the test item (AOO) were employed.

The positive control item (25 % (w/v) HCA in AOO) induced significant stimulation over the relevant control (SI = 20.9). Thus, confirming the sensitivity and validity of the assay.

No mortality was observed during the main test. No significant, treatment related effect on body weights or any other sign of systemic toxicity were observed in any treatment group. No signs of irritation (monitored by erythema scoring) or any other local effect were observed at the treatment site (ears) in any treatment group.

Significantly increased lymphoproliferation (indicated by an SI ≥ 3) compared to the control was noted for 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol at the applied test concentrations. Significant dose-response correlation was observed. The observed stimulation index values were 20.4, 12.0, 4.6 and 4.1 at test item concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. Significant dose-response correlation was observed (p = 0.01, r = 0.99, evaluated by linear regression using the SI values).

According to evaluation criteria of the relevant guidelines, the significantly increased lymphoproliferation (indicated by an SI ≥ 3) at the maximum feasible concentration of 10 % (w/v) and also at lower concentrations as well as the significant dose-response correlation are considered as evidence that 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol is a skin sensitizer.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Mar. 2018-Dec. 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
This in chemico test method was conducted to investigate skin sensitisation potential of the test item. The information needed for the classification or risk assessment of a substance has to be obtained through non-animal methods as a first step.
Non-animal methods are the default requirement.
Details on study design:
To quantify the sensitisation potential, the depletion of the cysteine and lysine containing peptides caused by known amounts of the test item was measured using HPLC.

The test item was found to be insoluble in acetonitrile, acetonitrile/water and water in pre-tests. Therefore, isopropanol was used as vehicle in the first experiment. As the Cys-peptide stability was affected by isopropanol (peptide concentrations of the reference controls C were below the acceptable range in two separate assays) acetone and acetone/acetonitrile 50/50 (% v/v) were additionally tested as vehicle.
Positive control results:
The mean peptide depletion and standard deviation of the three replicates of the positive control cinnamaldehyde were in the acceptable range of 60.8 – 100.0 % and ≤ 14.9 %, respectively, for the Cys-peptide.
The mean peptide depletion and standard deviation of the three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0 % and ≤ 11.6 %, respectively, for the Lys-peptide.
Parameter:
other: mean peptide depletion in the Lys-peptide and Cys-peptide
Run / experiment:
Batch 20180606, solvent acetone; Batch 20180606, solvent acetone/acetonitrile 5050 (% v/v)
Value:
0.92
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: mean peptide depletion in the Lys-peptide and Cys-peptide
Run / experiment:
Batch 20180412, solvent isopropanol; Batch 20180426, solvent isopropanol
Value:
0.52
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The test item was found to be insoluble in acetonitrile, acetonitrile/water and water in pre-tests. Therefore isopropanol was used as vehicle in the first experiments. As the Cys-peptide stability was affected by isopropanol (peptide concentration of the reference controls C were below the acceptable range in two separate assays) acetone and acetone/acetonitrile 50/50 (% v/v) were additionally tested as vehicle.
The test was performed three times for each peptide, during one experiment three solvents were used in parallel.
As two experiments for both peptides for which all acceptance criteria were fulfilled were available the test was considered valid.
The classification was based on peptide depletions based on Cys-peptide experiments using acetone and acetone/acetonitrile 50/50 (% v/v) as solvent and Lys-peptide experiments using isopropanol as solvent.
Interpretation of results:
other: The DPRA prediction is “negative” with minimal reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model.
Conclusions:
The DPRA prediction is “negative” with minimal reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model. As the lysine peptide samples were turbid in all experiments, the negative result is to be treated with caution according to the guideline. This is considered reasonable, because the evaluation using the Cysteine 1:10 prediction model alone also results in the prediction “negative”.
Executive summary:

The study was performed in order to evaluate the reactivity of the test item 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol towards cysteine (Cys-) and lysine (Lys-) containing peptides. A test item solution in Isopropanol, Acetone and Acetone/Acetonitrile (50/50) was incubated 24 ± 2 h at 25 °C together with cysteine and lysine peptides, respectively, and the peptide concentration after the incubation was measured using HPLC-UV.

Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured in parallel.

The DPRA prediction is “negative” with minimal reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model. As the lysine peptide samples were turbid in all experiments, the negative result is to be treated with caution according to the guideline. This is considered reasonable, because the evaluation using the Cysteine 1:10 prediction model alone also results in the prediction “negative”.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008.

4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol was shown to have skin sensitization potential in the Local Lymph Node Assay in mice.

As a result 4-ethyl-2-(8-heptadecenyl)-2-oxazoline-4-methanol is considered to be classified for skin sensitisation category 1 subcategory 1A, because a SI-Index of > 3 was observed in the 1 % test item concentration in the LLNA-Assay: May cause an allergic skin reaction (H317).