Phenethyl isobutyrate

Endpoint summary

Description of key information

Based on the available in vitro tests (DPRA and LuSens) the test item is not considered as skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015-06-18 to 2015-12-17

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:

2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In chemico test system)

Selection of concentrations:
The C-containing peptide was incubated with the test substance in a ration of 1:10 (0.5 mM peptide, 5mM test substance) and the K-containing peptide in a ration of 1:50 (0.5 mM peptide, 25 mM test substance).

Experimental procedure:
Test substance was formulated in a suitable vehicle (100 mM, in acetonitrile). Three samples of the test substance were incubated with each peptide for ca. 24 h at 25 °C. Additionally, triplicates of the concurrent vehicle control were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition, calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.
Prior to the assay the solubility of the test substance was determined.

HPLC conditions:
Device: Liquid chromatograph Agilent HP 1100 with DAD
Column: Phenomenex Luna 3µ C18 (2), 100 mm x 2mm with guard column “Security guard” C18, 4mm x 2 mm
Mobile phase A: 0.1 % (v/v) triflouracetic acid (99+%) in de-ionized water, HPLC grade
Mobile phase B: 0.085 % (v/v) triflouracetic acid (99+%) in acetonitrile, HPLC grade

Negative control: Acetonitrile
Positive control: p-Benzoquinone

The mean peptide depletion of a test substance was calculated as the mean value of C-containing peptide depletion and K-containing peptide depletion.

According to the classification tree model described by Gerberick et al. for substances with known molecular weight highly reactive test substance (mean peptide depletion > 42.47 %) is predicted to be a strong sensitizer, a moderately reactive test substance (22.62 % < mean peptide depletion < 42.47 %) a moderate sensitizer, a test substance of low reactivity (6.38 % < mean peptide depletion < 22.62 %) a weak sensitizer.

Positive control substance(s):

yes

Remarks:

Ethylene glycol dimethacrylate

Positive control results:

The positive control substance caused a mean peptide depletion of 59.77 %.

Key result

Parameter:

other: mean Cystein-peptide depletion in %

Value:

-1.02

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Parameter:

other: mean Lysine-peptide depletion in %

Value:

-1.19

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY: In-house validation was performed prior to guideline adoption (Bauch et al., 2012; Urbisch et al., 2015)

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Due to the insolubility of the test substance in the Lysine-peptide samples calculation of mean peptide depletion is not applicable for the test substance.

No co-elution of test substance and peptides was noticed. Complete depletion was caused by the positive control substance.

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance shows a minimal chemical reactivity in the DPRA under the test conditions chosen and the test result was therefore negative.

Executive summary:

The reactivity of the test substance towards synthetic cysteine or lysine-containing peptides was evaluated in the DPRA assay according to OECD guideline 442C. The test substance was incubated with synthetic peptides for ca. 24 hours at 25 °C and the remaining non-depleted peptide concentrations were determined by HPLC. 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 (Cysteine) or 1:50 (Lysine). The following results were obtained: The mean Cysteine-peptide depletion caused by the test substance was -1.02 %, the mean Lysine-peptide depletion caused by the test substance was -1.19 %. Due to the insolubility of the test substance in the Lysine-peptide samples calculation of mean peptide depletion is not applicable and the Cysteine 1:10 prediction model is used for evaluation. It was concluded that the test substance shows a minimal chemical reactivity under the test conditions chosen.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2015-06-18 to 2015-12-17

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:

2015

Deviations:

no

Principles of method if other than guideline:

Currently, the only in vitro ARE-Nrf2 luciferase test method covered by the OECD Test Guideline is the KeratinoSensTM test 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). This study used the LuSens cell line and protocol, which is in the process of being included as a "me too" assay.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

TEST SYSTEM:
- Source: LuSens cell line prepared in collaboration with RWTH Aachen

TEST PROCEDURE:
In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to nine concentrations of the test substance and cytotoxicity was determined by a MTT assay.

The test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 h at 37 °C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. The highest tested concentration in the main experiment was 1.22 fold of the concentration affording a viability of 75% (CV75). The additional concentrations were obtained by a 1:1exp2 dilution series of the CV75. Cytotoxicity was determined via the MTT assay.

Two independent experiments were performed. In each experiment, three duplicates of each treatment were tested.

Negative control (NC): D,L-Lactic acid (0.450 mg/mL)
Vehicle control: 1% DMSO in culture medium
Positive control: Ethylene glycol dimethacrylate (EGDMA, 18 µg/mL in 1 % DMSO in culture medium)

Acceptance criteria:
A tested concentration was not further evaluated when relative viability is less than 70%.
The cell viability of untreated cells must yield at least 90%. The positive control should be ~3 fold induction and lactic acid <2 and viability ~70%. The average standard of the variability in the vehicle control wells for each plate should be below 20%. A study is considered acceptable if the positive and negative and vehicle control data lies within the range of the historical data.

Evaluation criteria:
A test substance is concluded to exhibit an keratinocyte activating potential when the luciferase activity exceeds 2.0 fold induction with respect to the vehicle control and at concentrations that do not reduce a viability below 70%.

Positive control results:

The positive control substance led to a mean fold induction of luciferase activity of 7.5 and a relative viability of 120.6 %. Fold induction of 1.50 and a relative viability of >70 % are considered as a positve result.

Key result

Run / experiment:

other: Experiment 1 test item

Parameter:

other: fold induction of luciferase activity

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: for all tested concentrations

Key result

Run / experiment:

other: Experiment 1 test item

Parameter:

other: % cell viability

Value:

73.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: for concentrations of 491 - 1467 µg/mL

Key result

Run / experiment:

other: Experiment 1 test item

Parameter:

other: % cell viability

Value:

9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: concentration 1760 µg/mL

Key result

Run / experiment:

other: Experiment 2 test item

Parameter:

other: fold induction of luciferase activity

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: for concentrations of 491 -1222 µg/mL

Key result

Run / experiment:

other: Experiment 2 test item

Parameter:

other: fold induction of luciferase activity

Value:

1.57

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: concentrations of 1467 - 1760 µg/mL

Key result

Run / experiment:

other: Experiment 2 test item

Parameter:

other: % cell viability

Value:

92.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: for all tested concentrations

Key result

Run / experiment:

other: Experiment 3 test item

Parameter:

other: fold induction of luciferase activity

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: for all tested concentrations

Key result

Run / experiment:

other: Experiment 3 test item

Parameter:

other: % cell viability

Value:

71.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: for concentrations of 707 - 1222 µg/mL

Key result

Run / experiment:

other: Experiment 3 test item

Parameter:

other: % cell viability

Value:

0.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: for concentrations of 1467 - 2002 µg/mL

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none

DEMONSTRATION OF TECHNICAL PROFICIENCY: In-house validation was performed prior to guideline adoption (Bauch et al., 2012; Urbisch et al., 2015)

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Interpretation of results:

GHS criteria not met

Conclusions:

The test substance does not have a keratinocyte activating potential.

Executive summary:

The keratinocyte activating potential of the test item was evaluated in the LuSens assay. The test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 h 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-test was performed. Cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined by MTT assay. A total of 3 valid experiments (main test) were performed. The following results were observed: After 48 hours of exposure to the test substance luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70 % viability in two independent experiments. Therefore, it was concluded that the test item does not have a keratinocyte activating potential.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In vitro studies covering 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) were performed. These study types have initially undergone in-house validation in the test facility using 54 substances (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504). Recently, a validation performed with 213 substances has been published (Urbisch et al. 2015 Regul Toxicol Pharmacol. 71: 337-351).

 

Based on the results of the in-house validation (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504) 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. This was confirmed in the validation study with more substances (Urbisch 2015): 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 90%, a specificity of 90 % and an accuracy of 90 %.

 

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 (Bauchet 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-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 DPRA and the keratinocyte activation assay follow the procedures of the respective OECD testing guidelines (OECD 442C and D). An OECD testing guideline for the dentritic cell activation assay is under preparation.

 

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 and does not activate keratinocytes.

In accordance with the published evaluation scheme (Bauchet 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 (EC) No 1272/2008. Based on available data on skin sensitization the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the eighth time in Regulation (EU) No 2016/918.