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A skin sensitizing in-vivo study on guinea pigs showed no sensitizing potential. The overall result of the in-vitro DPRA test places the test item in the reactivity class of minimal and therefore it is predicted to be a non skin sensitizer. Also in the in-vitro h-CLAT test up to the highest soluble concentration of 250 μg/mL in DMSO the test substance was not a skin sensitiser under the test conditions of this study. The human patch test from Fuji showed no sensitizing effects. Therefore an overall conclusion was determined that the test substance is predicted to be a non skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
The open epicutaneous test represents an animal bioassay conducted with guinea pigs and designated to generate quantitative data. The generation of skin sensitization data includes a 3 step way. In the pretesting phase the primary irritating treshold concentration of the test substance is determined. The induction phase consists of a 3-week period of daily open applications. The final determination wheter sensitization has occured or not takes place in the final challenge phase.
GLP compliance:
no
Type of study:
open epicutaneous test
Justification for non-LLNA method:
A reliable test method (open epicutaneous test) was already performed in 1985 to fulfill the data requirements of this endpoint.
Species:
guinea pig
Strain:
not specified
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 300-450 g
Route:
epicutaneous, open
Vehicle:
other: not specified
Concentration / amount:
3 phases:
Irritation: 0.025 mL of each test concentration
Induction: 21 x 0.1 mL in the concentrations (if possible) 100, 30, 10, 3, 1 and 0.3 %
Challenge: 0.025 mL of minimal irritating concentration, max. non irritating concentration, lower primary non irritating concentrations (no data)
Route:
epicutaneous, open
Vehicle:
other: not specified
Concentration / amount:
3 phases:
Irritation: 0.025 mL of each test concentration
Induction: 21 x 0.1 mL in the concentrations (if possible) 100, 30, 10, 3, 1 and 0.3 %
Challenge: 0.025 mL of minimal irritating concentration, max. non irritating concentration, lower primary non irritating concentrations (no data)
No. of animals per dose:
5-7 groups of 6-8 males and females
Details on study design:
RANGE FINDING TESTS: 1 day before starting the induction procedure, the threshold irritating concentration of the test material is estimated on the guinea pigs subsequently used for the experimental group. A single application of 0.025 mL of each test concentration (e.g. 100, 30,10 and 3%) is simultaneously performed on one of the areas measuring 2cm2 of the flank skin previously clipped and marked with a circular stamp. Reactions are read 24 h after the application of the test material. The minimal irritating and the maximal nonirritating concentrations are determined by an all-or-none criterion. The minimal irritating concentralion is defined as the lowest one causing skin irritation. The maximal nonirritating concentration is defined as the highest one not causing macroscopic skin reactions in any of the animals.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 21 applications
- Exposure period: For 3 weeks
- Test groups: 5-7 groups of 6-8 animals
- Site: on an area of 8 cm2 on the clipped flank skin
- Frequency of applications: daily
- Duration: 3 weeks
- Concentrations: if possible 100, 30, 10, 3, 1 and 0.3 %

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: 21 and 35
- Test groups: Both the test and the control group
- Site: on the contralateral flank
- Concentration: 0.025 mL of minimal irritating concentration, max. non irritating concentration, lower primary non irritating concentrations (no data)
- Evaluation (hr after challenge): 24, 48 and 72 h
Challenge controls:
no data
Positive control substance(s):
not specified
Positive control results:
no data
Key result
Remarks on result:
no indication of skin sensitisation

The test substance was found to have no sensitizing effect on the skin.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
In the Open Epicutaneous Test the test substance did not induce skin reactions indicating a sensitising potential.
Executive summary:

The skin sensitizing potential of the test item was tested in the open epicutaneous test with guinea pigs. The test was conducted on groups of 6 -8 male and female animals weighting 300 -450 grams. Daily applications were made for 3 weeks to a clipped 8 cm2 area on the flank of each guinea pig. The test sites were not covered and the reactions were read 24 hours after each application. A total of 21 applications of 0.1 mL test material in an unspecified vehicle were made for 21 days. The 10 controls were either left untreated or treated with 0.1 mL of the vehicle for 21 days. At the challenge phase, both the test and control animals were treated at some lower primary non-irritating concentrations. On the basis of these resulst, the test substance was considered to be non sensitising. This result is supported by results of skin sensitization tests on humans, as described in this publication.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 442E (In Vitro Skin Sensitisation Human Cell Line Activation Test (h-CLAT))
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells
Details on study design:
Skin sensitisation (In vitro test system)
Cell line used: THP-1 cells, THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202.

Technical material and conditions:
- For cell culture standard laboratory equipment was used.

- Culture medium:RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L Glucose, 1% (v/v) sodium pyruvate and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) was used to culture the cells and during the assay.
- Buffer: FACS buffer
- Blocking Solution 0.01% Globulins Cohn fraction II,III
- Antibodies: Anti - CD86 antibody (Clone: Fun-1)
Anti – CD54 antibody (Clone: 6.5B5)
FITC labeled-mouse IgG1 (isotype control)

Controls used:
Medium Control: Culture medium
Solvent Control for the Test Item: Name: DMSO (final concentration for XTT cytotoxicity 0.2% and for h-CLAT 0.2%)
Positive Control: Name: DNCB
Solvent: DMSO, diluted with culture medium to an end concentration of 2 and 3 μg/mL
(DNCB).
Solvent Control for the Positive Control (h-CLAT): Name: DMSO (final concentration 0.2%)

Test substance preparation:
On the day of the experiment (immediately prior to start) the test item was solved in DMSO. The maximum concentration of test item was 250 μg/mL (DMSO) as tested by a solubility test.
Eight concentrations of the test item were analysed. Dilutions were prepared by 1:2 serial dilutions from the highest soluble concentration (250 μg/mL DMSO).

Selection of concentrations
The doses investigated in the main experiment (h-CLAT) were determined with a XTT test. Two independent cytotoxicity experiments were performed on different days to obtain a reliable
CV75. The CV75 could not be determined therefore the highest soluble test item concentration was used to calculate the dose-range for the main experiment (h-CLAT).
Two independent cytotoxicity experiments were performed on different days to obtain a reliable CV75. Since the CV75 could not be determined, a stock solution of the highest soluble dose ×
1.2 was prepared and seven further concentrations of the test item were prepared by serial 1:1.2 dilution from.

Experimental procedure
The test item was tested in four independent runs. Due to a technical problem with the flow cytometer, the second h-CLAT run was stopped before the start of the measurement and restarted
on another day.

- Cell preparation:
For the test item exposure the highest dose solution calculated from the XTT assay was prepared corresponding to 1.2 × the highest concentration of the XTT test instead of 1.2 × CV75, since no
CV75 could be determined. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment. Each solution was diluted with culture
medium before application of the test solution to the cells to reach a final concentration of 0.2% (v/v) in the medium.
Each volume (500 μL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 1 hours. Each concentration of the test item, medium control, and positive and DMSO control was tested in triplicates for the different staining (with FITC-labeled anti-CD86, CD54 antibody or mouse IgG1).

- Staining of the cells
The triplicates of each test item-treated and not test item treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5 min) and then washed twice with approx. 2 mL of FACS buffer. Thereafter, the cells were centrifuged, resuspended and blocked with 600 μL of blocking solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 μl FACS-buffer. The cells were stained with FITC-labeled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedure. The cells were gently mixed by hand and incubated light protected for 40 min for run 1 and 30 ±
5 min for run 2 to 4 at 2 - 8 °C (on ice).

- Flow cytometric analysis:
Before using the flow cytometer, the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions. The expression of cell surface antigens (CD54, CD86) was analyzed by flow cytometry. The FITC acquisition channel (FL-1) should be set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) should be set for the optimal detection of DNA-bound 7-AAD fluorescence signal.
Preparation of the acquisition
The following acquisition plots were prepared:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of each channel (FL-1 and FL-3, respectively)
The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC were not needed for the analysis, but the FSC/SSC plot should be checked to make sure that a
single population appeared without contamination or excessive debris. The FL-1 and FL-3 voltage was set and compensate to appropriate position. The FL-1 voltage was set using the
FITC labeled-mouse IgG1 medium-treated cells tube, such that the MFI of control cells was set in the range between approx. 1.0 and 3.0 (FACSCalibur, Becton Dickinson). A R1 gate was set at the middle position between the peak of the negative fraction. The negative fraction corresponded to the living cells and was kept for the subsequent analyzes. The percentage of R1-gated cells should exceed 95 %. The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing should be conducted very carefully since insoluble chemicals could flow in the flow line.

Acquisition
Dead cells were gated-out by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analyzed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample was used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was not calculated, if the cell viability was less than 50 % (due to diffuse labelling of cytoplasmic structures that were generated due to cell membrane destruction).

Data evaluation:
The test item is tested in 2 independent runs. If the RFI of CD86 is ≥ 150% or if the RFI of CD54 is ≥ 200% in both independent run data, the test item is considered to be a sensitizer.
Otherwise it is considered to be a non-sensitizer. In case of different results in both runs, a third run has to be performed. If the RFI of CD86 is ≥ 150% at any dose in at least 2 of 3 independent
run data, or if the RFI of CD54 is ≥ 200% in at least 2 of 3 independent run data, the test item is considered to be a sensitizer. Otherwise it is considered to be a non-sensitizer.

Acceptance criteria:
- A tested concentration is not to be further evaluated when relative viability is less than 50%.
- Cell viability of vehicle control cells must yield at least 90%.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should be over the positive criteria (CD86≥ 150% and CD54 ≥ 200%) and cell viability should be ≥ 50%.
- In the negative control (LA), RFI values of both CD86 and CD54 should not exceed the positive criteria (RFI CD86< 150% and RFI CD54 < 200%) and cell viability should be ≥ 50%.
- For all vehicle controls, the MFI ratio of both CD86 and CD54 to isotype controls should be ≥ 105%.
- The reactivity check of new thawed cells should produce a positive response in CD86 and CD54 for NiSO4 and DNCB and a negative response in CD86 and CD54 for LA.
- Positive, negative and vehicle control data should lie within the range of the historic data.
Positive control results:
The results of the positive control were within the historical control range.
Key result
Parameter:
other: EC200% (CD54)
Remarks:
(the concentration resulting in a RFI of 200%)
Run / experiment:
Experiment 1, 2, 3, 4
Value:
< 200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other:
Run / experiment:
EC150 % (CD86)/the concentration
Value:
< 150
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:

The test item with a log Pow (Partition coefficient: n-octanol/water) of 2.36 was tested in 4 independent runs. The RFI of CD86 and/or CD54 was not equal or greater than 150% and 200%,respectively at any dose in the run data of run 2 and 4. In addition, the RFI of CD54 exceeded the threshold of ≥ 200% in the cells treated with the highest test item concentration of 300 μg/mL in run 1 and in the cells treated with 250 μg/mL test item in run 3 (285.9 and 200%, respectively). However, phase separation was observed in the highest tested test item concentration (300 μg/mL) in all four runs. Therefore, the results of this tested concentration were excluded from the evaluation. With this exclusion the RFI of CD86 and CD54 was not equal or greater than 150% and 200% in three out of four h-CLAT runs. Even though four independent runs instead of maximum 3 runs were performed, the test item is considered to be a non-sensitiser up to the highest tested soluble test item concentration (250 μg/mL) in DMSO.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item tested up to the highest soluble concentration of 250 μg/mL in DMSO was not a skin sensitiser under the test conditions of this study.
Executive summary:

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the skin sensitizing potential of the test item dissolved in DMSO when administered to THP-1 cells for 24 ± 1 hours. The dose for the main experiment (h-CLAT) of the test item was previously determined by two XTT tests. Cytotoxic effects were not observed following incubation with the test item up to the highest

tested concentration (250 μg/mL). The viability of the cells in both XTT spaced within a range of 92.31 to 117.21% and 93.77% to 111.06%, respectively (threshold of cytotoxicity: < 75%).

Due to the lack of cytotoxicity, a CV75 value could not be calculated. The following concentrations of the test item (solved in DMSO) were tested in the main experiment (h-CLAT): 84, 100, 121, 145, 174, 208, 250 and 300 μg/mL. The test item with a log Pow (Partition coefficient: n-octanol/water) of 2.36 was tested in 4 independent runs. The RFI of CD86 and/or CD54 was not equal or greater than 150% and 200%, respectively at any dose in the run data of run 2 and 4. In addition, the RFI of CD54 exceeded the threshold of ≥ 200% in the cells treated with the highest test item concentration of 300 μg/mL in run 1 and in the cells treated with 250 μg/mL test item in run 3 (285.9 and 200%, respectively). However, phase separation was observed in the highest tested test item

concentration (300 μg/mL) in all four runs. Therefore, the results of this tested concentration were excluded from the evaluation. With this exclusion the RFI of CD86 and CD54 was not equal or greater than 150% and 200% in three out of four h-CLAT runs. Even though four independent runs instead of maximum 3 runs were performed, the test item is considered to be a non-sensitiser up to the highest tested soluble test item concentration (250 μg/mL) in DMSO. In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 did not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%). The RFI values of the positive control (DNCB) for CD86 and CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability was >50%. In conclusion, the test item tested up to the highest soluble concentration of 250 μg/mL in DMSO was not a skin sensitiser under the test conditions of this study.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016-03-09 to 2016-08-08
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Details on study design:
Skin sensitisation (In chemico test system) - Details on study design:

Assessment of Test Item Solubility
The solubility of the test item in acetonitrile was assessed at a concentration of 100mM.

Preparation of Peptide Stock Solutions
Stock solutions of each peptide were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2) so the peptide concentration of the stock solutions is 0.667 mM.

Preparation of Peptide Calibration Standards
Calibration standards of both peptides were prepared by diluting the stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was prepared as well.

Preparation of Stability Controls and Precision Controls
Stability controls and precision controls of both peptides were prepared at a concentration of 0.5 mM.

Preparation of Positive Control Solution
The positive control chemical (Cinnamic aldehyde) was prepared at a concentration of 100 mM.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Acetonitrile solutions of the test item and the positive control were diluted with the Cysteine peptide so as to prepare solutions containing 0.5 mM Cysteine and 5 mM of either the test item or the positive control. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls
Acetonitrile solutions of Ethyl Phenyl Acetate and the positive control were diluted with the Lysine peptide so as to prepare solutions containing 0.5 mM Lysine and 25 mM of either the test item or the positive control. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

Incubation
The appearance of the test item and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis
The concentration of the test item and the associated positive control was quantified by HPLC using UV detection as detailed in the chromatographic section

High performance liquid
chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Column: Agilent Zorbax SB C18, 3.5µm, 100 x 2.1 mm
Guard column: Phenomenex AJO4286
Column temperature: 30°C
Sample temperature: 25°C
Mobile Phase (MP) A: 0.1% TFA in Water
Mobile Phase (MP) B: 0.085% TFA in ACN
Gradient: Time (minutes) MP A(%) MP B (%)
0 90 10
20 75 25
21 10 90
23 10 90
23.5 90 10
30 90 10
Flow rate: 0.35 mL/minute
Stroke volume 25 μL
Detector wavelength: UV, 220 nm
Injection volume: 2 μL (slow draw rate)
Run time: 30 minutes
Approximate retention time (Cysteine) 11 minutes
Approximate retention time (Lysine) 7 minutes

According to the classification tree model described by Gerberick et al. for substances with known molecular weight highly reactive test substance (mean peptide depletion > 42.47 %) is predicted to be a strong sensitizer, a moderately reactive test substance (22.62 % < mean peptide depletion < 42.47 %) a moderate sensitizer, a test substance of low reactivity (6.38 % < mean peptide depletion < 22.62 %) a weak sensitizer.
Positive control substance(s):
yes
Remarks:
Cinnamic Aldehyde
Positive control results:
The positive control substance caused a mean peptide depletion of 56.3 %.
Key result
Parameter:
other: mean Cystein-peptide depletion in %
Value:
1.18
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Parameter:
other: mean Lysine-peptide depletion in %
Value:
-0.71
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:

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

No co-elution of test substance and peptides was noticed. Complete depletion was caused by the positive control substance.
Interpretation of results:
GHS criteria not met
Conclusions:
Solutions of test item were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine
containing synthetic peptides. The overall result places test item in the reactivity class of minimal and therefore it is predicted to be a non skin sensitizer.
Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of the test item. Solutions of the test item were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. The overall result places the test item in the reactivity class of minimal and therefore it is predicted to be a non skin sensitizer.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

For the evaluation of the skin sensitisation potential of the test substance a test battery of in vivo and non in vivo experiments was performed, as a single in vitro/in chemico test is not sufficient for adequate judgment. An evaluation of all tests in a Weight of Evidence approach and further human patch tests (see section 7.10.4) are used to reach a final conclusion on the skin sensitisation potential of the test substance and its need for classification.

 

Open epicutaneous test of G. Klecak, 1985 (Curr Probl Dermatol 14 pg 152-71).

The skin sensitizing potential of the test item was tested in the open epicutaneous test with guinea pigs. The test was conducted on groups of 6 -8 male and female animals weighting 300 -450 grams. Daily applications were made for 3 weeks to a clipped 8 cm2 area on the flank of each guinea pig. The test sites were not covered and the reactions were read 24 hours after each application. A total of 21 applications of 0.1 mL test material in an unspecified vehicle were made for 21 days. The 10 controls were either left untreated or treated with 0.1 mL of the vehicle for 21 days. At the challenge phase, both the test and control animals were treated at some lower primary non-irritating concentrations. On the basis of these resulst, the test substance was considered to be non sensitising. This result is supported by results of skin sensitization tests on humans, as described in this publication.

DPRA

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of the test item. Solutions of the test item were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. The overall result places the test item in the reactivity class of minimal and therefore it is predicted to be a non skin sensitizer.

 

h-CLAT

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the skin sensitizing potential of the test item dissolved in DMSO when administered to THP-1 cells for 24 ± 1 hours. The dose for the main experiment (h-CLAT) of the test item was previously determined by two XTT tests. Cytotoxic effects were not observed following incubation with the test item up to the highest tested concentration (250 μg/mL). The viability of the cells in both XTT spaced within a range of 92.31 to 117.21% and 93.77% to 111.06%, respectively (threshold of cytotoxicity: < 75%). Due to the lack of cytotoxicity, a CV75 value could not be calculated. The following concentrations of the test item (solved in DMSO) were tested in the main experiment (h-CLAT): 84, 100, 121, 145, 174, 208, 250 and 300 μg/mL. The test item with a log Pow (Partition coefficient: n-octanol/water) of 2.36 was tested in 4 independent runs. The RFI of CD86 and/or CD54 was not equal or greater than 150% and 200%, respectively at any dose in the run data of run 2 and 4. In addition, the RFI of CD54 exceeded the threshold of ≥ 200% in the cells treated with the highest test item concentration of 300 μg/mL in run 1 and in the cells treated with 250 μg/mL test item in run 3 (285.9 and 200%, respectively). However, phase separation was observed in the highest tested test item concentration (300 μg/mL) in all four runs. Therefore, the results of this tested concentration were excluded from the evaluation. With this exclusion the RFI of CD86 and CD54 was not equal or greater than 150% and 200% in three out of four h-CLAT runs. Even though four independent runs instead of maximum 3 runs were performed, the test item is considered to be a non-sensitiser up to the highest tested soluble test item concentration (250 μg/mL) in DMSO. In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 did not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%). The RFI values of the positive control (DNCB) for CD86 and CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability was >50%. In conclusion, the test item tested up to the highest soluble concentration of 250 μg/mL in DMSO was not a skin sensitiser under the test conditions of this study.

Human patch test

T. Fujii, S. Furukawa and S. Suzuki, 1972 (Yukugaku 21 (12) pg 904 -908).

In a closed human patch study the skin sensitizing potential of the test item was tested. The test was conducted with 7703 subjects suffering from dermatoses. The test item item was applicated to the upper inside of the arm. Ethanol 99% and a non-irritative cream base were used as vehicle. A concentration of 0.05 -0.5 % were applicated. Only in one subject erythema reactions were found. According to this result the test item can be considered as not sensitizing.

Conclusion:

A skin sensitizing in-vivo study on guinea pigs showed no sensitizing potential. The overall result of the in-vitro DPRA test places the test item in the reactivity class of minimal and therefore it is predicted to be a non skin sensitizer. Also in the in-vitro h-CLAT test up to the highest soluble concentration of 250 μg/mL in DMSO the test substance was not a skin sensitiser under the test conditions of this study. The human patch test from Fuji showed no sensitizing effects. Therefore an overall conclusion was determined that the test substance is predicted to be a non skin sensitizer.

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 (EC) No 1272/2008. As a result the substance is not considered to be classified for skin sensitisation under Regulation (EC) No 1272/2008.