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Description of key information

The skin sensitizing potential of the test substance was assessed using an in vitro OECD guideline testing strategy comprising the following assays:

- Direct Peptide Reactivity Assay (DPRA),

- Keratinocyte Activation Assay (LuSens) and

- Dendritic Cell Line Acti vation Assay (h-CLAT).

The DPRA and LuSens assay were conducted under GLP according to the respective OECD guideline. The h-CLAT was not performed since both other assays showed no potential for skin sensitization.

The results were as follows:

- DPRA: negative

- LuSens: negative

- h-CLAT: not needed

In addition, a quantitative structure-activity relationship (QSAR) system for the estimation of the skin sensitization potency that incorporates skin metabolism and considers the potential of parent chemicals and/or their activated metabolites to react with skin proteins was used. The QSAR programs TIMES-SS predicted the substance including its simulated skin metabolites not to be a skin sensitizer. The substance fulfilled the criteria for the applicability domain of the QSAR model.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-2018
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))
Version / remarks:
adopted 04 February 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Specific details on test material used for the study:
The test item was homogeneous by visual inspection.
Storage conditions: Room temperature
Batch No.: VFH-2016-08
Details on study design:
The test substance was prepared at a 100 mM concentration in acetonitrile considering a purity/contents of 99.0% and its molecular weight. The C-containing peptide was incubated with
the test substance in a ratio of 1:10 (0.5 mM peptide, 5 mM test substance) and the K-containing peptide in a ratio of 1:50 (0.5 mM peptide, 25 mM test substance).

Three samples of the test substance were incubated with each peptide. Additionally, triplicates of the concurrent vehicle control (= NC) 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.

The samples were prepared in suitable tubes, capped tightly and incubated at 25°C ± 2.5°C in the dark for 24 +/- 2 hours. Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis. Unsolved samples were centrifuged or filtrated prior to injection into the HPLC in order to remove any unsolved particles. The HLPC analysis of the batch of samples started about 24 hours after sample preparation and the analysis time itself did not exceed 30 hours.

Negative control (NC): vehicle control = acetonitrile
Positive control (PC): Ethylene glycol dimethacrylate prepared as a 50 mM solution in acetonitrile.
Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide.
Positive control results:
67.6% (Cys-peptide) was depleted by the positive control substance ethylene glycol dimethacrylate using acetonitrile as solvent.
13.6% (Lys-peptide) was depleted by the positive control substance ethylene glycol dimethacrylate using acetonitrile as solvent.
Parameter:
other: Peptide depletion [%] Cys
Run / experiment:
mean
Value:
0.67
Vehicle controls validity:
valid
Positive controls validity:
valid
Parameter:
other: Peptide depletion [%] Lys
Run / experiment:
mean
Value:
0.62
Vehicle controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
other: inactive in the direct peptide binding assay
Executive summary:

The reactivity of the UVCB-substance 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 ca. 25°C 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 formulated 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-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= VC) were incubated with the peptides. Additionally, triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. Further, a co-elution control was performed 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 mean C-peptide depletion, caused by the test substance was determined to be 0.67%.

The mean K-peptide depletion, caused by the test substance was determined to be 0.62%.

The mean peptide depletion was calculated to be 0.64% and therefore the substance was determined to be inactive in the direct peptide binding assay.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
adopted February 2015
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Specific details on test material used for the study:
The test item was homogeneous by visual inspection.
Storage conditions: Room temperature
Batch No.: VFH-2016-08
Details on 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.


Two independent experiments were performed. In each experiment, three duplicates of each treatment were tested.
LuSens cells from the working cell bank were thawed and cultured using culture containing antibiotics, under standard culture conditions for at least 2 weeks at passage >5 but not longer than 15 passages prior to testing.


Prior to substance incubation, cells were seeded in 96-well microtiter plates (0.12 mL of 0.83 x 10exp5 cells/ml cell suspensions), using culture medium without antibiotics for incubation for 24 hours.

Treatment was initiated by replacing regular cell culture medium with medium containing the test substance and a reduced content of FBS (1%).

Substance incubation was performed under standard cell culture conditions for 48 h and luciferase activity then determined using SteadyGlo™ (Promega, Germany) according to manufacturer’s instructions.
Positive control results:
see table below
Parameter:
other: EC1.5
Run / experiment:
mean of two runs
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Calculation of the EC1.5 could not be performed since there was no dose-dependent increase. The substance was inactive in this assay.
Other effects / acceptance of results:
The substance was not cytotox up to limit concentrations.

Concentration (test substance) (µM] mean, 1st experiment
fold induction
mean, 1st experiment
% viability
t-test, p-value markers Concentration (test substance) (µM] mean, 2nd experiment
fold induction
mean, 2nd experiment
% viability
t-test, p-value markers
564 0.59 92 0.003 ** 564 0.92 101 0.103 n.s.
676 0.99 87 0.420 n.s. 676 0.99 99 0.450 n.s.
812 1.16 90 0.038 * 812 1.02 99 0.339 n.s.
974 1.22 89 0.148 n.s. 974 1.03 98 0.369 n.s.
1169 1.13 94 0.103 n.s. 1169 1.07 93 0.146 n.s.
1403 1.18 82 0.096 n.s. 1403 1.08 89 0.145 n.s.
1683 1.27 82 0.014 * 1683 1.06 91 0.201 n.s.
2020 1.40 77 0.019 * 2020 1.15 85 0.003 **
VC 1.00 100 - - VC 1.00 100 - -
EGDMA 90.8 µM 5.29 82 0.000 ** EGDMA 90.8 µM 3.79 83 0.000 **
LA 5000 µM 1.09 105 0.044 * LA 5000 µM 1.12 93 0.026 *
Interpretation of results:
other: inactive in the LuSens assay
Conclusions:
After 48 hours of exposure the luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent
experiments. From this it has to be concluded that test substance does not have a keratinocyte activating potential.
Executive summary:

The keratinocyte activating potential of test material 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 1st main experiment, pre-tests (non- GLP) were performed. Cells were exposed to several concentrations of the test-substance preparations and cytotoxicity was determined by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) of the test substance was determined by linear regression from the concentration response curve.

Based on this result, 8 concentrations between 564 μg/mL and 2020 μg/mL were chosen for the 1st and 2nd main experiment. Luciferase activity was measured after 48-hour exposure.

In parallel a MTT assay was performed to assess cytotoxicity of the test substance.

The following results were observed: At concentrations used in the main experiment the test substance was soluble in 4% DMSO and in 1% DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 hours.

The 1st and 2nd experiments were valid and showed 8 evaluable concentrations (relative viability ≥ 70%). No relevant keratinocyte induction and no cytotoxicity was obtained up to the highest (limit) concentration.

Endpoint:
skin sensitisation: in chemico
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE
OASIS TIMES v2.27.19.13

2. MODEL (incl. version number)
Skin sensitization with autoxidation; v. 21.26

3. SMILES IDENTIFIERS USED AS INPUT FOR THE MODEL
CC(=O)OCC(C)(C)COC(C)=O

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Defined endpoint: In vivo: skin sensitization
- Unambiguous algorithm: refer to QMRF
- Defined domain of applicability:
1. General parametric requirements - includes ranges of variation of log KOW and MW. It specifies in the domain only those chemicals that fall in the range of variation of the MW and log Kow defined on the bases of the correctly predicted training set chemicals. This layer of the domain is applied only on parent chemicals.
2. Structural domain - it is represented by list of atom - centered fragments extracted from the chemicals in the training set. The training chemicals were split into two subsets: chemicals correctly predicted by the model and incorrectly predicted
chemicals. These two subsets of chemicals were used to extract characteristics determining the "good" and "bad" space of the domain. Extracted characteristics were split into three categories: unique characteristics of correct and incorrect chemicals (presented only in one of the subsets) and fuzzy characteristics presented in both subsets of chemicals. Structural domain is applied on parent chemicals, only.
3. Mechanistic domain - in SS model it includes: Interpolation space: this stage of the applicability domain of the model holds only for chemicals for which an additional COREPA model is required. It estimates the position of the target chemicals in the population density plot built in the parametric space defined by the explanatory variables of the model by making use the training set chemicals. Currently, the accepted threshold of population density is 10%.
The mechanistic domain is applied on the parent structures and on their metabolites.

- Appropriate measures of goodness-of-fit and robustness and predictivity:
External Validation: For substances in the applicability domain, a predictivity of 100% was found for 100 industrial chemicals for the distinction of non-sensitizers versus sensitizers of GHS Category 1. The evaluation has been published in W. Teubner, A. Mehling, P.X. Schuster, K.Guth, B. A. Worth, J. Burton, B. van Rawenzwaay, R. Landsiedel: Computer models versus reality: How well do in silico models currently predict the sensitization potential of a substance, Regulatory Toxicology and Pharmacology 67 (2013) 468-485

Statistics for goodness-of-fit: For 875 chemicals, the TIMES-SS model was able to predict correctly 90% of the strong sensitizers, 55% of the weak sensitizers and 77% of the non-sensitizers, i.e., an overall performance of 78 %. Sensitivity: 78 %, Specificity: 77 %

- Mechanistic interpretation:
The TIMES-SS (Tissue Metabolism Simulator for skin sensitization) model integrates a simulator of skin metabolism together with a number of “local” QSAR models for assessing the reactivity of specific alerts. A skin metabolism simulator was developed based on empirical and theoretical knowledge (not enough reported observed skin metabolism data). The transformation probabilities (defining the priority of their execution) were parameterized to reproduce skin sensitization data. The simulator comprises of about 420 transformations, which can be divided into four main types: abiotic transformations, covalent interaction with proteins, Phase I and Phase II reactions. Autoxidation (AU) of chemical is also accounted for. Interactions with skin proteins are grouped into three types: leading to strong or weak skin
sensitization effect and interactions requiring QSAR models to quantify the potency of sensitization of the alerting groups. The QSAR models were developed by the COmmon PAttern Recognition (COREPA) approach [3]. The skin sensitization model predicts skin sensitization effect in three classes: strong, weak and non-sensitizers.
Reliability of alerts in the TIMES-SS model has been also evaluated to provide transparent mechanistic reasoning for predicting sensitization potential. Alert performance was defined as the ratio between the number of correct (positive and negative) predictions and the total number of chemicals within the local training set that triggered the alert. The alert performance was assessed based on the predictions on parents, autoxidation products simulated by the external AU simulator and metabolites as simulated by the skin metabolism simulator embedded in TIMES-SS model. Four different categories of reliability were defined:
High reliability – alert performance higher than 60% and more than 5 chemical in local (transformation/alert) training set
Low reliability – performance less than 60% and more than 5 chemicals in training set
Undetermined reliability – less than 5 chemicals in training set
Undetermined (theoretical) – there are no chemicals supporting the alert in the local training set

5. APPLICABILITY DOMAIN
- Descriptor domain:
Log(Kow):
range = [ -13.2 .. 15.4 ]
calculated: 1.77 (In domain)
MOL._WEIGHT: range = [ 30 .. 738 ]Da
calculated: 188Da (In domain)
--> Conclusion: The chemical fulfils the general properties requirements.

- Structural fragment domain: The following ACF are identified: Fragments in correctly predicted training chemicals – 100.00%, Fragments in non-correctly predicted training chemicals – 0.00%, Fragments not present in the training chemicals – 0.00%
--> Conclusion: The chemical is in the interpolation structural space

- Mechanistic domain: Interpolation space
- Similarity with analogues in the training set: not reported

6. ADEQUACY OF THE RESULT
The substance falls in the applicability domain of the model. The model was found to give reliable predictions for industrial chemicals. It is therefore considered to be acceptable for REACH.

The substance is considered to be non skin seniziting.
Qualifier:
according to
Guideline:
other: REACH guidance on QSARs R.6, May/July 2008
Principles of method if other than guideline:
TIMES-SS v.2.27.19.13 - Skin sensitization with autoxidation v.21.26 (structure-toxicity and structure-metabolism relationships)
GLP compliance:
no
Specific details on test material used for the study:
SMILES used to enter: CC(=O)OCC(C)(C)COC(C)=O
Key result
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
The registrant considers this predication as valid because TIMES-SS was validated with 100 substances from the registrant's portfolio (Teubner et al., Regulatory Toxicology and Pharmacology 67 (2013) 468–485). All predictions that fullfilled all domain requirements were correct (Specificity 100%).

The QSAR program calculated a negative sensitization potential of the test substance. The substance is in domain of the system.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

To assess the skin sensitizing potential of the test substance an in vitro skin sensitization turnkey testing strategy was conducted. The following tests were used:

DPRA:

In order to assess the reactivity towards peptides, synthetic cysteine (C)- or lysine (K)-containing peptides were incubated with the testsubstance for ca. 24 hours at ca. 25°C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UVdetection at 220 nm according to the OECD guideline 442C.

Therefore, the test substance was dissolved at a 100 mM concentration in acetonitrile and three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide).

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

LuSens:

In order to assess the keratinocyte activating potential, a luciferase reporter cell line (LuSens cells) was incubated with the test substance for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer according to the OECD guidline 442D.

Calculation of an EC1.50 (the concentration resulting in a 1.50-fold luciferase induction) was not applicable due to the overall negative result of the study.

In summary, 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 at least two independent experiments. From this it has to be concluded that the test substance does not have a keratinocyte activating potential.

Examination of the test substance in two different non-animal methods addressing different key events of the skin sensitization Adverse Outcome Pathway resulted in two negative result in the LuSens and DPRA.

Applying the Adverse Outcome Pathway-based "2 out of 3" integrated testing strategy approach to skin hazard identification the skin sensitization potential the test substance is predicted to be not sensitizing.

To confirm this conclusion a quantitative structure-activity relationship (QSAR) system for the estimation of the skin sensitization potency that incorporates skin metabolism and considers the potential of parent chemicals and/or their activated metabolites to react with skin proteins was used. The QSAR programs TIMES-SS calculated a negative sensitization potential of the test substance. The test substance was in domain of the system. The registrant considers this prediction as valid, because TIMES-SS was validated from the registrant´s portfolio (Teubner et al., Regulatory Toxicology and Pharmaclogy 67 (2013) 468-485).

Based on this weight of evidence approach consisting of in vitro tests and QSAR modeling, that uniformly predict the test substance as a non-skin sensitizer, 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. As a result, the substance is not considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480.