Butyl toluene-4-sulphonate

Endpoint summary

Description of key information

in silico / in chemico / in vitro

DEREK

The test material is predicted to be sensitising to the skin. The predicted EC3 of 2.1 % is not considered to be reliable.

Direct Peptide Reactivity Assay

The test material was negative under the conditions of this 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 SPCL, it is not clear 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.

KeratinoSens™

Under the conditions of this study, the test material is positive according to the KeratinoSens™ prediction model.

Weight of evidence of data from in silico, in chemico and in vitro studies

As the current information obtained with the in vitro test strategy does not allow a conclusion on the skin sensitisation potential and classification of the substance, it is, as a last resort, justified to continue with in vivo testing (LLNA or GPMT). The result from this test will be used to determine the skin sensitising potential and classification for the test material for skin sensitisation according to Regulation (EC) No 1272/2008 and related amendments.

In vivo

Local Lymph Node Assay

Findings from the study indicate that the test material could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test material concentration that will give a SI = 3) of 100 % was calculated based on the mean values. The test material should therefore be classified as a skin sensitiser (category 1B) according to EU criteria.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Remarks:

skin sensitisation: in silico

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Study period:

26 September 2019

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: DEREK Nexus
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.
DEREK NEXUS contains an expert-derived functionality that can provide negative predictions for skin sensitisation. This functionality further evaluates those compounds which do not trigger any skin sensitisation alerts in DEREK NEXUS. The query compound is compared to a Lhasa reference set of 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 (misclassified features are those that have been derived from non-alerting substances in the Lhasa test reference set).
• 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 (unclassified features are those that have not been found in the Lhasa test reference set).
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.

2. MODEL: version 6.0.1

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL: CAS 778-28-9 (SMILES: C1=CC(=CC=C1C)S(=O)(=O)OCCCC)

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
Please see attached

5. APPLICABILITY DOMAIN
i. descriptor domain: The scopes of the structure-activity relationships describing the skin sensitisation endpoint are defined by the developer to be the applicability domain for the model. Therefore, if a chemical activates an alert describing a structure-activity for skin sensitisation it can be considered to be within the applicability domain. The applicability of potency predictions may be judged, and modified, by the user based on the displayed data for nearest neighbours. If a compound does not activate an alert of reasoning rule then Derek makes a negative prediction. The applicability f the negative prediction to the query compounds can be determined by an expert, if required, by investigating the presence (or absence) of misclassified and/or unclassified features.
ii. structural fragment domain: The substructure of the substance falls within the structural fragment domain for alert 414.
iii. mechanism domain: The structural fragment of the substance falls within the mechanistic domain of the molecules in the training set on which the alert is based.
iv. metabolic domain: The structural fragment of the substance falls within the metabolic domain of the molecules in the training set on which the alert is based.
- Considerations on structural analogues: All similar compounds contain an alkyl sulphate or sulphonate. These compounds fall within the scope of alert 414. Ten structural analogues (structural similarity of 11 - 40 %) are used for calculating the EC3 and vary from non-sensitisers to strong sensitisers. The query compound contains a sulfonic butyl ester and a toluene group. These structural fragments are not present simultaneously within its closest structurally related analogues. Both these structures are of influence on the alkylating capabilities of the compound.
- The uncertainty of the prediction: DEREK NEXUS predictive performance against a combined human dataset had an accuracy of 76 %.
https://www.lhasalimited.org/Public/Library/2014/Derek%20Nexus%20predicts%20human%20skin%20sensitisation%20accurately.pdf
- The chemical and biological mechanisms according to the model underpinning the predicted result: The potential mechanism for this alert is the substance acting as an SN2-reactive electrophile capable of direct alkylation with nucleophilic centres in skin proteins.

6. ADEQUACY OF THE RESULT
- Regulatory purpose: The present prediction may be used for preparing the REACH Registration Dossier on the substance for submission to ECHA, as required by Regulation (EC) 1907/2006 and related amendments.
- Approach for regulatory interpretation of the model result: This result can be directly used within a weight-of-evidence approach to complete the endpoint skin sensitisation.
- Outcome: Substance should be classified according to DEREK; however, this (Q)SAR prediction cannot be used as stand-alone for classification purposes or for covering the endpoint skin sensitisation for registration under REACH.
- Conclusion: The result is adequate to be used in a weight-of-evidence approach together with in chemico/in vitro studies to complete the endpoint skin sensitisation.

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSARs R.6

Principles of method if other than guideline:

The in silico model DEREK NEXUS (version 6.0.1) was used to obtain a prediction on the potential for skin sensitisation of the test material.

GLP compliance:

no

Remarks:

not applicable to QSAR

Justification for non-LLNA method:

Annex VII of the REACH Regulation includes a requirement for in chemico/in vitro tests as a first step for addressing skin sensitisation.

Key result

Remarks on result:

other: the test material is predicted to be sensitising to the skin

The result as generated by DEREK NEXUS is attached to this robust study summary within the 'Attached background material' section. The relevant QSAR Model Reporting Format (QMRF) and the QSAR Prediction Reporting Format (QPRF) are attached to this robust study summary within the 'Attached justification' section.

DEREK NEXUS version 6.0.1 did yield a skin sensitisation alert for the test material based on the presence of a sulphonate and predicted the test material to be sensitising to the skin (plausible). The potential mechanism behind the alert is that the substance can directly alkylate nucleophilic centres in skin proteins.

DEREK NEXUS predicted an EC3 of 2.1 % (moderate sensitiser) for the test material based on LLNA data from ten structurally related analogues (structural similarity of 11 - 40 %). The query compound contains a sulphonic butyl ester and a toluene group. These structural fragments are of influence on the reactivity of alkylating capabilities of the compound. Considering both structural fragments are not present simultaneously within its closest structurally related analogues, the EC3 prediction is considered not sufficiently reliable for classification purposes.

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, the test material is predicted to be sensitising to the skin. The predicted EC3 of 2.1 % is not considered to be reliable.

Executive summary:

The in silico model, DEREK NEXUS (version 6.0.1), was used to obtain a prediction on the potential for skin sensitisation of the test material.

DEREK NEXUS did yield a skin sensitisation alert for the test material based on the presence of a sulphonate and predicted the test material to be sensitising to the skin (plausible). The potential mechanism behind the alert is that the substance can directly alkylate nucleophilic centres in skin proteins.

DEREK NEXUS predicted an EC3 of 2.1 % (moderate sensitiser) for the test material based on LLNA data from ten structurally related analogues (structural similarity of 11 - 40 %). The query compound contains a sulphonic butyl ester and a toluene group. These structural fragments are of influence on the reactivity of alkylating capabilities of the compound. Considering both structural fragments are not present simultaneously within its closest structurally related analogues, the EC3 prediction is considered not sufficiently reliable for classification purposes.

In conclusion, the test material is predicted to be sensitising to the skin. The predicted EC3 of 2.1 % is not considered to be reliable.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

20 August 2019 to 23 August 2019

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))

Deviations:

no

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin sensitisation tests is the Direct Peptide Reactivity Assay (DPRA), which is recommended in international guidelines (e.g. OECD 442C).

Specific details on test material used for the study:

- Purity/Composition correction factor: No correction factor required

Details on the study design:

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.
- Batch number: 111016HS-MHeW0419 and 020517HS-MeW0219
- Storage: Stored in the freezer (≤-15 °C) for a maximum of 6 months.

PREPARATION OF SOLUTIONS
- Preparation of Test Material: For both the cysteine and lysine reactivity assay 35.28 mg of test material was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1545 μL acetonitrile 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.
> 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 10.5 mg of SPCC in 20.96 mL phosphate buffer pH 7.5. The mixture was stirred for 5 minutes followed by 5 minutes of 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 acetonitrile. An SPCC calibration curve was prepared.
- Co-elution Control, Test Material and Positive Control Samples: The co-elution control sample (CC) contained 750 μL phosphate buffer pH 7.5, 200 μL acetonitrile, and 50 μL test material test solution (100 mM). The test material control sample contained 750 μL stock solution of 0.667 mM SPCC, 200 μL acetonitrile, and 50 μL test material test solution (100 mM). The positive control sample contained 750 μL stock solution of 0.667 mM SPCC, 200 μL acetonitrile, and 50 μL cinnamic aldehyde solution (100 mM in acetonitrile).
> 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 10.3 mg of SPCL in 19.88 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 acetonitrile. A SPCL peptide calibration curve was prepared.
- Co-elution Control, Test Material and Positive Control Samples: The co-elution control sample (CC) contained 750 μL ammonium acetate buffer pH 10.2, and 250 μL test material test solution (100 mM). The test material control sample contained 750 μL stock solution of 0.667 mM SPCL, and 250 μL test material test solution (100 mM). The positive control sample contained 750 μL stock solution of 0.667 mM SPCL, and 250 μL cinnamic aldehyde solution (100 mM in acetonitrile).

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 24.2 hours. The time between the first RCcysB- or RClysB-injection and the last injection of a cysteine or lysine sequence did not exceed 30 hours.
Prior to HPLC 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 supernatant was transferred to a new vial.

HPLC ANALYSIS
SPCC and SPCL peak areas in the samples were measured by HPLC.
- System: Alliance separations module 2695 (Waters, Milford, MA, USA); Dual λ absorbance detector 2487 (Waters)
- Column: Zorbax SB-C18, 100 mm × 2.1 mm ID, 3.5 μm particle size (Agilent Technologies, Santa Clara, CA, USA)
- Guard column: SecurityGuard™ cartridge for C18, 4 × 2.0 mm (Phenomenex, Torrance, CA, USA)
- Column temperature: 30 °C
- Mobile phase: A: 0.1 % (v/v) TFA in Milli-Q water. B: 0.085 % (v/v) TFA in acetonitrile
- Gradient:
Cysteine: at 0 min: 10 % B; at 10 min: 25 % B; at 11 and 13 min: 90 % B; at 13.5 and 20 min: 10 % B.
Lysine: at 0 min: 10 % B; at 10 min: 20 % B; at 11 and 13 min: 90 % B; at 13.5 and 20 min: 10 % B.
- Flow: 0.35 mL/min
- Injection volume: 5 µL
- Sample tray temperature: 25 °C
- Detection: spectrophotometric detection, monitoring at 220 and 258 nm

DATA EVALUATION
The concentration of SPCC or SPCL was spectrophotometrically 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)] x 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 % < mean area ratio of control samples < 110 % gives a good indication that co-elution has not occurred.

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, as follows (Mean of cysteine and lysine % depletion; Reactivity class; DPRA prediction):
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

ACCEPTABILITY CRITERIA
The following criteria had to be met for a run to be considered valid:
a) The standard calibration curve had to have an r² > 0.99.
b) 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.
c) 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.
d) The mean peptide concentration of Reference Controls A had to be 0.50 ± 0.05 mM.
e) The Coefficient of Variation (CV) of peptide areas for the nine Reference Controls B and C in acetonitrile had to be < 15.0 %.
The following criteria had to be met for the results of the test material to be considered valid:
a) 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.
b) The mean peptide concentration of the three Reference Controls C in the appropriate solvent had to be 0.50 ± 0.05 mM.

Positive control results:

CYSTEINE REACTIVITY ASSAY
The Percent SPCC Depletion was calculated versus the mean SPCC peak area of Reference Controls C. The mean Percent SPCC Depletion for the positive control cinnamic aldehyde was 92.5 ± 0.4 %. This was within the acceptance range of 60.8 to 100 % with a SD that was below the maximum (SD < 14.9 %).

LYSINE REACTIVITY ASSAY
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 65.4 ± 0.6 %. This was within the acceptance range of 40.2 to 69.0 % with a SD that was below the maximum (SD < 11.6 %).

Key result

Run / experiment:

other: Mean of SPCC and SPCL depletion

Parameter:

other: SPCC and SPCL depletion (%)

Value:

5.4

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Negative: No or minimal reactivity

Other effects / acceptance of results:

ACCEPTABILITY OF THE CYSTEINE REACTIVITY ASSAY
The correlation coefficient (r²) of the SPCC standard calibration curve was 1.000. Since the r² was > 0.99, the SPCC standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.502 ± 0.001 mM, the mean peptide concentration of Reference Controls C was 0.500 ± 0.001 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 (acetonitrile) 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 0.3 %. 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 37.53. The mean A220/A258 ratio ± 10 % range was 33.78 - 41.29. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

ACCEPTABILITY OF THE LYSINE REACTIVITY ASSAY
The correlation coefficient (r²) of the SPCL standard calibration curve was 0.9999. Since the r² was > 0.99, the SPCL standard calibration curve was accepted.
The mean peptide concentration of Reference Controls A was 0.497 ± 0.001 mM, the mean peptide concentration of Reference Controls C was 0.516 ± 0.020 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 (acetonitrile) 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 2.9 %. 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 31.05. The mean A220/A258 ratio ± 10 % range was 27.94 - 34.15. Each sample showing an A220/A258 ratio within this range gives an indication that co-elution has not occurred.

Solubility Assessment of the Test Material

At a concentration of 100 mM, the test material was not soluble in Milli-Q water and acetonitrile:Milli-Q water (1:1, v/v), but was soluble in acetonitrile, isopropanol, acetone:acetonitrile (1:1, v/v), DMSO:acetonitrile (1:9, v/v), ethanol and methanol. Since acetonitrile 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 analysis, following 24.2 hours of incubation at 25 ± 2.5 °C.

Preparation of a 100 mM test material stock solution in acetonitrile 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. No precipitate or phase separation was observed in any of the samples.

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 test material-cys samples, the mean SPCC A220/A258 area ratio was 37.61. Since this was within the 33.78 - 41.29 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 7.3 ± 0.2 %.

All of the acceptability criteria were fulfilled for the Cysteine Reactivity Assay.

 

Lysine Reactivity Assay

The reactivity of the test material towards SPCL was determined by quantification of the remaining concentration of SPCL using HPLC analysis, following 24.2 hours of incubation at 25 ± 2.5 °C.

Preparation of a 100 mM test material stock solution in acetonitrile 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. After incubation a precipitate was observed in the CC and the 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 test material-lys samples, the mean SPCL A220/A258 area ratio was 31.05. Since this was within the 27.94 - 34.15 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 3.5 ± 0.6 %.

All of the acceptability criteria were fulfilled for the Lysine Reactivity Assay.

 

DPRA Prediction and Reactivity Classification

In the cysteine reactivity assay the test material showed 7.3 % SPCC depletion while in the lysine reactivity assay the test material showed 3.5 % SPCL depletion. The mean of the SPCC and SPCL depletion was 5.4 % 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.

Interpretation of results:

other: Negative: No or minimal reactivity in the DPRA

Conclusions:

The test material was negative under the conditions of this 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 SPCL, it is not clear 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.

Executive summary:

The reactivity of the test material towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was investigated in a study which was conducted in accordance with the standardised guideline OECD 442C and under GLP conditions.

During the study the test material was incubated with either SPCC or SPCL and the relative peptide concentration determined by High-Performance Liquid Chromatography (with gradient elution and spectrophotometric detection at 220 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.

Upon preparation in acetonitrile, as well as after incubation of the SPCC test material samples, no precipitate or phase separation was observed in any of the samples. After incubation of the SPCL test material samples, a precipitate was observed.

In the cysteine reactivity assay the test material showed 7.3 % SPCC depletion while in the lysine reactivity assay the test material showed 3.5 % SPCL depletion. The mean of the SPCC and SPCL depletion was 5.4 %. Cinnamic aldehyde was used as positive control and showed 92.5 % SPCC depletion while in the lysine reactivity assay it showed 65.4 % SPCL depletion.

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.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. The mean value of SPCC and SPCL depletion of 5.4 % indicates that the test material is 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. However, since precipitation was observed after the incubation period for SPCL, it is not clear 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.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

02 August 2019 to 16 August 2019

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

Justification for non-LLNA method:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin sensitisation tests is the KeratinoSens™ assay, which is recommended in international guidelines (e.g. OECD 442D).

Specific details on test material used for the study:

Purity/Composition correction factor: No purity correction factor required

Details on the study design:

TEST SYSTEM
A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock.
Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

CONTROLS
- Vehicle Control: 1 % DMSO in exposure medium. Eighteen wells were tested per plate.
- Positive Control: Ethylene dimethacrylate glycol (2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO such that the final concentrations ranged from 7.8 to 250 μM; final concentration of DMSO of 1 %). All concentrations of the positive control were tested in triplicate.
- Blanks: On each plate three blank wells were tested (no cells and no treatment).

DOSE FORMULATION
A solubility test was performed. The test material was dissolved in dimethyl sulfoxide (DMSO) to a final concentration of 200 mM (clear colourless solution). The 100-fold dilution of the 200 mM DMSO stock in DMEM glutamax formed a homogeneous solution (no precipitation). 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 (clear 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 with exposure medium 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 of DMSO of 1 %). All concentrations of the test material were tested in triplicate. All formulations formed a clear solution.
Precipitation was observed at the start of the incubation period at concentrations of 1000 μM and upwards.
At the end of the incubation period, no precipitate was observed at any dose level tested in the first experiment and at the highest dose level tested in the second experiment.
Test material concentrations were used within 3 hours after preparation.

ENVIRONMENTAL CONDITIONS
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100 % (actual range 63 – 96 %), containing 5.0 ± 0.5 % CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.2 – 36.7 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.

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.

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).

MAIN STUDY
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+5 in experiment 1 and P+8 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 material and control items 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 valid experiments were performed.

LUCIFERASE ACTIVITY MEASUREMENT
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) were mixed together. The assay plates were removed from the incubator and the medium was 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 5 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, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 - 4 hours at 37 ± 1.0 °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.

CALCULATIONS
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 test material 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.

Fold luciferase activity induction is calculated by Equation 1, and the overall maximal fold induction (Imax) is calculated as the average of the individual repetitions.
Equation 1: 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 Equation 2, and the overall EC1.5 is calculated as the mean of the individual repetitions.
Equation 2: 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 Equation 3:
Equation 3: Viability = [(Vsample - Vblank) / (Vvehicle - Vblank)] × 100
Where:
Vsample is the MTT-absorbance reading in the test material 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.
Equation 4: ICx = (Cb – Ca) x [((100 − x) - Va) / (Vb − Va + Ca)]
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 equal or higher 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.

ACCEPTABILITY CRITERIA
The KeratinoSens™ test is considered acceptable if it meets the following criteria:
a) The luciferase activity induction obtained with the positive control, ethylene dimethacrylate glycol, should be statistically significantly equal to or 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
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 KeratinoSens™ prediction is considered negative:
1. The Imax is equal or 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 ≥ 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.
Negative results obtained with concentrations <1000 μM or 200 μg/mL and which do not reach cytotoxicity (< 70 % viability) at the maximal tested concentration should be considered as inconclusive.

Positive control results:

EXPERIMENT 1
- The positive control, ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.44 and the EC1.5 was 68 μM.

EXPERIMENT 2
- The positive control, ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.72 and the EC1.5 was 37 μM.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: EC1.5 (μM)

Value:

82

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: EC1.5 (μM)

Value:

68

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

8.83

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

8.39

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Run / experiment:

other: Experiment 1

Parameter:

other: IC30 (μM)

Value:

353

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

Two independent experiments were performed. The cells were in these experiments incubated with test material in a concentration range of 0.98 – 2000 μM (2-fold dilution steps) for 48 hours ± 1 h. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

EXPERIMENT 1
- Precipitation was observed at the start of the incubation period at concentrations of 1000 μM and upwards and no precipitation was observed at the end of the incubation period.
- The test material showed toxicity. The calculated IC30 was 353 μM. Fifty percent toxicity could not accurately be calculated due to the biphasic nature of the toxicity curve.
- A dose related luminescence activity induction was observed after treatment with test material. The Imax was 8.83 and the EC1.5 was 82 μM.

EXPERIMENT 2
- Precipitation was observed at the start of the incubation period at concentrations of 1000 μM and upwards and at the end of the incubation period at the top dose level of 2000 μM.
- The test material showed no toxicity. The viability of the cells was higher than 70 % at all test concentrations and therefore no IC30 and IC50 values could be calculated.
- A dose related luminescence activity induction was observed after treatment with the test material. The Imax was 8.39 and the EC1.5 was 68 μM.

ACCEPTANCE CRITERIA
Both tests passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control, ethylene dimethacrylate glycol, was statistically significantly 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 (68 and 37 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.44-fold and 2.72-fold in experiment 1 and 2, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20 % (5.6 and 3.6 % in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Discussion

The test material showed toxicity only in the first experiment (IC30 value of 353 μM). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 82 and 68 μM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 8.83-fold and 8.39-fold in experiment 1 and 2 respectively. The test material is therefore classified as positive in the KeratinoSens™ assay since positive results (> 1.5-fold induction) were observed at test concentrations < 1000 μM with a cell viability of > 70 % compared to the vehicle control.

Interpretation of results:

other: Positive according to the KeratinoSens™ prediction model.

Conclusions:

Under the conditions of this study, the test material is positive according to the KeratinoSens™ prediction model.

Executive summary:

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

The objective of the study was to evaluate the ability of the test material to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens™ assay.

During the study, the 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. The test material precipitated at the highest dose level tested in the second experiment only. Two independent experiments were performed.

Both experiments passed the acceptance criteria. It is therefore concluded that the test conditions were adequate and that the test system functioned properly.

Under the conditions of the study, the test material showed toxicity only in the first experiment (IC30 value of 353 μM). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 82 and 68 μM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 8.83-fold and 8.39-fold in experiment 1 and 2, respectively. The test material therefore fulfils the criteria for a positive classification in the KeratinoSens™ assay since positive results (> 1.5-fold induction) were observed at test concentrations < 1000 μM with a cell viability of > 70 % compared to the vehicle control.

In conclusion, the test material is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

other: Expert review of available in silico, in chemico and in vitro data.

Adequacy of study:

weight of evidence

Study period:

not applicable

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

according to guideline

Guideline:

other: ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a Endpoint specific guidance v.6.0

Version / remarks:

July 2017

Principles of method if other than guideline:

The ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a Endpoint specific guidance v.6.0 makes reference to REACH Annex VI, and notes that "the registrant should gather and evaluate all existing available information before considering further testing", which "includes structural considerations, physico-chemical properties, (Q)SAR ((Quantitative) Structure-Activity Relationship), information from structurally similar substances, in vitro/in chemico data, animal studies, and human data." Specifically, "Information from in vitro/in chemico test method(s) recognised according to Article 13(3), addressing each of the following key events of skin sensitisation:
(a) molecular interaction with skin proteins;
(b) inflammatory response in keratinocytes;
(c) activation of dendritic cells."
As such, the aim of this study report was to reach an overall conclusion on the endpoint of skin sensitisation based on all available relevant information, including in silico (DEREK), in chemico (DPRA) and in vitro (KeratinoSens™) data.
The objective of this study was to evaluate whether sufficient information is available to meet the information requirements for skin sensitisation of Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the relevant classification in accordance with Regulation (EC) No 1272/2008 (CLP) and related amendments. A weight of evidence approach according to Annex XI, sections 1.2 - 1.5, to the REACH Regulation is used.

The weight of evidence approach is based on in silico, in chemico and in vitro data, addressing each of the following key events of skin sensitisation on its own or together:
1. Key event 1: Covalent binding of the electrophilic substance to proteins; tested by OECD 442C: Direct Peptide Reactivity Assay (DPRA)
2. Key event 2: Release of pro-inflammatory cytokines and induction of cyto-protective pathways in keratinocytes; tested by OECD 442D: ARE-Nrf2 Luciferase Test Method or KeratinoSens™ assay
3. Key event 3: Activation and maturation of dendritic cells; OECD 442E Myeloid U937 Skin Sensitisation Test (U-SENS™)
4. Key event 4: Presentation of the chemical allergen by dendritic cells to naïve T-cells, which leads to their differentiation and proliferation into allergen-specific memory Tcells; no generally accepted in vitro test available yet.
If information from test method(s) addressing one or two of these key events allows classification and risk assessment according to point 8.3 of Annex VII of the REACH Regulation, studies addressing the other key event(s) need not be conducted.
According to the Guidance on information requirements and chemical safety assessment R7a (v.6.0 July 2017), to reach a conclusion on (non-)classification, the following questions should be addressed:
- Does the evidence enable to conclude that the substance is not a skin sensitiser? If so, conclude on no classification.
- Does the evidence enable to conclude that the substance is presumed to produce significant sensitisation in humans i.e. Cat. 1A? If so, classify accordingly.
- Does the evidence enable to conclude that the substance is a skin sensitiser and significant sensitisation in humans i.e. Cat. 1A can be excluded? If so, it is presumed that the substance would be a moderate skin sensitiser i.e. Cat. 1B and it is recommended to classify accordingly.
In case none of these conditions are met, e.g. when Cat. 1A cannot be excluded, further testing needs to be performed, in vivo testing being the last resort. At the moment no accepted in vitro studies are available to discern between Cat. 1A and 1B.
In case of positive in chemico/in vitro skin sensitisation tests and absence of reliable indication for potency by DEREK, for the time being performance of an in vivo study is the only option to determine the degree of potency (see CLP Regulation 3.4 Respiratory or skin sensitisation).

GLP compliance:

yes

Remarks:

studies, upon which the assessment was based, were all conducted under GLP conditions (with the exception of the in silico prediction)

Type of study:

other: expert review of available data

Justification for non-LLNA method:

The objective of this study was to evaluate whether sufficient information is available to meet the information requirements for skin sensitisation.

Positive control results:

Not applicable - refer to individual study records

Key result

Remarks on result:

not determinable

No data were available that would preclude performance of the studies to determine the potential for skin sensitisation. Therefore, STEP 1 studies were performed, i.e. a DEREK assessment, a DPRA assay, and a KeratinoSens™ assay.

 

DEREK Assessment

DEREK NEXUS version 6.0.1 yielded a skin sensitisation alert for the test material based on the presence of a sulphonate group and predicted the test material to be sensitising to the skin (plausible). DEREK NEXUS predicted an EC3 of 2.1 % (moderate sensitiser) for the test material based on LLNA data from ten structurally related analogues (structural similarity of 11 - 40 %). The EC3 prediction is considered not sufficiently reliable for classification purposes.

 

DPRA Assay

A valid DPRA test was performed according to OECD 442C and in accordance with GLP principles. For the DPRA assay, the test material was dissolved completely in acetonitrile at 100 mM. Upon preparation of the synthetic peptides containing cysteine (SPCC) or lysine (SPCL) with test material no precipitate or phase separation was observed in any of the samples. However, in the SPCL samples a precipitate was observed after incubation with the test material samples. No co-elution of the test material with SPCC or SPCL was observed. In the cysteine reactivity assay the test material showed 7.3 % SPCC depletion while in the lysine reactivity assay the test material showed 3.5 % SPCL depletion. The mean of the SPCC and SPCL depletion was 5.4 % 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. However, since precipitation was observed after the incubation period for 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 care.

 

KeratinoSens™ Assay

A valid KeratinoSens™ assay was performed according to OECD TG 442D and in accordance with GLP principles. Two valid independent tests were performed. For the KeratinoSens™ tests the test material was dissolved in DMSO to a final concentration of 200 mM (clear colourless solutions by visual observation). The cells were incubated with the test material for 48 ± 1 hours in a concentration range of 0.98 – 2000 μM in both experiments (2-fold dilution series). Precipitation was observed at the start of the incubation period at concentrations of > 1000 μM and upwards. The test material showed toxicity only in the first experiment (IC30 value of 353 μM). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 82 and 68 μM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 8.83-fold and 8.39-fold in experiment 1 and 2, respectively. The test material is classified as positive in the KeratinoSens™ assay since positive results (> 1.5-fold induction) were observed at test concentrations < 1000 μM with a cell viability of > 70 % compared to the vehicle control under the experimental conditions described.

 

Data Evaluation

A DEREK assessment predicted the test material to be a skin sensitiser based on the presence of a sulphonate. Sulphonates are known for directly alkylating nucleophilic centres in skin proteins. The DPRA showed a minor depletion of SPCC and SPCL but not significantly according to the Cysteine 1:10 / Lysine 1:50 prediction model. However, since precipitation was observed in the SPCL samples the result may be underestimated and should be interpreted with care. The KeratinoSens™ assay outcome was positive as a clear dose-dependent increase of luciferase in both experiments was measured. This indicates that the cells may have become activated after exposure to the test material, and the test material may exhibit skin sensitising properties. As contradicting results were found for the DPRA and KeratinoSens™ assay, performance of a confirmatory additional in vitro assay, a U-SENS™ assay, addressing the activation of dendritic cells should be considered. In case the outcome of the U-SENS™ assay is negative, the U-SENS™ assay confirms the DPRA assay, but contradicts the DEREK and KeratinoSens™ assay, and no unambiguous conclusion can be determined. In case of a positive outcome the substance is confirmed to be a sensitiser, however, potency would still need to be determined. The DEREK assessment is considered not sufficiently reliable to determine potency and at this juncture no accepted in vitro studies are available to determine potency and differentiate between skin sensitiser categories. As the current information obtained with the in vitro test strategy does not allow a conclusion on the skin sensitisation potential and classification of the substance, it is, as a last resort, justified to continue with in vivo testing (LLNA or GPMT). The result from this test will be used to determine the skin sensitising potential and classification for the test material for skin sensitisation according to Regulation (EC) No 1272/2008 and related amendments.

Interpretation of results:

study cannot be used for classification

Conclusions:

As the current information obtained with the in vitro test strategy does not allow a conclusion on the skin sensitisation potential and classification of the substance, it is, as a last resort, justified to continue with in vivo testing (LLNA or GPMT). The result from this test will be used to determine the skin sensitising potential and classification for the test material for skin sensitisation according to Regulation (EC) No 1272/2008 and related amendments.

Executive summary:

The objective of this study was to reach an overall conclusion on the endpoint skin sensitisation based on all available relevant information, including in silico, in chemico and in vitro data.

A DEREK assessment, a DPRA and a KeratinoSens™ assay were performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a.

A DEREK prediction on the skin sensitising potential of the test material was positive based on the presence of a sulphonate. The DPRA was negative as the test material did not significantly deplete the SPCC and SPCL moieties. However, since precipitation was observed in the SPCL samples the result may be under-estimated and should be interpreted with care. The KeratinoSens™ assay outcome was positive as a dose-dependent increase of luciferase was measured indicating that the cells may have become sensitised after exposure to the test material. Irrespective of the outcome of an additional U-SENS™ assay an unambiguous conclusion on skin sensitisation potential or potency cannot be drawn. Therefore, as a last resort, it is considered justified to continue with an in vivo test. The result will be used to determine and classify the skin sensitisation properties of the test material according to Regulation (EC) No 1272/2008 and related amendments.

Reference 5

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 October 2019 to 05 February 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

July 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

- Purity/Composition correction factor: No correction factor required

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: inbred, SPF-quality
- Age at study initiation: Young adult animals (approximately 10 - 11 weeks old)
- Weight at study initiation: 18.7 - 24.6 g
- Housing: group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages containing sterilised sawdust as bedding material and equipped with water bottles. For psychological/environmental enrichment, animals were provided with paper and shelters.
- Diet: pelleted rodent diet, ad libitum
- Water: municipal tap-water, ad libitum
- Acclimation period: at least 5 days before the commencement of dosing

ENVIRONMENTAL CONDITIONS
- Temperature: 22 - 23 °C
- Humidity: 42 - 51 % (relative)
- Air changes: 10 or more air changes per hour with 100 % fresh air (no air recirculation)
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES
- From: Not reported
- To: 03 February 2020

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

0 (vehicle control), 25, 50 and 100 % (w/w)

No. of animals per dose:

Five females per group

Details on study design:

PRE-SCREEN TESTS
A pre-screen test was conducted in order to select the highest test material concentration to be used in the main study. Two test material concentrations were tested: 50 and 100 % concentrations. The highest concentration was the maximum concentration as required in the test guidelines. As hunched posture was noted in the main study following the first pre-screen test, another pre-screen was performed at test material concentrations of 50 and 100 % to clarify the clinical signs as seen for the main study.
The test system, procedures and techniques were identical to those used in the main study except that the assessment of lymph node proliferation and necropsy were not performed. Two young adult females per concentration were selected. Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

MAIN STUDY
- Induction: Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test material, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test material.
- Excision of the Nodes: Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline containing 20 μCi of 3H-methyl thymidine. After five hours, all animals were euthanised by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20 %. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in PBS.
- Tissue Processing for Radioactivity: Day 6
Following excision of the nodes, a single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (maze size: 200 μm, diameter: ± 1.5 cm). LNC were washed twice with an excess of PBS by centrifugation at 200 g for 10 minutes at 4 °C. To precipitate the DNA, the LNC were exposed to 5 % trichloroacetic acid (TCA) and then stored in the refrigerator until the next day.
- Radioactivity Measurements: Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2910TR). Counting time was to a statistical precision of ± 0.2 % or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).
- Observations and measurements
Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from their cage during observation, unless necessary for identification or confirmation of possible findings.
All the animals were examined for reaction to dosing (once daily on Days 1-6; on Days 1-3 between 3 and 4 hours after dosing). The onset, intensity and duration of these signs was recorded (if appropriate), particular attention being paid to the animals during and for the first hour after dosing. Post-dose observations were performed once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Animals were weighed individually on Day 1 (pre-dose) and 6 (prior to necropsy).
Erythema and eschar formation observations were performed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) and scored as follows:
No erythema: 0
Very slight erythema (barely perceptible): 1
Well-defined erythema: 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth): 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema: 4

No necropsy was performed, since all animals survived until the end of the observation period.

ANIMAL ASSIGNMENT
- Animal assignment: Animals were assigned to the study at the discretion of the co-ordinating biotechnician, with all animals within ± 20 % of the sex mean body weights. Animals in poor health or at extremes of body weight range were not assigned to the study.

TREATMENT PREPARATION AND ADMINISTRATION
Test material dosing formulations (w/w) were homogenised to visually acceptable levels at appropriate concentrations to meet dose level requirements.
The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test material.
The dosing formulations were kept at room temperature until dosing. The dosing formulations were stirred until and during dosing.
No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/composition of the test material, since the test method requires a logical concentration range rather than specific dose levels.
Any residual volumes were discarded.

DATA EVALUATION
DPM values were recorded for each animal and for each dose group. A Stimulation Index (SI) was calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
If the results indicate a SI ≥ 3, the test material may be regarded as a skin sensitiser.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Not performed

Positive control results:

A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by the Test Facility. In this study, performed in December 2019, females of the CBA/J mouse strain were checked for sensitivity to Alpha-Hexylcinnamaldehyde, technical grade (HCA). The females were approximately 10 weeks old at commencement of the study. The study was based on the OECD 429, EU Method B.42 and EPA OPPTS 870.2600. Alpha-Hexylcinnamaldehyde, technical grade was tested at concentrations of 5, 10 and 25 % in acetone/olive oil (4:1 v/v; AcOO) and afforded the following results:

0 % HCA: mean DPM ± SEM = 433 ± 73; SI ± SEM = 1.0 ± 0.2
5 % HCA: mean DPM ± SEM = 726 ± 55; SI ± SEM = 1.3 ± 0.3
10 % HCA: mean DPM ± SEM = 1481 ± 324; SI ± SEM = 3.4 ± 0.7
25 % HCA: mean DPM ± SEM = 2385 ± 583; SI ± SEM = 5.5 ± 1.3

The SI values calculated for the HCA concentrations 5, 10 and 25 % were 1.3, 3.4 and 5.5, respectively. An EC3 value of 9.0 % was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5 %.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at the Test Facility is an appropriate model for testing for contact hypersensitivity.

Parameter:

SI

Remarks:

(mean SI, based on mean DPM)

Value:

1

Variability:

± 0.3 (Standard Error of the Mean)

Test group / Remarks:

vehicle control (0 % test material in vehicle)

Key result

Parameter:

SI

Remarks:

(mean SI, based on mean DPM)

Value:

1.7

Variability:

± 0.3 (Standard Error of the Mean)

Test group / Remarks:

25 % test material in vehicle

Key result

Parameter:

SI

Remarks:

(mean SI, based on mean DPM)

Value:

2

Variability:

± 0.2 (Standard Error of the Mean)

Test group / Remarks:

50 % test material in vehicle

Key result

Parameter:

SI

Remarks:

(mean SI, based on mean DPM)

Value:

3

Variability:

± 0.7 (Standard Error of the Mean)

Test group / Remarks:

100 % test material in vehicle

Key result

Parameter:

SI

Remarks:

(mean SI, based on median DPM)

Value:

2.6

Test group / Remarks:

25 % test material in vehicle

Key result

Parameter:

SI

Remarks:

(mean SI, based on median DPM)

Value:

3.2

Test group / Remarks:

50 % test material in vehicle

Key result

Parameter:

SI

Remarks:

(mean SI, based on median DPM)

Value:

4.1

Test group / Remarks:

100 % test material in vehicle

Cellular proliferation data / Observations:

PRE-SCREEN TEST
Initially, a pre-screen test was performed at 50 and 100 % test material concentrations. Because no signs of systemic toxicity were noted and only very slight irritation was observed, a 100 % concentration was selected as highest concentration for the main study. The second pre-screen study confirmed the results of the first pre-screen test.

MAIN STUDY
Initially, a main study was performed by dosing three groups of five animals with one test material concentration per group. The highest test item concentration was selected from the pre-screen test. One group of five animals was treated with the vehicle. Unexpectedly, clinical signs (hunched posture on Days 2 and 3 for the majority of test material dosed animals and for one test material dosed animal on Day 4) were present in the animals of the main study and because the test guideline states that “doses should be used that do not induce systemic toxicity”, the results obtained were declared invalid and not used for interpretation. The main study was repeated using the same concentrations because the final results of the pre-screen study did indicate that no clinical signs were to be expected.

MACROSCOPIC EXAMINATION OF THE LYMPH NODES AND SURROUNDING AREA
The majority of auricular lymph nodes were considered normal in size, except for the nodes of one animal dosed at 50 % test material concentration and in all animals dosed at 100 % test material concentration, which were slightly enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

DETAILS ON STIMULATION INDEX
Mean DPM/animal values for the experimental groups treated with test material concentrations 25, 50 and 100 % were 1282, 1458 and 2210 DPM, respectively. The mean DPM/animal value for the vehicle control group was 738 DPM. The SI values calculated for the test material concentrations 25, 50 and 100 % were 1.7, 2.0 and 3.0, respectively.
As the mean values showed a large variation, the SI values were also calculated based on the median values. Median DPM/animal values for the experimental groups treated with test material concentrations 25, 50 and 100 % were 1277, 1584 and 2022 DPM, respectively. The median DPM/animal value for the vehicle control group was 495 DPM. The SI values calculated for the test material concentrations 25, 50 and 100 % were 2.6, 3.2 and 4.1, respectively.
The data showed a dose-response and an EC3 value (the estimated test material concentration that will give a SI = 3) of 100 % was calculated based on the mean values.

CLINICAL OBSERVATIONS
- Mortality/Moribundity Checks: No mortality occurred.
- Clinical Observations (Post-dose Observations): No clinical signs of systemic toxicity were observed in the animals.
- Irritation: Very slight irritation of the ears was shown by all animals dosed at 100 and 50 % test material concentrations between Days 1 and 4; this was considered not to have a toxicologically significant effect on the activity of the nodes.
- Body weight: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Interpretation of results:

other: Skin Sensitiser (Category 1B) according to EU criteria

Conclusions:

Findings from the study indicate that the test material could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test material concentration that will give a SI = 3) of 100 % was calculated based on the mean values. The test material should therefore be classified as a skin sensitiser (category 1B) according to EU criteria.

Executive summary:

The skin sensitisation potential of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 429, EU Method B.42 and EPA OPPTS 870.2600, and under GLP conditions.

Test material concentrations selected for the main study were based on the results of a pre-screen test. Based on the results, the highest concentration required according to the guidelines was selected.

In the main study, three experimental groups of five female CBA/J mice were treated with test material concentrations of 25, 50 or 100 % w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (acetone/olive oil). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

The majority of auricular lymph nodes were considered normal in size, except for the nodes of one animal dosed at 50 % test material concentration and in all animals dosed at 100 % test material concentration, which were slightly enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Mean DPM/animal values for the experimental groups treated with test material concentrations 25, 50 and 100 % were 1282, 1458 and 2210 DPM, respectively. The mean DPM/animal value for the vehicle control group was 738 DPM. The SI values calculated for the test material concentrations 25, 50 and 100 % were 1.7, 2.0 and 3.0, respectively.

Because the mean values showed a large variation, the SI values were also calculated based on the median values. Median DPM/animal values for the experimental groups treated with test material concentrations 25, 50 and 100 % were 1277, 1584 and 2022 DPM, respectively. The median DPM/animal value for the vehicle control group was 495 DPM. The SI values calculated for the test material concentrations 25, 50 and 100 % were 2.6, 3.2 and 4.1, respectively.

The six-month reliability check with alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at the Test Facility is an appropriate model for testing for contact hypersensitivity.

The results indicate that the test material could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test material concentration that will give a SI = 3) of 100 % was calculated based on the mean values. Accordingly, the test material is classified as a skin sensitiser (category 1B) according to EU criteria.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

in silico / in chemico / in vitro

DEREK

The in silico model, DEREK NEXUS (version 6.0.1), was used to obtain a prediction on the potential for skin sensitisation of the test material. The study was awarded a reliability score of 2 in accordance with the criteria set forth by Klimisch et al. (1997).

DEREK NEXUS did yield a skin sensitisation alert for the test material based on the presence of a sulphonate and predicted the test material to be sensitising to the skin (plausible). The potential mechanism behind the alert is that the substance can directly alkylate nucleophilic centres in skin proteins.

DEREK NEXUS predicted an EC3 of 2.1 % (moderate sensitiser) for the test material based on LLNA data from ten structurally related analogues (structural similarity of 11 - 40 %). The query compound contains a sulphonic butyl ester and a toluene group. These structural fragments are of influence on the reactivity of alkylating capabilities of the compound. Considering both structural fragments are not present simultaneously within its closest structurally related analogues, the EC3 prediction is considered not sufficiently reliable for classification purposes.

In conclusion, the test material is predicted to be sensitising to the skin. The predicted EC3 of 2.1 % is not considered to be reliable.

Direct Peptide Reactivity Assay

The reactivity of the test material towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was investigated in a study which was conducted in accordance with the standardised guideline OECD 442C and under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

During the study the test material was incubated with either SPCC or SPCL and the relative peptide concentration determined by High-Performance Liquid Chromatography (with gradient elution and spectrophotometric detection at 220 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.

Upon preparation in acetonitrile, as well as after incubation of the SPCC test material samples, no precipitate or phase separation was observed in any of the samples. After incubation of the SPCL test material samples, a precipitate was observed.

In the cysteine reactivity assay the test material showed 7.3 % SPCC depletion while in the lysine reactivity assay the test material showed 3.5 % SPCL depletion. The mean of the SPCC and SPCL depletion was 5.4 %. Cinnamic aldehyde was used as positive control and showed 92.5 % SPCC depletion while in the lysine reactivity assay it showed 65.4 % SPCL depletion.

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.

In conclusion, since all acceptability criteria were met this DPRA is considered to be valid. The mean value of SPCC and SPCL depletion of 5.4 % indicates that the test material is 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. However, since precipitation was observed after the incubation period for SPCL, it is not clear 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.

KeratinoSens™

The skin sensitisation potential of the test material was investigated in vitro 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 objective of the study was to evaluate the ability of the test material to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens™ assay.

During the study, the 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. The test material precipitated at the highest dose level tested in the second experiment only. Two independent experiments were performed.

Both experiments passed the acceptance criteria. It is therefore concluded that the test conditions were adequate and that the test system functioned properly.

Under the conditions of the study, the test material showed toxicity only in the first experiment (IC30 value of 353 μM). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 82 and 68 μM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 8.83-fold and 8.39-fold in experiment 1 and 2, respectively. The test material therefore fulfils the criteria for a positive classification in the KeratinoSens™ assay since positive results (> 1.5-fold induction) were observed at test concentrations < 1000 μM with a cell viability of > 70 % compared to the vehicle control.

In conclusion, the test material is classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described.

Weight of evidence of data from in silico, in chemico and in vitro studies

The objective of this study was to reach an overall conclusion on the endpoint skin sensitisation based on all available relevant information, including in silico, in chemico and in vitro data.

A DEREK assessment, a DPRA and a KeratinoSens™ assay were performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a.

A DEREK prediction on the skin sensitising potential of the test material was positive based on the presence of a sulphonate. The DPRA was negative as the test material did not significantly deplete the SPCC and SPCL moieties. However, since precipitation was observed in the SPCL samples the result may be under-estimated and should be interpreted with care. The KeratinoSens™ assay outcome was positive as a dose-dependent increase of luciferase was measured indicating that the cells may have become sensitised after exposure to the test material. Irrespective of the outcome of an additional U-SENS™ assay an unambiguous conclusion on skin sensitisation potential or potency cannot be drawn. Therefore, as a last resort, it is considered justified to continue with an in vivo test. The result will be used to determine and classify the skin sensitisation properties of the test material according to Regulation (EC) No 1272/2008 and related amendments.

In vivo

Local Lymph Node Assay

The skin sensitisation potential of the test material was investigated in a study which was conducted in accordance with the standardised guidelines OECD 429, EU Method B.42 and EPA OPPTS 870.2600, and under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Test material concentrations selected for the main study were based on the results of a pre-screen test. Based on the results, the highest concentration required according to the guidelines was selected.

In the main study, three experimental groups of five female CBA/J mice were treated with test material concentrations of 25, 50 or 100 % w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (acetone/olive oil). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

The majority of auricular lymph nodes were considered normal in size, except for the nodes of one animal dosed at 50 % test material concentration and in all animals dosed at 100 % test material concentration, which were slightly enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals. Mean DPM/animal values for the experimental groups treated with test material concentrations 25, 50 and 100 % were 1282, 1458 and 2210 DPM, respectively. The mean DPM/animal value for the vehicle control group was 738 DPM. The SI values calculated for the test material concentrations 25, 50 and 100 % were 1.7, 2.0 and 3.0, respectively.

Because the mean values showed a large variation, the SI values were also calculated based on the median values. Median DPM/animal values for the experimental groups treated with test material concentrations 25, 50 and 100 % were 1277, 1584 and 2022 DPM, respectively. The median DPM/animal value for the vehicle control group was 495 DPM. The SI values calculated for the test material concentrations 25, 50 and 100 % were 2.6, 3.2 and 4.1, respectively.

The six-month reliability check with alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at the Test Facility is an appropriate model for testing for contact hypersensitivity.

The results indicate that the test material could elicit a SI ≥ 3. The data showed a dose-response and an EC3 value (the estimated test material concentration that will give a SI = 3) of 100 % was calculated based on the mean values. Accordingly, the test material is classified as a skin sensitiser (category 1B) according to EU criteria.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance requires classification with respect to skin sensitisation (category 1B) and is assigned the hazard statement H317 (May cause an allergic skin reaction).