Etocrilene

Endpoint summary

Description of key information

Sensitization involves a number of key steps in order to take place, and can be described in terms of an adverse outcome pathway (AOP). These include reactivity with skin proteins (peptide reactivity), activation of skin cells (keratinocyte activation) and immune cells (dendritic cell activation). The studies carried out for this substance address these three key steps. The results are then used in a predefined evaluation scheme to determine hazard classification as a sensitizer by a weight-of-evidence approach.

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:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-compliant study, validated method

Qualifier:

according to guideline

Guideline:

other: Dentritic cell activation assay (MUSST) performed as described in Python F. et al (2007), Toxicol Appl. Pharmacol. 220(2), 113-24 and Bauch C. et al. (2011), Toxicology in Vitro 25, 1162 – 1168.

Principles of method if other than guideline:

Measurement of CD 86 expression as marker for stimulation of human dentritic cells (Myeloid U937 Skin Sensitization Test, MUSST)

GLP compliance:

yes

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

In accordance with Annex XI of EC legislation 1907/2006 in-vivo testing is not required if the endpoint is adequately covered by in-vitro testing.

Details on the study design:

Provider of U937 cells: German Resource Center for Biological Material (DSMZ, Germany, catalog no.: ACC 5).

Culture medium: RPMI 1640 (Gibco): with L-Glutamine, 25mM HEPES + 10% FBS + 1% Penicillin/Streptomycin

Vehicle: DMSO (final conc. 0.25%)

Culture conditions: 37°C, ca. 5% CO2, ≥ 90% humidity

Number of passages prior to testing: 5-13

For substance incubation, cells were seeded in 96-well microtiter plates (100 μL of 0.5x10exp6 cells/mL cell suspensions).


Treatment was performed by adding 100 μL of test-substance preparation to the cells, thus diluting the 2x concentrated test-substance preparations to their final concentration and the cells to 0.25x10exp6 cells/mL. After application the plates were sealed with semi-permeable plate
sealers in order to prevent evaporation of the test substance.
The plates were placed into the incubator under standard culture conditions for the exposure period of 48 hours.

Two independent experiments were performed. In each experiment, duplicates of each treatment were tested. If contradictory results were obtained in the first and second experiments, further experiments were conducted.

A visual inspection for test-substance precipitates was performed for each test-substance concentration prior to application. In addition, each well was inspected under a microscope after the exposure period of 48 hours in order to detect irregularities in cell morphology or test-substance precipitates.

After visual inspection the cells were transferred into V-shaped 96-well microtiter plates and centrifuged. Supernatants were discarded. The cells were washed once with PBS with 5% FBS at 4°C. Cells were resuspended in 100 μL PBS (with 5% FBS) and labeled for 30 min at 4°C in the dark with 5 μL IgG-FITC (isotype control) or 5 μL anti-CD86-FITC antibody.
Following incubation, cells were washed twice with PBS (with 5% FBS) and once with PBS and were then resuspended in 200 μL PBS. For cell viability analysis, cells were stained with propidium iodide (1.25 μg/mL final concentration in PBS) for 5 min at 4°C in darkness. Fluorescence intensity was analyzed using flow cytometry.

Positive control:

other: Ethylene diamine (70 μg/mL)

Positive control results:

Ethylene diamine gave the expected induction of CD86. For details, see table 3

Remarks on result:

other: CD86 value = 1

Outcome of the prediction model:

negative [in vitro/in chemico]

The test substance was tested in a concentration range of 0.88 to 28.00 µg/mL. No decrease in cell viability below 70% was observed. In both experiments no induction of the expression of CD 86 was observed. The test substance was not active in the MUSST assay. Precipitation occurred after 48 h at 28.0 µg/ml.

Interpretation of results:

other: no activation of dentritic cells in vitro

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: In-house validated study

Qualifier:

according to guideline

Guideline:

other: Keratinocyte activation assay (LuSens) as described in Bauch et al. (2012) Regul Toxicol Pharmacal, 63(3):489-504.

Principles of method if other than guideline:

The LuSens assay is an in vitro method for the identification of keratinocyte activating substances using the genetically modified keratinocytes (LuSens, Bauch et al. 2012). It employs the reporter gene for luciferase under the control of an antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor activity. The endpoint measurement is the up-regulation of the luciferase activity after 48 hour incubation with test substances. This up-regulation is an indicator for the activation of the Keap1/Nrf2/ARE signaling pathway (Ade et al. 2009, Natsch 2012, Natsch & Emter 2008, Vandebriel et al. 2010).

GLP compliance:

yes

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

In accordance with Annex XI of EC legislation 1907/2006 in-vivo testing is not required if the endpoint is adequately covered by in-vitro testing.

Details on the study design:

LuSens cells (derived from human keratinocyte cell line HaCaT)

The reporter gene cell line LuSens was prepared in collaboration with the RWTH Aachen University. This keratinocyte cell line derived from HaCaT cells carries a reporter gene for luciferase under the control of an antioxidant-response-element (ARE) and hence monitors Nrf2 transcription factor activity. The sequence of the ARE promoter originates from the NADPH:quinone oxidoreductase1 gene from rats.

LuSens cells are routinely cultured in complete DMEM culture medium with high glucose supplemented with 10% fetal bovine serum (FBS),100 U/mL penicillin – 100 ug/mL streptomycin and 0.5 ug/mL puromycin in T75 culture flasks.

The substance was diluted in DMSO (1% final concentration) and culture medium. ln the main test, test substance was used at six final concentrations determined with regard to the CV75 value: CV75 x 1.2^3 ,CV75 x 1.2^2 , CV75 x 1.2, CV75, CV75/1.2, CV75/1.2^2, CV75/1.2^3 and CV75/1.2^4. The CV75 is the estimated concentration that affords 75% cell viability and was determined to be 5.68 µg/ml for the test substance under the chosen exposure conditions on LuSens cells.

At least two independent experiments were performed. In each experiment, three duplicates of each treatment were tested.

Remarks on result:

other: luciferase activity = 1

After 48 hours of exposure to test substance luciferase activity in LuSens cells was induced at concentration between 3.94 µg/ml and 9.81 µg/ml affording at least 70% viability. From this it has to be concluded that test substance has an keratinocyte activating potential.

Concentration µg/ml	1stexperiment		2ndexperiment		3rd experiment
	fold induction	rel. viability	fold induction	rel. viability	fold induction	rel. viability
9.81	n.d.	n.d.	2.61	87.4	2.24	73.6
8.18	n.d.	n.d.	1.77	91.0	1.65	82.4
6.81	1.29	95.3	1.53	95.0	1.00	80.2
5.68	1.18	94.6	1.66	93.2	0.99	87.1
4.73	1.00	94.5	1.68	91.6	1.08	86.4
3.94	1.22	107.4	1.61	99.3	1.03	84.5
3.28	1.22	109.5	n.d.	n.d.	n.d.	n.d.
2.74	1.34	103.3	n.d.	n.d.	n.d.	n.d.
EGDMA	3.80	96.7	3.61	104.6	4.56	98.5
LA	0.96	108.0	1.25	108.7	1.06	107.3

Interpretation of results:

other: induction of antioxidant response genes in a keratinocyte cell line

Executive summary:

The keratinocyte activating potential of test substance was evaluated in the LuSens assay. For this purpose the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment a pre-tests were performed. In each pre-test cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined by MTT assay. In the main test luciferase activity was measured after 48 hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of three valid experiments were performed.

After 48 hours of exposure to test substance luciferase activity in LuSens cells was induced at concentration between 3.94 µg/ml and 9.81 µg/ml affording at least 70% viability. From this it has to be concluded that test substance has an keratinocyte activating potential.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study, in-house validated procedure

Qualifier:

according to guideline

Guideline:

other: Direct peptide binding assay (DPRA) performed as described in Bauch C. et al. (2011), Toxicology in Vitro 25, 1162 – 1168.

Principles of method if other than guideline:

In the DPRA the reactivity of a test item (=test substance) towards synthetic cysteine (C)- or lysine (K)-containing peptides is evaluated. For this purpose the test substance is incubated with synthetic peptides for 24 hours at room temperature and the remaining non-depleted peptide concentration is determined thereafter by high performance liquid chromatography with gradient elution and UV-detection at 220 nm. The peptide depletion of test-substance incubated samples is compared to the peptide depletion of the NC samples and expressed as relative peptide depletion.

GLP compliance:

yes

Type of study:

other: Direct peptide reactivity assay

Justification for non-LLNA method:

In accordance with Annex XI of EC legislation 1907/2006 in-vivo testing is not required if the endpoint is adequately covered by in-vitro testing.

Details on the study design:

synthetic peptides (Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH; Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH)

Positive control results:

The mean peptide depletion by the positive control was 94.8%.

Parameter:

other: Cysteine peptide depletion [%]

Value:

0.4 %

Parameter:

other: Lysine peptide depletion [%]

Value:

12.1 %

Key result

Parameter:

other: Mean peptide depletion [%]

Value:

6.2 %

The mean Cysteine-peptide depletion, caused by the test substance was determined to be 0.4 %. The mean Lysine-peptide depletion, caused by the test substance was determined to be 12.1 %. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 6.2 %.

No co-elution of test substance and peptides was noticed.

Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) it was concluded that the test article shows a minimal chemical reactivity in the DPRA under the test conditions chosen. Thus according to the classification tree model proposed by Gerberick et al. the test substance is predicted to be a non-sensitizer.

Interpretation of results:

other: minimal chemical reactivity

Executive summary:

The reactivity of the test article towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose the test substance was incubated with synthetic peptides for ca. 24 hours at room temperature and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was dissolved at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. Further, a co-elution control was assessed in order to detect possible interference of the test substance with the peptides.

The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 nm / 258 nm was calculated as a measure of peak purity. The following results were obtained in the DPRA: The test substance was solved in acetonitrile. However, when mixed with the peptide stock solutions the samples became opaque. Thus the samples were centrifuged prior to HPLC analysis. The mean C-peptide depletion, caused by the test substance was determined to be 0.4%. The mean K-peptide depletion, caused by the test substance was determined to be 12.1%. Negative depletions were considered to be “zero” for calculation of the mean peptide depletion, which was thus calculated to be 6.2%. No co-elution of test substance and peptides was noticed. Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) it was concluded that the test article shows a minimal chemical reactivity in the DPRA under the test conditions chosen.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Due to the complexity of the skin sensitization process a single in vitro assay is not sufficient to adequately assess this toxicological endpoint. Therefore, a combination of several methods addressing key steps of the sensitization process: protein reactivity (DPRA), activation of keratinocytes (LuSens or KeratinoSens) and activation of dendritic cells (MUSST or h-CLAT) were used (test battery).

These in vitro studies cover three key steps of the adverse outcome pathway for skin sensitization as identified by the OECD (OECD Publication No.168; ENV/JM/MONO(2012)10). These study types have undergone in-house validation using 54 substances (Bauch et al., 2012, Regul Toxicol Pharmacol. 63: 489-504). The methods for the dendritic cell activation is in the final stages of validation at ECVAM (http://ihcp.jrc.ec.europa.eu/our_labs/eurl-ecvam/validation-regulatory-acceptance/topical-toxicity/skin-sensitisation). The ECVAM opinion on the direct peptide binding assay has been published on Dec 13, 2013 and the opinion on the KeratinoSens™ assay has been published in Feb 2014. Based on the results of the in house validation (Bauch et al., 2012) the predictive capacity of the evaluation scheme using DPRA (peptide reactivity), LuSens/KeratinoSens™ (keratinocyte activation) and (m)MUSST/h-CLAT (dendritic cell activation) is comparable to that of the local lymph node assay. When compared to the sensitization potential of the substances in humans, the classification based on an evaluation scheme using results obtained from the DPRA, LuSens and mMUSST had a sensitivity of 93%, a specificity of 95% and an accuracy of 94%. This publication was also referenced by the OECD in the AOP guidance document. Furthermore, it is also in line with the recently published strategy by ECVAM (EURL ECVAM, JRC79446, doi:10.2788/84214; 2013).

In the evaluation scheme used here any two of the three test results determine the overall classification, i.e. any two positive test results drive the prediction of a sensitizer, while any two negative test results drive the prediction of a test substance to be a non-sensitizer (Bauch et al. 2012; Table 1). If two assays (DPRA ,LuSens or KeratinoSens, MUSST or h-CLAT) yield concordant results, the result of the third assay is not necessarily required to determine the overall outcome of the evaluation scheme.

 

Table 1: Decision matrix for combinations of DPRA, LuSens/KeratinoSens and MUSST/ h-CLAT assays.

DPRA	LuSens/ KeratinoSens^TM	MUSST/ h-CLAT	Test battery evaluation
positive	positive	positive	sensitizer
positive	positive	negative	sensitizer
positive	negative	positive	sensitizer
positive	negative	negative	non-sensitizer
negative	positive	positive	sensitizer
negative	positive	negative	non-sensitizer
negative	negative	positive	non-sensitizer
negative	negative	negative	non-sensitizer

Each individual assay was performed under GLP like conditions and the cell based assays LuSens and MUSST consisted of at least two independent experiments. Positive and negative controls were included in each experiment and confirmed the functionality and validity of the individual assays.

 

The test battery applicability is limited when testing substances insoluble in the commonly used vehicles and highly volatile substances. Substances susceptible to base-catalyzed hydrolysis cannot be evaluated reliably for binding to lysine as the incubation is performed at pH 10.2. Also substances that interfere with the experimental measurements (e.g., co-elution with the peptide in the DPRA-assay) are not or only partially suitable for testing using in vitro methods. The substance under evaluation did not have any of the above mentioned limitations and the outcome of this assay is therefore considered adequate for hazard identification for the endpoint of skin sensitization.

The test substance is not peptide reactive, activates keratinocytes and does not activate dendritic cells. In accordance with the published evaluation scheme (Bauch et al., 2012, Regul Toxicol Pharmacol. 63: 489-504) and Sections 1.2 and 1.4 of Annex XI of EC regulation 1907/2006, the test substance is judged not to be a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008 (CLP). As a result the substance is not considered to be classified for skin sensitization under Regulation (EC) No 1272/2008.