3-methyl-1-isobutylbutyl acetate

Endpoint summary

Description of key information

DPRA: Reinen (2019)

Under the conditions of this study, the DPRA prediction of the test material was negative (no or minimal reactivity).

DEREK NEXUS Prediction: Jonis (2019)

The test material is predicted to be not sensitising to the skin.

KeratinoSens™ Assay: Woutersen (2019)

Under the conditions of this study, the test material is considered to be negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes).

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:

key study

Study period:

19 September 2018 to 02 October 2018

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

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The objective of this study was to determine the reactivity of the teat material towards model synthetic peptides containing either cysteine or lysine, and to categorise the test material in one of four classes of reactivity for supporting the discrimination between skin sensitisers and non-sensitisers.
The Direct Peptide Reactivity Assay (DPRA) is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test item at 25 °C. The synthetic peptides contain phenylalanine to aid in the detection. The relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. Cysteine and lysine peptide Percent Depletion Values are calculated and used in a prediction model which allows assigning the test material to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

Details on the study design:

DOSE FORMULATION AND ANALYSIS
- Preparation of Test Material: No correction for the purity/composition of the test material was performed.
- Solubility of the test material in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test material completely, i.e. by visual inspection the solution had to be not cloudy nor have noticeable precipitate. The following solvents were evaluated: Acetonitrile (ACN), Milli-Q water (MQ), ACN:MQ (1:1, v/v), isopropanol (IPA), acetone:ACN (1:1, v/v), dimethylsulfoxide (DMSO):ACN (1:9, v/v), ethanol (EtOH) and methanol (MeOH).
- Test material stock solutions were prepared freshly for each reactivity assay.
- For both the cysteine and lysine reactivity assay 34.49 mg of test material was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1851 µL ACN after vortex mixing to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test material was dissolved. The test material, positive control and peptide samples were prepared less than 4 hours before starting the incubation of the cysteine (cys) or lysine (lys) reactivity assay, respectively. Any residual volumes were discarded.
 
TEST SYSTEM
- Test system: Synthetic peptides containing cysteine (SPCC) (Ac RFAACAA-COOH) or synthetic peptides containing lysine (SPCL) (Ac RFAAKAA-COOH). The molecular weight is 750.9 g/mol for SPCC and 775.9 g/mol for SPCL.  
- Rationale: Recommended test system in the international OECD guideline for DPRA studies.
- Source: JPT Peptide Technologies GmbH, Berlin, Germany.
- Storage: The peptides were stored in the freezer (≤ 15 °C) for a maximum of 6 months.
 
EXPERIMENTAL DESIGN
Preparation of Solutions for Cysteine Reactivity Assay:
- Synthetic Peptide Containing Cysteine (SPCC) Stock Solution: A stock solution of 0.667 mM SPCC (0.501 mg SPCC/mL) was prepared by dissolving 10.4 mg of SPCC in 20.76 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes sonication.
- SPCC Reference Control Solutions: Three 0.5 mM SPCC reference control (RC) solutions (RCcysA, RCcysB and RCcysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCC stock solution with 250 µL ACN.
- SPCC Calibration Curve: A SPCC calibration curve was prepared as follows:
STDcys1: 0.534 mM (1600 µL stock solution of 0.667 mM SPCC + 400 µL ACN)
STDcys2: 0.267 mM (1 mL STDcys1 + 1 mL STDcys7)
STDcys3: 0.133 mM (1 mL STDcys2 + 1 mL STDcys7)
STDcys4: 0.067 mM (1 mL STDcys3 + 1 mL STDcys7)
STDcys5: 0.033 mM (1 mL STDcys4 + 1 mL STDcys7)
STDcys6: 0.017 mM (1 mL STDcys5 + 1 mL STDcys7)
STDcys7: 0 mM (8 mL phosphate buffer (pH 7.5) + 2 mL ACN)
- Co-elution Control, Test Material and Positive Control Samples: The co-elution control (CC) samples, test material samples and the cinnamic aldehyde positive control samples (PC) were prepared as follows:
Co-elution control (CC) (n=1): CCcys-209427/A (750 µL Phosphate buffer pH 7.5, 200 µL ACN and  50 µL 209427/A test solution (100 mM)).
Cinnamic aldehyde (PC) (n=3): PCcys-1 to PCcys-3 (750 µL Stock solution of 0.667 mM SPCC, 200 µL ACN and 50 µL Cinnamic aldehyde solution (100 mM in ACN)).
Test material 209427/A (n=3): 209427/A-cys-1 to 209427/A-cys-3 (750 µL Stock solution of 0.667 mM SPCC, 200 µL ACN and 50 µL 209427/A test solution (100 mM)).
 
Preparation of Solutions for Lysine Reactivity Assay
- Synthetic Peptide Containing Lysine (SPCL) Stock Solution: A stock solution of 0.667 mM SPCL (0.518 mg SPCL/mL) was prepared by dissolving 11.3 mg of SPCL in 21.81 mL of ammonium acetate buffer pH 10.2 followed by stirring for 5 minutes.
- SPCL Reference Control Solutions: Three 0.5 mM SPCL reference control (RC) solutions (RClysA, RClysB and RClysC) were prepared in amber vials by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL ACN.
- SPCL Calibration Curve: A SPCL peptide calibration curve was prepared as follows:
STDlys1: 0.534 mM (1600 µL stock solution of 0.667 mM SPCL + 400 µL ACN)
STDlys2: 0.267 mM (1 mL STDlys1 + 1 mL STDlys7)
STDlys3: 0.133 mM (1 mL STDlys2 + 1 mL STDlys7)
STDlys4: 0.067 mM (1 mL STDlys3 + 1 mL STDlys7)
STDlys5: 0.033 mM (1 mL STDlys4 + 1 mL STDlys7)
STDlys6: 0.017 mM (1 mL STDlys5 + 1 mL STDlys7)
STDlys7: 0 mM (8 mL ammonium acetate buffer (pH 10.2) + 2 mL ACN)
- Co-elution Control, Test Material and Positive Control Samples: The co-elution control (CC) samples, test material samples and the cinnamic aldehyde positive control samples (PC) were prepared as follows:
Co-elution control (CC) (n=1): CClys-209427/A (750 µL Ammonium acetate buffer pH 10.2 and 250 µL 209427/A test solution (100 mM)).
Cinnamic aldehyde (PC) (n=3): PClys-1 to PClys-3 (750 µL Stock solution of 0.667 mM SPCL and 250 µL Cinnamic aldehyde solution (100 mM in ACN)).
Test material 209427/A (n=3): 209427/A-lys-1 to 209427/A-lys-3 (750 µL Stock solution of 0.667 mM SPCL and 250 µL 209427/A test solution (100 mM)).

Sample Incubations:
- After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test material samples) were placed in the autosampler in the dark and incubated at 25 ± 2.5 °C. The incubation time between placement of the samples in the autosampler and analysis of the first RCcysB- or RClysB-sample was 25.5 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence, respectively, did not exceed 30 hours.
- Prior to HPLC PDA analysis the samples were visually inspected for precipitation. The samples that showed precipitation were centrifuged (at 400 g) for 5 minutes at room temperature and transferred to a new vial.
 
HPLC-PDA Analysis
- SPCC and SPCL peak areas in the samples were measured by HPLC PDA. Sample analysis was performed using the following systems:
- System 1 (used for Cysteine Reactivity Assay):
Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
MPS 3C autosampler (DaVinci, Rotterdam, The Netherlands)
LC Column oven 300 (Thermo Scientific)
Surveyor PDA detector (Thermo Scientific)

- System 2 (used for Lysine Reactivity Assay):
Surveyor MS HPLC pump (Thermo Scientific, Breda, The Netherlands)
HTC PAL autosampler (DaVinci, Rotterdam, The Netherlands)
Column Oven #151006 (Grace, Worms, Germany)
Surveyor PDA detector (Thermo Scientific)
 
Mobile phase: A: 0.1% (v/v) TFA in Milli-Q water. B: 0.085% (v/v) TFA in ACN
Gradient:
Cysteine: 0 min: 10 % B, 10 min: 25 % B, 11 min: 90 % B, 13 min: 90 % B, 13.5 min: 10 % B, 20 min: 10 % B
Lysine: 0 min: 10 % B, 10 min: 20 % B, 11 min 90 % B, 13 min: 90 % B, 13.5 min: 10 % B, 20 min 10 % B
Flow: 0.35 mL/min
Injection volume: 3 µL
Sample tray temperature: Set at 25 °C
Column: Zorbax SB-C18, 100 mm x 2.1 mm, df = 3.5 µm (Agilent Technologies, Santa Clara, CA, USA)
Guard column: SecurityGuard™ cartridge for C18, 4 x 2.0 mm (Phenomenex, Torrance, CA, USA)
Column temperature: Set at 30 °C
Detection: Photodiode array detection, monitoring at 220 and 258 nm
 
ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
- The standard calibration curve had to have an r^2 > 0.99.
- The mean Percent Peptide Depletion value of the three replicates for the positive control cinnamic aldehyde had to be between 60.8 % and 100 % for SPCC and between 40.2 % and 69.0 % for SPCL.
- The maximum standard deviation (SD) for the positive control replicates had to be <14.9 % for the Percent Cysteine Peptide Depletion and <11.6% for the Percent Lysine Peptide Depletion.
- The mean peptide concentration of Reference Controls A had to be 0.50 ± 0.05 mM.
- The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in ACN had to be <15.0 %.
The following criteria had to be met for a test material’s results to be considered valid:
- The maximum SD for the test material replicates had to be <14.9 % for the Percent Cysteine Depletion and <11.6 % for the Percent Lysine Depletion.
- The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50 ± 0.05 mM.
 
ANALYSIS
Data Evaluation:
- The concentration of SPCC or SPCL was photometrically determined at 220 nm in each sample by measuring the peak area of the appropriate peaks by peak integration and by calculating the concentration of peptide using the linear calibration curve derived from the standards.
- The Percent Peptide Depletion was determined in each sample by measuring the peak area and dividing it by the mean peak area of the relevant reference controls C according to the following formula:

Percent Peptide Depletion= [1-((Peptide Peak Area in Replicate Injection (at 220 nm))/(Mean Peptide Peak Area in Reference Controls (at 220 nm)))]×100

- In addition, the absorbance at 258 nm was determined in each sample by measuring the peak area of the appropriate peaks by peak integration. The ratio of the 220 nm peak area and the 258 nm peak was used as an indicator of co-elution. For each sample, a ratio in the range of 90 %  
Data Interpretation
- The mean Percent Cysteine Depletion and Percent Lysine Depletion were calculated for the test material. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model, the threshold of 6.38 % average peptide depletion was used to support the discrimination between a skin sensitiser and a non-sensitiser:
0 % ≤ Mean % depletion ≤ 6.38 %: No or minimal reactivity = NEGATIVE
6.38 % < Mean % depletion ≤ 22.62 %: Low reactivity = POSITIVE
22.62 % < Mean % depletion ≤ 42.47 %: Moderate reactivity = POSITIVE
42.47 % < Mean % depletion ≤ 100 %: High reactivity = POSITIVE

Key result

Run / experiment:

mean

Parameter:

other: SPCC depletion (%)

Value:

2.1

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: ± SD 3.7 %

Key result

Run / experiment:

mean

Parameter:

other: SPCL depletion (%)

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: ± SD 0.0 %

Key result

Run / experiment:

mean

Parameter:

other: Mean of SPCC and SPCL depletion (%)

Value:

1.1

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Other effects / acceptance of results:

SOLUBILITY OF THE TEST MATERIAL
- At a concentration of 100 mM, the test material was not soluble in MQ and ACN:MQ (1:1, v/v), but was soluble in ACN, IPA, acetone:ACN (1:1, v/v), DMSO:ACN (1:9, v/v), EtOH and MeOH. Since ACN is the preferred solvent for the DPRA, this solvent was used to dissolve the test material in this study.

CYSTEINE REACTIVITY ASSAY
- The reactivity of the test material towards SPCC was determined by quantification of the remaining concentration of SPCC using HPLC-PDA analysis, following 25.5 hours of incubation at 25 ± 2.5 °C.
- Acceptability of the Cysteine Reactivity Assay: The correlation coefficient (r^2) of the SPCC standard calibration curve was 0.998. Since the r^2 was >0.99, the SPCC standard calibration curve was accepted. The mean peptide concentration of Reference Controls A was 0.513 ±0.004 mM while the mean peptide concentration of Reference Controls C was 0.504 ± 0.004 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test material did not impact the Percent SPCC Depletion.
The Coefficient of Variation (CV) of the peptide areas for the nine Reference Controls B and C was 1.5 %. This was within the acceptance criteria (CV <15.0 %) and confirms the stability of the HPLC run over time.
The mean area ratio (A220/A258) of the Reference Control samples was 17.26. The mean A220/A258 ratio ± 10 % range was 15.54-18.99. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 72.0 % ± 0.2 %. This was within the acceptance range of 60.8 % to 100 % with a SD that was below the maximum (SD <14.9 %).
- Results Cysteine Reactivity Assay for the Test Material: Preparation of a 100 mM Alicate stock solution in ACN showed that the test material was dissolved completely. Upon preparation and after incubation, both the co-elution control (CC) as well as the test material samples were visually inspected. Upon preparation and after incubation a precipitate was observed in the co-elution control (CC) and test material samples. In this case one cannot be sure how much test material remained in the solution to react with the peptide.
In the CC sample no peak was observed at the retention time of SPCC. This demonstrated that there was no co-elution of the test material with SPCC. For the 209427/A-cys samples, the mean SPCC A220/A258 area ratio was 17.29. Since this was within the 15.54-18.99 range, this again indicated that there was no co elution of the test material with SPCC.
The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the test material was 2.1 % ± 3.7 %.

LYSINE REQCTIVITY ASSAY
- The reactivity of Alicate towards SPCL was determined by quantification of the remaining concentration of SPCL using HPLC-PDA analysis, following 25.5 hours of incubation at 2 5± 2.5 °C.
- Acceptability of the Lysine Reactivity Assay: The correlation coefficient (r^2) of the SPCL standard calibration curve was 0.997. Since the r^2 was >0.99, the SPCL standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.482 ± 0.022 mM while the mean peptide concentration of Reference Controls C was 0.457 ± 0.034 mM. The means of Reference Control samples A and C were both within the acceptance criteria of 0.50 ± 0.05 mM. This confirms the suitability of the HPLC system and indicates that the solvent (ACN) used to dissolve the test material did not impact the Percent SPCL Depletion.
The CV of the peptide areas for the nine Reference Controls B and C was 5.2 %. This was within the acceptance criteria (CV <15.0 %) and confirms the stability of the HPLC run over time.
The mean area ratio (A220/A258) of the Reference Control samples was 16.55. The mean A220/A258 ratio ± 10 % range was 14.89-18.20. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.
The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the positive control cinnamic aldehyde was 48.3 % ± 3.1 %. This was within the acceptance range of 40.2 % to 69.0 % with a SD that was below the maximum (SD <11.6 %).
- Results Lysine Reactivity Assay for the Test Material: Preparation of a 100 mM test material stock solution in ACN showed that the test material was dissolved completely. Upon preparation and after incubation, both the CC as well as the test material samples were visually inspected. Upon preparation and after incubation a precipitate was observed in the CC and test material samples. In this case one cannot be sure how much test material remained in the solution to react with the peptide.
In the CC sample no peak was observed at the retention time of SPCL. This demonstrated that there was no co-elution of the test material with SPCL. For the 209427/A-lys samples, the mean SPCL A220/A258 area ratio was 16.33. Since this was within the 14.89-18.20 range, this again indicated that there was no co-elution of the test material with SPCL.
The Percent SPCL Depletion was calculated versus the mean SPCL peak area of Reference Controls C. The mean Percent SPCL Depletion for the Test Material was 0.0 % ± 0.0 %.

DPRA PREDICTION AND REACTIVITY CLASSIFICATION
- Upon preparation and after incubation of the SPCC and SPCL test material samples, a precipitate was observed.
- In the cysteine reactivity assay the test material showed 2.1 % SPCC depletion while in the lysine reactivity assay the test material showed 0.0 % SPCL depletion. The mean of the SPCC and SPCL depletion was 1.1 % and as a result the test material was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test material remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.

SPCC and SPCL Depletion, DPRA Prediction and Reactivity Classification for the Test Material

	SPCC depletion		SPCL depletion		Mean of SPCC and SPCL depletion	DPRA prediction and reactivity classification
	Mean	± SD	Mean	± SD		Cysteine 1:10 / Lysine 1:50 prediction model
Test material	2.1 %	± 3.7 %	0.0 %	± 0.0 %	1.1 %	Negative: No or minimal reactivity

SD = Standard Deviation

Interpretation of results:

other: Negative in the DPRA Assay

Conclusions:

Under the conditions of this study, the DPRA prediction of the test material was negative (no or minimal reactivity).

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442C, under GLP conditions.

The objective of this study was to determine the reactivity of the test material towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test material with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test material to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test material and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. 

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test material, were all within the acceptability criteria for the DPRA.

Upon preparation and after incubation of the SPCC and SPCL test material samples, a precipitate was observed.

In the cysteine reactivity assay the test material showed 2.1 % SPCC depletion while in the lysine reactivity assay the test material showed 0.0 % SPCL depletion. The mean of the SPCC and SPCL depletion was 1.1 % and as a result the test material was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. The test material was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test material remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.

Under the conditions of this study, the DPRA prediction of the test material was negative (no or minimal reactivity).

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

other: DEREK NEXUS prediction

Adequacy of study:

supporting study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Qualifier:

no guideline followed

Principles of method if other than guideline:

The objective of this study was to obtain a prediction on the potential for skin sensitisation of the test material with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used.

GLP compliance:

no

Remarks:

No laboratory work was performed

Type of study:

other: DEREK NEXUS prediction

Details on the study design:

DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitisation based on the presence of molecular substructures. LHASA has inserted validation comments for the skin sensitisation alerts.
 
The level of likelihood of a structure being sensitising to skin is expressed in terms of:
- Certain: There is proof that the proposition is true.
- Probable: There is at least one strong argument that the proposition is true and there are no arguments against it.
- Plausible: The weight of evidence supports the proposition.
- Equivocal: There is an equal weight of evidence for and against the proposition.
 
The default of DEREK NEXUS for the level of likelihood, mentioning all alerts which are evaluated as being equivocal or greater was used in this assessment. 
 
If a substance is predicted to be no skin sensitiser, DEREK NEXUS contains an expert-derived functionality to provide negative predictions for skin sensitisation. This functionality further evaluates those compounds which do not fire any skin sensitisation alerts in DEREK NEXUS. The query compound is compared to a Lhasa reference set of Ames test or skin sensitisation data, producing the following outcomes:
- In compounds where all features in the molecule are found in accurately classified compounds from the reference set, a negative prediction is displayed: Inactive.
- For those query compounds where features in the molecule are found in non-alerting skin sensitisers in the Lhasa reference set, the prediction remains negative and the misclassified features are highlighted to enable the negative prediction to be verified by expert assessment.
- In cases where features in the molecule are not found in the Lhasa reference set, the prediction remains negative and the unclassified features are highlighted to enable the negative prediction to be verified by expert assessment.
 
If a substance is predicted to be a skin sensitiser, its potency is predicted by DEREK NEXUS by calculating an EC3 value based on experimental data from the closest structurally-related substances (at least 3 substances should be present) using the following equation:

EC3Q = MWQ /(Σ ωNN/ Σ TNN)

Where:
MW = molecular weight
T = Tanimoto similarity score
ω = weighting factor = (MWNN/EC3) * TNN
Q = query compound
NN = nearest neighbour
 
The EC3 is the estimated concentration needed to produce a stimulation index of 3.

Parameter:

other: DEREK NEXUS prediction

Remarks on result:

no indication of skin sensitisation

Remarks:

The test material is predicted to be not sensitising to the skin.

Other effects / acceptance of results:

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitisation for the test material. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitiser. The test material is predicted to be not sensitising to the skin.

Interpretation of results:

other: The test material is predicted to be not sensitising to the skin.

Conclusions:

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitisation for the test material. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitiser. The test material is predicted to be not sensitising to the skin.

Executive summary:

The objective of this study was to obtain a prediction on the potential for skin sensitisation of the test material with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used.

DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitisation based on the presence of molecular substructures. LHASA has inserted validation comments for the skin sensitisation alerts.

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitisation for the test material. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitiser. The test material is predicted to be not sensitising to the skin.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 August 2018 to 31 August 2018

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:

June 2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 155: KeratinoSens™

Version / remarks:

March 2018

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

PREPARATION OF TEST MATERIAL STOCK, SPIKING AND WORKING SOLUTIONS
No correction was made for the composition/purity of the test material.
A solubility test was performed. The test material was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 200 mM (colourless solution). The 100-fold dilution of the 200 mM DMSO stock in DMEM glutamax formed a clear solution (2000 μM). This concentration was selected as highest concentration for the main assay (highest dose required in the current guideline).
In the main experiments the test material was dissolved in DMSO at 200 mM (colourless solution). From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series).
The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (final concentration DMSO of 1 %). All concentrations of the test material were tested in triplicate. All formulations formed a clear solution.
No precipitation was observed at the start and end of the incubation period in the 96-well plates.
Test material concentrations were used within 2.5 hours after preparation. Any residual volumes were discarded.

Details on the study design:

CELL CULTURE
- Basic medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1 % (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum.
- Maintenance medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1 % (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum and geneticin (500 μg/mL).
- Exposure medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 1 % (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum.
- Environmental conditions: All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100 % (actual range 79 - 99 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 36.4 - 37.0 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

SUBCULTURING
Cells were subcultured upon reaching 80 - 90 % confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock and were not cultured for more than 25 passages from the frozen stock (P+25).

EXPERIMENTAL DESIGN
- Plating of Cells: For testing, cells were 80 - 90 % confluent. One day prior to testing cells were harvested and distributed into 96-well plates (10 000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was P+13 in experiment 1 and P+4 in experiment 2.
- Treatment of Cells: The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control materials were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37 ± 1.0 °C in the presence of 5 % CO2. In total 2 experiments were performed.
- Luciferase Activity Measurement: Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilised) were mixed together. The assay plates were removed from the incubator and the medium removed. Then 200 μL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).
- Cytotoxicity Assessment: For the KeratinoSens™ cell viability assay, medium was replaced after the 48-hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and cells were incubated for 3 - 4 hours at 37 °C in the presence of 5 % CO2. The MTT medium was then removed and cells were lysed overnight by adding 10 % SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSens™ test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 μM).
b) The EC1.5 should be within two standard deviations of the historical mean. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
c) Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO should be below 20 % in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded. If the variability is higher, a maximum of three of the eighteen wells may be excluded based on the Dixon’s Q-test. If the variability is still higher, the results should be discarded.

INTERPRETATION
> Data analysis
The following parameters are calculated in the KeratinoSens™ test method:
- The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control;
- The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5-fold threshold (i.e. 50 % enhanced luciferase activity) was obtained;
- The IC50 and IC30 concentration values for 50 and 30 % reduction of cellular viability, respectively.

Fold luciferase activity induction is calculated by the following equation, and the overall maximal fold induction (Imax) is calculated as the average of the individual repetitions.
Fold induction = (Lsample - Lblank) / (Lvehicle - Lblank)
Where:
Lsample is the luminescence reading in the test chemical well
Lblank is the luminescence reading in the blank well containing no cells and no treatment
Lvehicle is the average luminescence reading in the wells containing cells and vehicle (negative) control

The EC1.5 is calculated by linear interpolation according to the following equation, and the overall EC1.5 is calculated as the mean of the individual repetitions.
EC1.5 = (Cb - Ca) x [(1.5 - Ia) / (Ib - Ia)] + Ca
Where:
Ca is the lowest concentration in μM (or μg/mL) with > 1.5-fold induction
Cb is the highest concentration in μM (or μg/mL) with < 1.5-fold induction
Ia is the fold induction measured at the lowest concentration with > 1.5-fold induction (mean of three replicate wells)
Ib is the fold induction at the highest concentration with < 1.5-fold induction (mean of three replicate wells)

Viability is calculated by the following equation:
Viability = [(Vsample - Vblank) / (Vvehicle - Vblank)] x 200
Where:
Vsample is the MTT-absorbance reading in the test chemical well
Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment
Vvehicle is the average MTT-absorbance reading in the wells containing cells and vehicle (negative) control

Control IC50 and IC30 are calculated by linear interpolation and the overall IC50 and IC30 are calculated as the mean of the individual repetitions.
ICx = (Cb - Ca) x [((100 - x) - Va) / (Vb - Va)] + Ca
Where:
x is the % reduction at the concentration to be calculated (50 and 30 for IC50 and IC30)
Ca is the lowest concentration in μM (or μg/mL) with > x % reduction in viability
Cb is the highest concentration in μM (or μg/mL) with < x % reduction in viability
Va is the % viability at the lowest concentration with > x % reduction in viability
Vb is the % viability at the highest concentration with < x % reduction in viability

In case the luciferase activity induction is larger than 1.5-fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the vehicle (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05).
ToxRat Professional v 3.2.1 was used for statistical analysis of the data. The lowest concentration with > 1.5-fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30 % reduction in cellular viability at the EC1.5 determining concentration.

DATA INTERPRETATION
A KeratinoSens™ prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the prediction is considered negative:
1. The Imax is higher than (>) 1.5-fold and statistically significantly different as compared to the vehicle (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
2. The cellular viability is higher than (>) 70 % at the lowest concentration with induction of luciferase activity above 1.5-fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 μg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction

Positive control results:

EXPERIMENT 1
The positive control caused a dose related induction of the luciferase activity. The Imax was 2.69 and the EC1.5 63 μM.

EXPERIMENT 2
The positive control caused a dose related induction of the luciferase activity. The Imax was 3.38 and the EC1.5 25 μM.

ACCEPTANCE CRITERIA
Both tests passed:
- The luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was within two standard deviations of the historical mean (63 μM and 25 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.69-fold and 3.38-fold in experiment 1 and 2, respectively).

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

1.04

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

1.12

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

An overview of the viability and luciferase activity induction is summarised in Table 1. An overview of EC1.5, Imax, IC30 and IC50 values is given in Table 2.

EXPERIMENT 1
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- The test material showed toxicity at 500 and 1000 μM but not at 2000 μM. So, no dose-response was observed, and therefore, no IC30 and IC50 were calculated.
- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test material. The Imax was 1.04 and therefore no EC1.5 could be calculated.

EXPERIMENT 2
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- The test material showed only toxicity at 500 μM. So, no dose-response was observed and therefore no IC30 and IC50 were calculated.
- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test material. The Imax was 1.12 and therefore no EC1.5 could be calculated.

ACCEPTANCE CRITERIA
Both tests passed. In addition to the positive control passing the acceptance criteria, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20 % (7.2 and 6.7 % in experiment 1 and 2, respectively).
Overall it was concluded that the test conditions were adequate and that the test system functioned properly.

DISCUSSION
The test material showed toxicity, but no clear dose-response was observed. Therefore, no IC30 and IC50 values were calculated in both experiments. No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments.
The maximum luciferase activity induction (Imax) was 1.04-fold and 1.12-fold in experiment 1 and 2, respectively. The test material is classified as negative in the KeratinoSens™ assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.

Table 1: Overview Luminescence Induction and Cell Viability of the Test Material in Experiment 1 and 2

Experiment	Parameter	Concentration (μM)
		0.98	2.0	3.9	7.8	16	31	63	125	250	500	1000	2000
1	Luminescence	0.92	0.91	0.97	1.02	1.02	1.03	1.04	1.00	0.93	0.35	0.72	0.89
	Viability (%)	106.5	108.1	98.2	93.2	90.9	85.2	84.2	86.1	88.8	5.5	35.7	99.3
2	Luminescence	1.05	1.12	1.12	1.10	1.03	1.04	1.00	1.04	0.97	0.67	0.94	0.97
	Viability (%)	103.3	95.0	88.3	87.2	94.1	84.7	83.3	83.7	80.0	6.4	88.9	73.9

 

Table 2: Overview EC1.5, Imax, IC30 and IC50 Values

	EC1.5 (µM)	Imax	IC30 (µM)	IC50 (µM)
Test material Experiment 1	NA	1.04	NA	NA
Test material Experiment 2	NA	1.12	NA	NA
Positive Control Experiment 1	63	2.69	NA	NA
Positive Control Experiment 2	25	3.38	NA	NA

NA = Not applicable

Interpretation of results:

other: Negative in the KeratinoSens™ assay

Conclusions:

Under the conditions of this study, the test material is considered to be negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes).

Executive summary:

The objective of this study was to evaluate the ability of the test material to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens™ assay. The study was conducted in accordance with the standardised guideline OECD 442D under GLP conditions.

The liquid test material was dissolved in dimethyl sulfoxide (DMSO) at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 - 2000 μM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed.

Both experiments passed the acceptance criteria:

- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.

- The EC1.5 of the positive control was within two standard deviations of the historical mean (63 μM and 25 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.69-fold and 3.38-fold in experiment 1 and 2, respectively).

- The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20 % (7.2 and 6.7 % in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

The test material showed reproducible cytotoxicity, but no clear dose-response was observed, therefore no IC30 and IC50 values were calculated in both experiments. No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.04-fold and 1.12-fold in experiment 1 and 2, respectively.

The test material is classified as negative in the KeratinoSens™ assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.

Under the conditions of this study, the test material is considered to be negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

DPRA: Reinen (2019)

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442C, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The objective of this study was to determine the reactivity of the test material towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL). After incubation of the test material with either SPCC or SPCL, the relative peptide concentration was determined by High-Performance Liquid Chromatography (HPLC) with gradient elution and photodiode array (PDA) detection at 220 nm and 258 nm. SPCC and SPCL Percent Depletion Values were calculated and used in a prediction model which allows assigning the test material to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

Acetonitrile (ACN) was found to be an appropriate solvent to dissolve the test material and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. 

The validation parameters, i.e. calibration curve, mean concentration of Reference Control (RC) samples A and C, the CV for RC samples B and C, the mean percent peptide depletion values for the positive control with its standard deviation value and the standard deviation value of the peptide depletion for the test material, were all within the acceptability criteria for the DPRA.

Upon preparation and after incubation of the SPCC and SPCL test material samples, a precipitate was observed.

In the cysteine reactivity assay the test material showed 2.1 % SPCC depletion while in the lysine reactivity assay the test material showed 0.0 % SPCL depletion. The mean of the SPCC and SPCL depletion was 1.1 % and as a result the test material was considered to be negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. The test material was negative in the DPRA and was classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed after the incubation period for both SPCC and SPCL, one cannot be sure how much test material remained in the solution to react with the peptides. Consequently, this negative result is uncertain and should be interpreted with due care.

Under the conditions of this study, the DPRA prediction of the test material was negative (no or minimal reactivity).

DEREK NEXUS Prediction: Jonis (2019)

The objective of this study was to obtain a prediction on the potential for skin sensitisation of the test material with the in silico model DEREK NEXUS. In this assessment version 6.0.1 of DEREK NEXUS was used.

DEREK NEXUS is a knowledge-based system that contains 90 alerts for skin sensitisation based on the presence of molecular substructures. LHASA has inserted validation comments for the skin sensitisation alerts.

DEREK NEXUS version 6.0.1 did not yield any alerts for skin sensitisation for the test material. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitiser. The test material is predicted to be not sensitising to the skin.

KeratinoSens™ Assay: Woutersen (2019)

The objective of this study was to evaluate the ability of the test material to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens™ assay. The study was conducted in accordance with the standardised guideline OECD 442D under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

The liquid test material was dissolved in dimethyl sulfoxide (DMSO) at 200 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.98 - 2000 μM (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed.

Both experiments passed the acceptance criteria:

- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.

- The EC1.5 of the positive control was within two standard deviations of the historical mean (63 μM and 25 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.69-fold and 3.38-fold in experiment 1 and 2, respectively).

- The average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20 % (7.2 and 6.7 % in experiment 1 and 2, respectively).

Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

The test material showed reproducible cytotoxicity, but no clear dose-response was observed, therefore no IC30 and IC50 values were calculated in both experiments. No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.04-fold and 1.12-fold in experiment 1 and 2, respectively.

The test material is classified as negative in the KeratinoSens™ assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 μM.

Under the conditions of this study, the test material is considered to be negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes).

Discussion

A DEREK prediction on the skin sensitising potential of the test material was negative. The test material did not show to be reactive with cysteine and lysine containing peptides in the DPRA. Precipitation was observed after incubation and therefore it is not known how much test material remained in the solution during incubation. However, the structure of the test material does not possess substructures that would be reactive with nucleophilic sidechains of lysine and/or cysteine which supports the negative DPRA results observed. The KeratinoSens™ assay was negative, as it did not show a biologically relevant activation of keratinocytes when exposed to the test material.

Based on the outcomes of these assays, it is concluded that the test material does not possess skin sensitising properties. Therefore, no further testing is needed and the substance is not classified for skin sensitisation according to Regulation (EC) No 1272/2008 and related amendments.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008 based on the data available, the substance does not require classification with respect to skin sensitisation.