2,4-dimethylpentan-3-one

Endpoint summary

Description of key information

Sensitization involves a number of key steps in order to take place, and can be described in terms of an adverse outcome pathway (AOP). These include reactivity with skin proteins (peptide (cysteine and lysine) reactivity), activation of skin cells (keratinocyte activation) and immune cells (dendritic cell activation). The studies carried out for this substance address these three key steps. The results are used in a predefined evaluation scheme ("2 out of 3"-approach) to determine hazard classification as a sensitizer by a weight-of-evidence approach. Based on the in vitro results, the substance is considered to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

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:

2015

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

Turnkey Testing Strategy

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: BASF SE

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: After short stirring the test substance was soluble in the vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was prepared as a 100 mM preparation in acetonitrile. After short stirring the test substance was soluble in the vehicle.

OTHER SPECIFICS: The OECD toolbox did not indicate an alert for protein binding for either the substance or its predicted metabolites (auto-oxidation, hydrolysis, and skin metabolism).

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
The test substance was dissolved in a suitable vehicle. Per run three samples of the test substance were incubated with each peptide. Additionally, triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. The remaining non-depleted peptide concentration was determined thereafter by HPLC with gradient elution and UV-detection at 220 nm. In addition, calibration samples of known peptide concentration, prepared from the respective peptide stock solution used for test-substance incubation, were measured in parallel with the same analytical method.

Prior to the assay the solubilty of the test substance at a concentration of 100 mM was tested. A suitable non-reactive, water-miscible solvent which dissolves the test substance completely (no visible precipitation or cloudyness of the test substance preparation) should be used. The preferred solvent was acetonitrile. When not soluble in acetonitrile solutions in water, isopropanol, acetone, propanol, methanol or mixtures of these solvents were tried.

Peptide stock solutions in a concentration of 0.667 mM were prepared in pH 7.5 phosphate buffer (C-containing peptide) or pH 10.2 ammonium acetate buffer (K-containing peptide). The peptide stock solution was used for preparing the calibration samples and the test substance and control samples.

The test substance samples were prepare din triplicates for each peptide.
C-peptide: 750 µL C-peptide stock-solution, 200 µL solvent (vehicle), 50 µL test substance preparation (or PC preparation or solvent (VC)).
K-peptide: 750 µL K-peptide stock-solution, 250 µL test substance preparation (or PC preparation or solvent (VC)).
The samples were prepared in suitable tubes, capped tightly and incubated at 25 °C ± 2.5 °C in the dark for 24 ± 2 h. Visual inspection for solubility was performed directly after sample preparation and prior to HPLC analysis. Unsolved samples were centrifuged or filtrated prior to injection into the HPLC in order to remove any unsolved particles. The HPLC analysis of the batch of samples started about 24 h after sample preparation and the analysis time itself did not exceed 30 h.

Several vehicle controls were prepared in triplicates in the same way as the test substance samples described above with the vehicle (acetonitrile) instead of the test substance: One set (set A) was analyzed together with the calibration samples without incubation and serves as a performance control. Another three sets (two sets B and set C) were prepared and incubated with the samples. Sets B were placed at the very start and ending of the sample list and serve as stability control of the peptide over the analysis time. Set C was analyzed with the samples and serves for calculation of the peptide depletion of any chemical formulated in the vehicle.

Co-elution control: One sample per peptide was prepared in the same way as the test substance samples described above but without the peptides. Instead the respective peptide buffer was used. The samples were analyzed together with the calibration samples. Samples which were visually turbid or display precipitates were centrifuged or filtrated prior to injection into the HPLC in order to remove any unsolved particles.

Controls:
- Negative control (NC): vehicle control = acetonitrile
- Positive control (PC): Ethylene glycol dimethacrylate, prepared as a 50 mM solution in acetonitrile.
- Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide.

Key result

Run / experiment:

other: mean of 2 runs

Parameter:

other: mean peptide depletion

Value:

8.57

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: low chemical reactivity

Key result

Run / experiment:

other: 2

Parameter:

other: mean C-peptide depletion

Value:

15.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: mean K-peptide depletion

Value:

1.24

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: Two test runs were performed for the C-containing peptide due to relative high standard deviation of the three values measured in the first test run and as the result was close to the evaluation threshold.

Interpretation of results:

other: low chemical reactivity

Conclusions:

The mean peptide depletion was calculated to be 8.57 %. Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance shows low chemical reactivity in the DPRA under the test conditions chosen.

Executive summary:

The reactivity of the test substance towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose, the test substance was incubated with the synthetic peptides for ca. 24 h at ca. 25 °C and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm.

The test substance was dissolved at a 100 mM concentration in acetonitrile. One test run was performed for the K-containing peptide and two test runs for the C-containing peptide. Per test run three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-containing peptide) or 1:50 (for K-containing peptide). Additionally, triplicates of the concurrent vehicle control (= NC) were incubated with the peptides.

Further, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover, the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 nm / 258 nm was calculated as a measure of peak purity.

The following results were obtained in the DPRA:

The test substance was dissolved in acetonitrile at a concentration of 100 mM. The samples of the test substance with the peptides were solutions at the time of preparation. Visual observation after the 24 h incubation time did not reveal precipitates in any samples of the test substance with the peptides.

No co-elution of test substance and peptides was present.

Two test runs were performed for the C-containing peptide due to relative high standard deviation of the three values measured in the first test run and as the result was close to the evaluation threshold.

The mean C-peptide depletion, caused by the test substance was determined to be 14.64 % in the first test run and 17.17 % in the second test run. The mean C-peptide depletion of both test runs was determined to be 15.90 %.

The mean K-peptide depletion, caused by the test substance was determined to be 1.24 %.

Thus, the mean peptide depletion was calculated to be 8.57 %.

Based on the observed results and applying the cysteine 1:10 / lysine 1:50 prediction model it was concluded that the test substance shows low chemical reactivity in the DPRA under the test conditions chosen.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E: In Vitro Skin Sensitization (Activation of Dendritic Cells)

Version / remarks:

2016

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

Turnkey Testing System

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: BASF SE

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: After short stirring the test substance was soluble in the vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was weighed and topped up with the chosen vehicle (culture medium) to achieve the required 2 x concentration of the highest concentration (stock solution). Further concentrations were prepared as 2 x concentrations by serila 1:1.2 dilution according to the planned concentrations in culture medium. The test substance preparations were prepared by stirring and ultra-sonication.

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
THP-1 cells from the working cell bank were thawed and cultured in suspension using complete RPMI 1640 medium supplemented with 10 % fetal bovine serum (heat inactivated), 100 U/mL penicillin, 100 µg/mL streptomycin and 0.05 mM 2-mercaptoethanol under standard culture conditions (37 °C, ca. 5 % CO2, ≥ 90 % humidity) until for 5 passages but no longer than passage 30 prior to testing.
Prior to use of the cells for a study, a reactivity check is performed with each new-thawed cells, as proposed in the OECD test guideline, using Nickel(II)sulfate hexahydrate, lactic acid and 1-chloro-2,4-dinitrobenzene in order to demonstrate qualification of the cells for the assay.
For substance incubation, cells were seeded in 24-well plates (500 µL of 2.0 x 10^6 cells/mL cell suspensions). Three independent, valid experiments were performed. In each experiment, duplicates of each test substance concentration were tested.

Treatment was performed by adding 500 µL of test substance preparation to the cells, thus diluting the 2 x concentrated test substance preparations to their final concentration and the cells to 1.0 x 10^6 cells/mL. After test substance application the plates were sealed with semi-permeable plate sealers in order to prevent evaporation of the test substance. The plates were placed into the incubator under standard culture conditions for the exposure period of 24 h.

Each test substance concentration was visually inspected directly after application and after the exposure period of 24 h in order to detect test substance precipitates.

After visual inspection the cells were transferred into safe-lock tubes, collected by centrifugation and washed twice with 1 mL buffer. Cells were incubated with 600 µL of 0.01 % Globulins Chon fraction II, III at 4 °C for 15 min to block FC receptors (FcR). After FcR blocking, cells of each treatment condition were divided into 3 aliquots (approximately 0.3 x 10^6 cells/180 µL/group) in 96-well microtiter plates. Cells were centrifuged, supernatant was discarded and 50 µL working antibody solution was added to each pellet. Working solutions of the antibodies were prepared as follows:
- Anti-CD86 antibody: 6 µL antibody, 44 µL buffer (total volume: 50 µL)
- Anti-CD54 antibody: 3 µL antibody, 47 µL buffer (total volume: 50 µL)
- Mouse IgG1: 3 µL antibody, 47 µL buffer (total volume: 50 µL)

Cell staining was performed at 4 °C for 30 min in the dark. After staining the cells were washed twice with 200 µL buffer and finally re-suspended in 200 µL buffer. Before analysis in flow cytometer the cells were stained with 5 µL of PI (50 µg/mL diluted in buffer) to yield a final concentration of 1.22 µg/mL PI.

Controls:
- Negative control (NC): Lactic acid, 1000 µg/mL in culture medium
- Positive control (PC): 1-chloro-2,4-dinitrobenzene, 4.0 µg/mL in 0.2 % DMSO in culture medium
- Vehicle control (VC): culture medium
- Isotype control: In order to help distinguish non-specific ("background") staining from specific antibody staining each test substance concentration and control is additionally incubated with mouse IgG1.

Key result

Run / experiment:

other: 2 experiments

Parameter:

other: relative viability

Value:

50

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: test substance induces dendritic cell activation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes

Interpretation of results:

other: test substance induces dendritic cell activation

Conclusions:

After 24 h of exposure to the test substance CD86 and CD54 expression was induced in THP-1 cells affording at least 50 % viability in at least two independent experiments .From this it has to be concluded that the test substance induces dendritic cell activation.

Executive summary:

The potential of the test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose the test substance was incubated with human monocytic leukemia cell line THP-1 for ca. 24 h at 37 °C and membrane marker expression (CD86 / CD54) was measured by flow cytometry.

In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP) was performed. Cells were exposed to 11 concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. No cytotoxicity was observed.

In the main test after 24 h exposure THP-1 cells were stained with FITC labeled anti-human-CD86 / anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analyzed using flow cytometry. A total of 3 valid and evaluable experiments were performed. A 1st experiment was not evaluable due to a technical error. The following results were observed:

In the main experiments, the test substance was soluble in culture medium (2 x stock preparations) at concentrations of 2917 µg/mL and below. Higher concentrations were unsolved (homogeneously). Final concentrations in culture medium were solutions at all concentrations. No precipitates were noticed in any concentration after 24 h.

In the 2nd experiment a positive result was obtained in both markers, however, in the 3rd experiment the positive result was not clearly reproduced due to high standard deviations. Hence, in order to clarify the result of the study, a 4th experiment was performed under the same conditions, although formally two valid and evaluable experiments with a positive result were available already.

The EC150% (the concentration resulting in a RFI of 150 %) for CD86 was calculated to be 3144 µg/mL (experiment 4). Calculation of an EC150% from experiments 2 and 3 is not applicable as the lowest tested concentration was positive, each. The EC200% (the concentration resulting in a RFI of 200 %) for CD54 was calculated to be 1756 µg/mL (experiment 3). Calculation of an EC200% from experiments 2 and 4 is not applicable as the lowest tested concentration was positive, each.

In summary, after 24 h of exposure to the test substance CD86 and CD54 expression was induced in THP-1 cells affording at least 50 % viability in at least two independent experiments. From this it has to be concluded that the test substance induces dendritic cell activation.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

2015

GLP compliance:

yes (incl. QA statement)

Remarks:

(from the competent authority) Landesamt für Umwelt Rheinland-Pfalz

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

Turnkey Testing Strategy

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: BASF SE

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Solubility and stability of the test substance in the solvent/vehicle: After short stirring the test substance was soluble in the vehicle.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was weighed and topped up with the chosen vehicle (DMSO) to achieve the required 100 x concentration of the highest concentration (stock solution). Further concentrations were prepared as 100 x concentrations by serial 1:1.2 dilution according to the planned concentrations (master plate) and were further diluted (1:25) in culture medium 3 to obtain 4 x concentrations (dilution plate). The test substance preparations were prepared by stirring.

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
LuSens cells from the working cell bank were thawed and cultured using culture medium 1, under standard culture conditions (37 °C, ca. 5 % CO2, ≥ 90 % humidity) for at least passage ≥ 5 but not longer than 15 passages prior to testing.
Before substance incubation, cells were seeded in 96-well microtiter plates (120 µL of 0.83 * 10^5 cells/mL cell suspensions), using culture medium 2 for incubation for 24 h.
Two independent, valid experiments were performed. In each experiment, three replicates of each test substance concentration were tested.

After cell adaption for 24 h cell culture medium 2 was aspirated and replaced with 150 µL medium 3. The test substance was prepared. Each preparation of the dilution plate was then applied in a ratio of 1:4 (50 µL) to the cells (final DMSO concentration in the test medium = 1 %). After test substance application the plates were sealed with semi-permeable plate sealers in order to prevent evaporation of the test substance. The plates were placed into the incubator under standard culture conditions for the exposure period of 48 h. For the luciferase assay a white plate (luminescence compatible plate) was used. In addition, a clear plate was treated in parallel for the determination of cell viability.

Each test substance concentration was visually inspected directly after application and after the exposure period of 48 h in order to detect test substance precipitates.

Luciferase assay: After visual inspection of the cells, the supernatant was aspirated from the white assay plate and discarded. The cells were washed twice with 300 µL PBS (with Ca2+ / Mg2+). Subsequently 200 µL of One-Glo-preparation (= 100 µL One-Glo-Mix and 100 µL PBS (with Ca2+ / Mg2+)) per well was added and cells were shaken on a plate shaker for 10 min at room temperature in darkness. After the incubation the luminescence was measured in the luminometer.

Cell viability assay MTT: Cell culture medium was aspirated from all wells. The cells were washed twice with 300 µL PBS (with Ca2+ / Mg2+). Thereafter 200 µL of a 0.5 mg/mL thiazolyl blue tetrazolium bromide (MTT) solution (prepared 1:10 from a 5 mg/mL (MTT) stock solution in PBS (without Ca2+ / Mg2+) and medium 3) was added to each well of the 96-well microtiter plate and incubated for further 2 h after sealing the plates in the incubator. For analysis, medium was aspirated and cells were lysed by adding 100 µL of lysis solution (99.6 mL DMSO; 10 g sodium dodecyl sulfate, SDS; and 0.4 mL glacial acetic acid). Absorbance was measured at 570 nm with reference wavelength 690 nm using a spectral-photometer.

Controls:
- Negative control (NC): DL-Lactic acid, 5000 µM (= 450 µg/mL) in 1 % DMSO in culture medium 3
- Positive control (PC): ethylene glycol dimethacrylate, 90.8 µM (= 18 µg/mL) in 1 % DMSO in culture medium 3
- Vehicle control (VC): 1 % DMSO in culture medium 3
- Blank control: culture medium 3 without cells
- Basal control: culture medium 3 with cells

Key result

Run / experiment:

other: 2 experiments

Parameter:

other: relative viability

Value:

71

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

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

Interpretation of results:

other: no keratinocyte activating potential

Conclusions:

After 48 h of exposure to the test substance luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70 % viability in at least two independent experiments. From this it has to be concluded that the test substance does not have a keratinocyte activating potential.

Executive summary:

The keratinocyte activating potential of the test substance was evaluated in the LuSens assay. For this purpose, the test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 h at 37 °C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer.

In order to determine the concentrations suitable for the main experiment a pre-test (non-GLP) was performed. Cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined by MTT assay. The CV75 value (= estimated concentration that affords 75 % cell viability) was determined by linear regression from the concentration response curve to be 1543 µM (corresponding to test substance as provided by the sponsor).

In the main test luciferase activity was measured after 48 h exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 2 valid experiments were performed. Two further experiments were invalid as the PC did not meet the acceptance criteria. The following results were observed:

At concentrations used in the main experiment the test substance was soluble in DMSO (100 x stock preparation) and in 1 % DMSO in culture medium 3 (final concentrations). No precipitates were noticed in any concentration after 48 h.

Calculation of an EC1.50 (the concentration resulting in a 1.50 -fold luciferase induction) was not applicable.

In summary, after 48 h of exposure to the test substance luciferase activity in LuSens cells was not induced in at least two consecutive concentrations with statistical significance affording at least 70 % viability in at least two independent experiments. From this it has to be concluded that the test substance does not have a keratinocyte activating potential.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In vitro studies covering three key steps of the adverse outcome pathway for skin sensitization as identified by the OECD (OECD publication No. 168; ENV/JM/MONO(2012)10) are required as standard information for skin sensitization in Regulation (EC) No. 1907/2006 and were performed. These study types have initially undergone in-house validation using 54 substances (Bauch et al., 2012 Regul. Toxicol. Pharmacol. 63: 489 - 504). Recently, a validation performed with 213 substances has been published (Urbisch et al., 2015 Regul. Toxicol. Pharmacol. 71: 337 - 351).

Based on the results of the in house validation (Bauch et al., 2012 Regul. Toxicol. Pharmacol. 63: 489 - 504) the predictive capacity of the evaluation scheme using DPRA (peptide reactivity), LuSens / KeratinoSens (keratinocyte activation) and h-CLAT (dendritic cell activation) is comparable to that of the local lymph node assay. This was confirmed in the validation study with more substances (Urbisch, 2015): When compared to the sensitization potential of the substances in humans, the classification based on an evaluation scheme using results obtained from the DPRA, LuSens and h-CLAT had a sensitivity of 90 %, a specificity of 90 % and an accuracy of 90 %.

In the evaluation scheme used here any two of the three test results determine the overall classification, i.e. any two positive test results drive the prediction of a sensitizer, while any two negative test results drive the prediction of a test substance to be a non-sensitizer (Bauch et al., 2012; Table 1). If two assays (DPRA, LuSens or KeratinoSens, h-CLAT) yield concordant results, the result of the third assay is not necesserily required to determine the overall outcome of the evaluation scheme.

Table 1: Decision matrix for combinations of DPRA,LuSens/KeratinoSensand h-CLAT assays.

DPRA	LuSens / KeratinoSens	h-CLAT	Test battery evaluation
Positive	Positive	Positive	Sensitizer
Positive	Positive	Negative	Sensitizer
Positive	Negative	Positive	Sensitizer
Positive	Negative	Negative	Non-sensitizer
Negative	Positive	Positive	Sensitizer
Negative	Positive	Negative	Non-sensitizer
Negative	Negative	Positive	Non-sensitizer
Negative	Negative	Negative	Non-sensitizer

Each individual assay was performed under GLP. All three assays follow the procedures of the respective OECD testing guidelines (OECD 442C, OECD 442D and OECD 442E). Positive and negative controls were included in each experiment and confirmed the functionality and validity of the individual assays.

The test battery applicability is limited when testing substances insoluble in the commonly used vehicles and highly volatile substances. Substances susceptible to base-catalyzed hydrolysis cannot be evaluated reliably for binding to lysine as the incubation is performed at pH 10.2. Also substances that interfere with the experimental measurements (e.g., co-elution with the peptide in the DPRA-assay) are not or only partially suitable for testing using in vitro methods. The substance under evaluation did not have any of the above mentioned limitations and the outcome of this assay is therefore considered adequate for hazard identification for the endpoint of skin sensitization.

The test substance is peptide reactive and activates dendritic cells. In accordance with the published evaluation scheme (Bauch et al., 2012 Regul. Toxicol. Pharmacol. 63: 489 - 504) and Sections 1.2 and 1.4 of Annex XI of EC Regulation 1907/2006, the substance is judged to be a skin sensitizer in a weight-of-evidence approach.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result, the substance is considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008 (GHS Category 1, H317), as amended for the 5th time in Directive EC 944/2013. A subcategorization for potency is not possible from the existing experimental data.