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Description of key information

The test substance was not considered to be sensitizing to the skin in an OECD 442C study and an OECD 442D study. Based on the current data-set, there are no indications that the test substance has skin sensitising properties. Therefore it is concluded that it is scientifically not necessary to conduct further testing for skin sensitizing properties and that the test substance is not classified for this endpoint.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
6 Mar 2017 to 17 Mar 2017
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:
Februari 2015
GLP compliance:
yes
Type of study:
other: in vitro ARE-Nrf2 luciferase test method (KeratinoSensTM)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Identification: 1184 ZITRONENSÄURE, TEA-SALZ
- Source of test material: Zschimmer and Schwarz GmbH + Co. KG.
- Batch: SEALS 2016-198-001
- Purity/Composition: UVCB

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Test item storage: At room temperature
- Stable under storage conditions until: 03 January 2018 (expiry date)

OTHER
- Purity/Composition correction factor: Yes, according to the purity 1.28
Details on the study design:
CONTROLS
- vehicle control: DMSO
- positive control: Ethylene dimethacrylate glycol (CAS 97-90-5)

PREPARATION OF TEST ITEM STOCK, SPIKING AND WORKING SOLUTIONS
- A correction was made for the purity/composition of the test item. A correction factor of 1.28 was used. The test item solutions were prepared based on the molecular weight specified in gram dry matter/mol (639.71 gram dry weight/mol).
- A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 200 mM (study plan deviation). This stock was a clear colourless solution. The 100-fold dilution of the 200 mM DMSO stock in DMEM showed no precipitation (final concentration 2000 μM). This concentration was selected as highest concentration for the main assay.
- In the main assay a 200 mM stock solution in DMSO was prepared. From the stock 11 spike solutions were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium (final concentration DMSO of 4%). These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95 and 0.977 μM (final concentration DMSO of 1%; study plan deviation). All concentrations of the test item 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 (MTT-assay; study plan deviation).

PREPARATION OF THE POSITIVE CONTROL
For ethylene dimethacrylate glycol a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted such that the final concentration of the positive control ranges from 7.81 to 250 μM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.

SOLVENT CONTROL
The solvent control was 1% DMSO in exposure medium. Eighteen wells were tested per plate.

BLANK
On each plate three blank wells were tested (no cells and no treatment; study plan deviation).

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). 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 (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

CELL CULTURE
- Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum.
- Maintenance medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum and geneticin (500 μg/mL).
- Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal 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 51 – 86 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.4 – 37.3°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.
- 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 (actual passage number used 22 and 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.
- 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 items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37±1.0°C in the presence of 5% CO2. In total 2 experiments were performed.
- Luciferase Activity Measurement: The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is 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 luminometer to assess the quantity of luciferase (integration time one second).
- Cytotoxicity Assessment: For the KeratinoSensTM 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 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 with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSensTM test is considered acceptable if it meets the following criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.81 to 250 μM).
- The EC1.5 should be between 5 and 125 μM. 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.
- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.

DATA ANALYSIS
The following parameters are calculated in the KeratinoSensTM 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.

DATA INTERPRETATION
A KeratinoSensTM 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 KeratinoSensTM prediction is considered negative:
- The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s ttest)
- 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)
- The EC1.5 value is less than (<) 1000 μM (or < 200 μg/mL for test chemicals with no defined MW
- There is an apparent overall dose-response for luciferase induction
Negative results obtained with concentrations <1000 μM or 200 μg/mL should be considered as inconclusive.
Positive control results:
EXPERIMENT 1
- Imax: 2.02 μM
- EC1.5: 100.6 μM

EXPERIMENT 2
- Imax: 4.03 μM
- EC1.5: 35.7 μM
Key result
Run / experiment:
other: Experiment 1 and 2
Parameter:
other: viability (%)
Value:
70
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
other: no toxicity
Key result
Run / experiment:
other: Experiment 1
Parameter:
other: Imax (µM)
Remarks:
represents the maximal average fold induction of luciferase activity
Value:
0.92
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
no EC1.5 could be calculated
Key result
Run / experiment:
other: Experiment 2
Parameter:
other: Imax (µM)
Remarks:
represents the maximal average fold induction of luciferase activity
Value:
1.09
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
no EC1.5 could be calculated
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS
- Acceptance criteria for negative control are met.
- Acceptance criteria for positive control are met.
- Acceptance criteria for variability between replicate measurements are met.
Interpretation of results:
GHS criteria not met
Remarks:
In combination with the DPRA Assay (OECD 442C)
Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
6 Mar 2017 to 9 Mar 2017
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:
4 February 2015
GLP compliance:
yes
Type of study:
direct peptide reactivity assay (DPRA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Identification: 1184 ZITRONENSÄURE, TEA-SALZ
- Source of test material: Zschimmer and Schwarz GmbH + Co. KG.
- Batch: SEALS 2016-198-001
- Purity/Composition: UVCB
- Stable under storage conditions until: 03 January 2018 (expiry date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Test item storage: At room temperature - Stability under test conditions:

OTHER SPECIFICS:
- Appearance: Clear colourless liquid (determined by Charles River Den Bosch)
- Purity/Composition correction factor: Yes, according to the purity: 1.28
- pH (1% in water, indicative range): 6.67 – 6.65 (determined by Charles River Den Bosch)
- Test Item Characterization: The Sponsor provided to the Test Facility documentation of the identity, purity, composition, and stability for the test item. A Certificate of Analysis or equivalent document was provided to the Test Facility.
Details on the study design:
SAMPLE COLLECTION AND ANALYSIS
No analysis of the formulated test item was conducted for this study with respect to either test item concentration, homogeneity or of the test item in the vehicle. Nevertheless, according to OECD Guidelines relative to the application of the Good Laboratory Practice Principles to short-term studies, the preparations were performed with approved procedures and documented in detail. Preparations were visually homogenous prior to use and all preparations were used within 4 hours after adding the vehicle to the test item. This GLP exception was therefore considered as being minor with no impact on the outcomes and the integrity and the achievement of the objective of the study.

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 SPCC: JE.#, 30737_1; PO.#, 20164384
- Batch SPCL: JE.#, 31276_1; PO.#, 20165265
- Storage: The peptides were stored in the freezer (≤ 15°C) for a maximum of 6 months.

TEST ITEM PREPARATION
- A correction factor of 1.28 was used to correct for purity/composition of the test item. Test item solutions were prepared based on the molecular weight specified in gram dry matter/mol.
- Solubility of the test item in an appropriate solvent was assessed before performing the DPRA. An appropriate solvent dissolved the test item 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) and MQ/ACN (1:1, v/v).
- Test substance stock solutions were prepared freshly for each reactivity assay.
- For the cysteine and lysine reactivity assay 110.9 mg of test substance was pre-weighed into a clean amber glass vial and dissolved, just before use, in 1354 µL MQ/ACN (1:1, v/v) to obtain a 100 mM solution. Visual inspection of the forming of a clear solution was considered sufficient to ascertain that the test item was dissolved. The test item, 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.

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 mg of SPCC in 19.96 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. In addition, a RCcysCMQ/ACN sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCcysCMQ/ACN sample was prepared by mixing 750 µL of the 0.667 mM SPCC stock solution with 200 µL ACN and 50 µL MQ/ACN (1:1, v/v).
- SPCC Calibration Curve: A SPCC calibration curve was prepared as described in Table 1 under ‘Any other information on materials and methods incl. tables’.
- Co-elution Control, Test Item and Positive Control Samples: The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in Table 2 under ‘Any other information on materials and methods incl. tables’.

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 mg of SPCL in 19.31 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. In addition, a RCclysCMQ/ACN sample was included to evaluate the effect of the solvent that was used to dissolve the test item on the Percent Peptide Depletion. The RCclysCMQ/ACN sample was prepared by mixing 750 µL of the 0.667 mM SPCL stock solution with 250 µL MQ/ACN (1:1, v/v).
- SPCL Calibration Curve: A SPCL peptide calibration curve was prepared as described in Table 3 under ‘Any other information on materials and methods incl. tables’.
- Co-elution Control, Test Item and Positive Control Samples: The co-elution control (CC) samples, test item samples and the cinnamic aldehyde positive control samples (PC) were prepared as described in Table 4 under ‘Any other information on materials and methods incl. tables’.

SAMPLE INCUBATIONS
- After preparation, the samples (reference controls, calibration solutions, co-elution control, positive controls and test item 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 26.5-31.5 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.

HPLC-PDA ANALYSIS
- SPCC and SPCL peak areas in the samples were measured by HPLC PDA. Sample analysis was performed using the following system:
- 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)
- All samples were analyzed according to the HPLC-PDA method presented in Table 5 (‘Any other information on materials and methods incl. tables’)

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 item’s results to be considered valid:
- The maximum SD for the test item 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.

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.
- 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 substance. Negative depletion was considered as “0” when calculating the mean. By using the Cysteine 1:10 / Lysine 1:50 prediction model (see Table 6 under ‘Any other information on materials and methods incl. tables’), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer.
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 71.8% ± 1.5%. 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 58.5% ± 2.1%. 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
Parameter:
other: Mean of SPCC and SPCL depletion
Value:
0.4
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: Mean Percent SPCC Depletion
Parameter:
other: Cysteine reactivity
Value:
0.1
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Run / experiment:
other: Mean Percent SPCL Depletion
Parameter:
other: Lysine reactivity
Value:
0.7
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
CYSTEINE REACTIVITY ASSAY
- Upon preparation and after approximately 24 hours of incubation , no precipitate was observed in any of the samples
- There was no co-elution of the test item with SPCC
- The acceptance criteria for the calibration curve are met
- The acceptance criteria for the positive control are met
- The acceptance criteria for the Reference controls are met
- The acceptance criteria for variability are met

LYSINE REACTIVITY ASSAY
- Upon preparation and after approximately 24 hours of incubation , no precipitate was observed in any of the samples
- There was no co-elution of the test item with SPCL
- The acceptance criteria for the calibration curve are met
- The acceptance criteria for the positive control are met
- The acceptance criteria for the Reference controls are met
- The acceptance criteria for variability are met

Table. SPCC and SPCL Depletion and Reactivity Classification for the test substance

Test item

SPCC depletion

SPCL depletion

Mean of SPCC and SPCL depletion

Reactivity class

Mean

± SD

Mean

± SD

Cysteine 1:10 / Lysine 1:50 prediction model

test substance

0.1%

±0.2%

0.7%

±1.2%

0.4%

No or minimal reactivity

SD = Standard Deviation.

Interpretation of results:
GHS criteria not met
Remarks:
In combination with the KeratinoSensTM Assay (OECD 442D)
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Skin sensitisation in vitro

In a GLP compliant study according to OECD 442D the ability of the test substance to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay was evaluated. The batch of the test item was a clear colourless liquid. A correction factor of 1.28 was used to correct for the purity. The test item was dissolved in dimethyl sulfoxide 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 0.977 – 2000 μM (2-fold dilution series). Two independent experiments were performed.

Both experiments passed the acceptance criteria: 1) The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration. 2) The EC1.5 of the positive control was between 5 and 125 μM (101 μM and 35.7 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.02-fold and 4.03-fold in experiment 1 and 2, respectively). 3) The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (15.7% and 13.8% 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 item showed no toxicity (no IC30 and IC50 value) and 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 0.92-fold and 1.09-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations of ≥1000 μM with a cell viability of >70% compared to the vehicle control. In conclusion, the test substance is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described.

 

Skin sensitisation in chemico

The reactivity of the test substance towards model synthetic peptides containing either cysteine (SPCC) or lysine (SPCL) was determined in a GLP compliantin chemico study according to OECD 442C. After incubation of the test substance 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 chemical to one of four reactivity classes used to support the discrimination between sensitizers and non-sensitizers. MQ/ACN (1:1, v/v) was found to be an appropriate solvent to dissolve the test substance and was therefore used in this Direct Peptide Reactivity Assay (DPRA) study. The validation parameters, i.e. calibration curve, mean concentration and the Coefficient of Variation for Reference Control samples, 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 substance, were all within the acceptability criteria for the DPRA. No co-elution of the test item with SPPC or SPCL was observed. In the cysteine reactivity assay the test item showed 0.1% SPCC depletion while in the lysine reactivity assay the test item showed 0.7% SPCL depletion. The mean of the SPCC and SPCL depletion was 0.4%. As a result the test substance was 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 substance 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.

Overall conclusion

A Weight of Evidence evaluation was prepared to reach an overall conclusion on the endpoint skin sensitization based on all available relevant information (see attached document for the comprehensive Weight of Evidence evaluation).

A DPRA assay and KeratinoSensTM assay were performed in accordance with the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a. The test substance was negative in the DPRA and classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model. Furthermore, the test substance gave a negative result in the KeratinoSensTM assay (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes).

Based on the current data-set there are no indications that the test substance has skin sensitizing properties. Therefore it is concluded that it is scientifically not necessary to conduct further testing for skin sensitizing properties and that the test substance is not classified for this endpoint.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results of the available studies, classification for skin sensitisation is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.