Methyl o-toluate

Endpoint summary

Description of key information

Sensitisation LLNA (OECD TG 429): Negative up to 30% (maximum dose in view of skin irritation in the skin irritation test)

Sensitisation in DPRA (OECD TG 442C): Negative

Sensitisation KeratinoSens (OECD TG 442D): Negative

Sensitisation h-CLAT (OECD TG 442E): Positive

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study was conducted between 2004-09-15 and 2004-09-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be a reliability 1 as it has been conducted according to OECD Test Guideline 429 using the Skin Sensitisation: Local Lymph Node Assay method and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

- Age Range: 11 weeks at start of dosing; records of dates of birth for animals used in this study are retained in the Calvert archives.
- Body Weight Range: 20 - 26 grams at the outset (Day 1) of the study.
- Animal Source: Jackson Laboratories, Bar Harbor, ME 04609
- Experimental History: Purpose-bred and experimentally naive at the onset of the study.
- Identification: Tail marked with an indelible marker and cage card.
- Housing: Animals were group housed (5 per cage) upon receipt in compliance with National Research Council "Guide for the Care and Use of Laboratory Animals". The room in which the animals were kept was documented in the study records. No other species were kept in the same room
- Lighting: 12 hours light/12 hours dark
- Room Temperature: 21.1 to 25.6°C
- Relative Humidity: 30 - 54%
- Food: Animals had access to Certified Rodent Chow 7012C ad libitum.
- Water: Tap water was available ad libitum.
- Acclimation: Study animals were acclimated to their housing for six days prior to their first day of dosing.

All animals used in this study were assessed as to their general health by a member of the veterinary staff or other authorized personnel. During the acclimation period, each animal was observed at least once daily for any abnormalities or for the development of infectious disease. Only animals that were determined by the veterinary staff and/or Study Director to be suitable for use were assigned to this study.

Animals were assigned to study groups by a computerized randomization program (LABCAT Randomization module version 2.48, developed by Innovative Program Associates, Inc. 303 Wall Street, Princeton, NJ 08540-1515) designed to achieve similar group mean body weights.

Treatment of animals was in accordance with the study protocol and also in accordance with Calvert SOP's which adhere to the regulations outlined in the USDA Animal Welfare Act (9 CFR Parts 1, 2 and 3) and the conditions specified in the Guide for the Care and Use of Laboratory Animals (ILAR publication, 1996, National Academy Press). The Calvert IACUC approved the study protocol prior to dose administration, outlined in the USDA Animal Welfare Act (9 CFR Parts 1, 2 and 3) and the conditions specified in the Guide for the Care and Use of Laboratory Animals (ILAR publication, 1996, National Academy Press). The Calvert IACUC approved the study protocol prior to dose administration.

Vehicle:

other: Diethyl phthalate/ethanol 3:1

Concentration:

Concentrations of 0, 7.5, 15, and, 30% v/v in vehicle 3:1 were tested.

No. of animals per dose:

Groups of five mice were treated

Details on study design:

Test Item Administration:
- Route: Topically on the dorsal surface of both ears
- Frequency: Once daily for 3 consecutive days (Days 1 - 3). The timing of dose administration remained consistent(± 2 hours) during the dosing phase.
- Procedure: A volume of 25 µL/ear was applied using a micropipette.

Observations and Measurements:
- Mortality/Morbidity: Daily on Days 1 to 6
- Clinical Observations: Prior to dose administration and once post-dose on Days 1 to 3. Clinical observations were performed once daily on Days 4 to 6. Particular attention was given to the application sites. Any significant alterations (e.g., erythema and edema) to the application sites, and the general appearance of the pinnae, including build up of test article, was recorded.
- Body Weight: Animals were weighed at the time of randomization/selection, and on Days 1 and 6.

Method of Performance:
Mice were treated on the dorsal surface of both ears, once per day on Days 1, 2, and, 3. Approximately 24 ± 2 hours between applications of test article was maintained. On Day 6 the mice were injected i.v. with 20 µCi of 3H­ thymidine in 250 µL of sterile saline. Five hours later the mice were euthanized by CO2 asphyxiation. Ear thickness measurements of each mouse were recorded and the draining auricular lymph nodes removed. At removal, the number of nodes collected per animal was recorded, and the nodes were examined for size/appearance and the data recorded. Any unexpected observations were noted in study records. A single cell suspension was prepared from the lymph nodes of each mouse. Cells were washed twice with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) overnight at 2 - 8°C. The pellets were recovered by centrifugation and resuspended in 1 mL of TCA and transferred to a vial containing scintillation fluid. An additional 1 mL of TCA was used to rinse the tube, and it was also transferred to the scintillation fluid. Incorporation of 3H­ thymidine was measured in a B-scintillation counter.

Positive control substance(s):

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

Statistics:

The mean DPM for each group was evaluated using SYSTAT version 9.01, developed by SPSS, Inc. Increases in 3H-thymidine incorporation relative to the vehicle-treated control were derived for each group and recorded as stimulation indices (SI). The criterion for a positive response is that one or more concentrations of a test article elicits a 3-fold or greater increase in isotope incorporation relative to the vehicle control. Body weight data and ear thickness measurements were also evaluated. Individual DPM values were analyzed by log transformation (base 10) of the data. The evaluation of the equality of means for the DPM, body weight and ear thickness data was made by a one-way analysis of variance using the F distribution to assess statistical significance. If statistically significant differences between the means are found, a Dunnett's test was used to determine the degree of significance from the control means.

Positive control results:

The positive control, 35% (v/v) HCA, resulted in a stimulation index (SI) of 5.03. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control is considered a positive response. In addition, the response with the positive control in this study was also statistically significant (p<0.001) when compared to the vehicle control group.

Parameter:

EC3

Remarks on result:

other: see Remark

Remarks:

The following SI values were derived at 7.5, 15, and 30% (w/v): 1.13, 1.59, and 1.22, respectively. A test material is considered to have skin sensitizing activity if, at one or more concentrations, it induces a 3-fold or greater increase in proliferative activity relative to the concurrent vehicle treated control. Thus, a stimulation index 2 3.0 is regarded as a positive response. However, treatment with the test substance at 7.5, 15 and 30% (w/v) did not result in a stimulation index of 3 or greater.

Key result

Parameter:

SI

Remarks on result:

other: at 7.5, 15 and 30%: 1.13, 1.59 and 1.22

Interpretation of Results: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non‑sensitiser". For chemicals, which the lowest concentration tested resulted in a stimulation index of greater than 3, an EC3 value was extrapolated from the two lowest doses utilized. The extrapolated EC3 value was calculated by log‑linear interpolation between the two points on a plane where the x-axis represents the dose level and the y‑axis represents the stimulation index. The point with the higher stimulation index was denoted (a, b) and the point with the lower stimulation index was denoted (c, d). The formula for the extrapolated EC3 value was as follows: EC3= 2^ {log2(c) + (3-d)/(b-d)] x [log2(a)-log2(c)]} a= mid concentration giving a stimulation index >3 b = actual stimulation index caused by ‘a’ c = lowest concentration giving a stimulation index of >3 d = actual stimulation index caused by ‘c’ The radioactive disintegrations per minute per animal and the stimulation index were recorded. The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are provided.

Interpretation of results:

other: not sensitising

Remarks:

according to the CLP Regulation EC 1272/2008 and its updates

Conclusions:

The test item was considered not to be a sensitiser under the conditions of the test.

Executive summary:

The skin sensitisation potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay method. At 7.5, 15, and 30% the substance showed SI values of 1.13, 1.59, and 1.22, respectively. No statistically significant differences in the ear measurements were observed in any of the treatment groups when compared to the vehicle group. The test item was considered not to be a sensitiser under the conditions of the test.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14-19th November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Study is part of 3 studies that model the adverse outcome pathway for skin sensitisation: the DPRA reflects the 1st molecular event that is the binding to proteins

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
Synthetic peptide containing cysteine: Ac-RFAACAA-COOH, lot number 1556171, purity 95% (by HPLC), supplied by AnaSpec, stored frozen (-10°C to -30°C).
Synthetic peptide containing lysine: AC-RFAAKAA-COOH, lot number 1556172, purity 94% (by HPLC), suppoed by AnaSpec,stored frozen (-10°C to -30°C).

Positive control: cinnamic aldehyde, purity > 95%, was prepared at a concentration of 100 mM in acetonitrile

Preparation of peptide stock solutions:
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (for cysteine, 100 mM phosphate buffer pH 7.5, for lysine 100 mM ammonium acetate buffer pH 10.2).

Preparation of peptide calibration standards:
Calibration standards of both peptides were prepared by diluting the requisite 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 also prepared.

Preparation of Reference (Stability) Controls and Precision Controls:
Reference (stability) controls and precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile. These were injected throughout the analytical run to confirm consistency of peptide response throughout each analytical run.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls:
A 100 mM solution in acetonitrile of the test substance was prepared and further diluted in HPLC vials. Cysteine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM test substance solution in more acetonitrile and cysteine peptide stock solution. The final sample concentration was 5 mM of the test substance, 0.5 mM cysteine.
In place of the test substance, the positive control solution contained cinnamic aldehyde at a concentration of 5 mM with 0.5 mM cysteine.
The co-elution control sample contained 5 mM of the test substance in phosphate buffer solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls:
A 100 mM solution in acetonitrile of the test substance was prepared and further diluted in HPLC vials. Lysine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM
test substance solution in lysine peptide stock solution. The final sample concentration was 25 mM of the test substance, 0.5 mM lysine.
In place of the test substance, the positive control solution contained cinnamic aldehyde at a concentration of 25 mM with 0.5 mM lysine. The co-elution control sample contained 25 mM of the test substance in ammonium acetate buffer solution.

Incubation:
The appearance of the test substance, positive control samples and co-elution controls in the HPLC vials was documented following preparation with the vials then 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 both the cysteine and lysine peptides in the presence of the test substance and the associated positive controls were quantified by HPLC using UV detection.
Equipment: HPLC Waters Alliance 2695 separation module an d2487 dual wavelength detector.
Column: Agilent Zorbax SB C18, 3.5 µm, 100 × 2.1 mm
Guard column: Phenomenex AJO4286
Column temperature: 30 °C
Sample temperature: 25 °C
Mobile phase A: 0.1% trifluoroacetic acid in water
Mobile phase B: 0.085% trifluoroacetic acid in acetonitrile
Flow rate: 0.35 mL/minute
Detector wavelength: UV, 220 nm
Injection volume: 2 μL
Run time: 30 minutes
Approximate retention time (cysteine): 11 minutes
Approximate retention time (lysine): 7 minutes

Calculations:
The peak area response for each peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:

% peptide depletion = 100 - [(Peptide peak area in replicate depletion samples x 100) / (Mean peptide peak area of reference (stability) control samples)]

Acceptance criteria for analytical measurements are presented in Table 1 in the section "Any other information on materials and methods incl. tables". Information on the interpretation of the results is presented in the same section in Table 2.

Positive control results:

73.1% depletion (CV 0.44%, n = 3) and 59.8% depletion (CV 0.47%, n = 3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.

Key result

Run / experiment:

other: 1

Parameter:

other: cysteine depletion, %

Value:

-1.53

Vehicle controls validity:

valid

Remarks:

stability and precision controls

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: lysine depletion (%)

Value:

-0.47

Vehicle controls validity:

valid

Remarks:

stability and precision controls

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

coelution peak of the test substance and lysine

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met forr eference (stability) controls and precision controls of both peptides were met (CV 1.06%, n = 6 and CV 0.47%, n = 6, for cysteine and lysine, respectively, at 0.50 mM).
- Acceptance criteria met for positive control: yes, 73.1% depletion (CV 0.71%, n = 3) and 59.8% depletion (CV1.98%, n = 3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.
- Acceptance criteria met for variability between replicate measurements: yes, CV 0.44% (n=3) and 0.47% (n=3), respectively, for cysteine and lysine depletion by the test item.

Interpretation of results:

other: DPRA is negative

Conclusions:

It can be concluded that this DPRA test is valid and the test substance is negative because the substance showed mean depletion of cysteine and lysine of -1.53 and -0.47%, respectively, which is is classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

In a GLP-compliant OECD guideline 442C study, Direct Peptide Reactivity Assay (DPRA) was used to assess the reactivity and sensitizing potential of the test substance. In the cysteine and lysine reactivity assay all analytical acceptance criteria of the test were met. The test substance caused -1.53% (CV 0.44) and -0.473% (CV 0.47) cysteine and lysine peptide depletion, respectively. These results are categorised as “no to minimal reactivity” based on the DPRA prediction model and is thus considered to be negative in the DPRA.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 November, 2016 - 25 November, 2016

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: human Cell Line Activation Test (h-CLAT)

Version / remarks:

July 2016

GLP compliance:

yes (incl. QA statement)

Type of study:

other: human Cell Line Activation Test (h-CLAT)

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
Solvent: DMSO (final concentration 0.2% in culture medium, also used as a solvent for positive control)
Positive control: DNCB in DMSO diluted with culture medium (2 and 3 μg DNCB/mL)

XTT is used instead of flow cytometry for the cytotoxicity measurement and estimation of the CV75 value in the dose range finding assay

Cell line: THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202. THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitisers. The cell density did not exceed 1 × 10^6 cells/mL. The passage numbers of the used THP-1 cells was 9 in both XTT assays and 10 and 11 in the h-CLAT for runs 1 and 2, respectively.
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, 1% (v/v) L-glutamine and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) was used to culture the cells during the assay.
Preparation and seeding of THP-1 cells: On the day of the cytotoxicity experiment (XTT) directly before the application of the test item, solvent and medium control, a volume of 100 μL with a cell density of 0.9 - 1 × 10^6 THP-1 cells/mL was seeded in each well of a 96-well flat bottom plate. For the main experiment (h-CLAT) 0.9- 1 × 10^6 cells/well in a volume of 500 μL was seeded in a 24-well plate before the treatment.

Dose finding assay: Two cytotoxicity XTT tests were conducted to find the concentrations to be used in the h-CLAT test.

The XTT test is based on the cleavage of the yellow tetrazolium salt XTT [= (sodium 3'-(1-phenylaminocarbonyl)-(3,4-tetrazolium)–bis-(4–methoxy–6-nitro)-benzenesulfonic acid hydrate)] to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria. A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of orange formazan formed, as monitored by the absorbance.

Two independent cytotoxicity experiments were performed with different cell cultures days to obtain a reliable CV75. The mean of two CV75 values was used to determine the dose-range for the main experiment (h-CLAT). CV75 is defined as the concentration of toxicant required to reduce the relative absorbance to 75% of the solvent control and is calculated as:
CV75 = Conc.>75 - [(Conc.>75 - Conc.<75) x (%>75 - 75)]/(%>75 - %<75), where:
a) Conc.>75 = maximal meausred concentration with the % of solvent control > 75%
b) Conc.<75 = minimal measured concentration with the % of solvent control < 75%
c) %>75 = relative absorpbance at a) in %
d) %<75 = relative absorpbance at b) in %

Test item preparation: immediately prior to start the substance was dissolved in culture medium. The maximum concentration of test item was 625 μg/mL in culture medium, as tested by a solubility test. For the XTT test (dose finding assay) eight concentrations of the test item were analysed. Therefore, dilutions were prepared by 1:2 serial dilutions from 625 μg/mL in culture medium.
XTT Labelling and Measurement: At the end of the incubation period, 50 µL of the XTT labelling mixture were added to each well. The cells were incubated and subsequently transferred to a microplate reader (Versamax® Molecular Devices). The absorbance was measured at 450 nm (reference wavelength 690 nm). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).

Acceptability criteria of XTT assay (Cytotoxicity):
The XTT test is considered to be acceptable if it meets the following criteria:
• mean absorbance of the medium control is ≥ 0.5
• mean viability of the solvent control is ≥ 90% in comparison to the medium control

Main test:
The test item was tested in two independent runs.
Test item preparation: For the test item exposure the highest dose solution calculated from the XTT assay was prepared corresponding to 1.2 × CV75. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment. The following concentrations of the test item (solved in culture medium) were tested in the main experiment (h-CLAT): 4.9; 9.8; 19.5; 39.1; 78.2; 156.3; 312.5;; 625 μg/mL.
Treatment of the cells: Each volume (100 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, positive an4.9d DMSO control was tested in triplicates for the different staining (with FITC-labelled 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 (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended 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-labelled 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 30 ± 5 min. at 2 - 8 °C (on ice).
Sample preparation for measurement: After staining with the antibodies, the cells were washed twice (2-8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-aminoactinomycin D (7-AAD) solution were added.
Flow cytometry acquisition: The expression of cell surface antigens (CD54, CD86) was analyzed by flow cytometry. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.
Acquisition: A total of 10,000 living cells were analyzed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. 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 are generated due to cell membrane destruction).
Data analysis and interpretation:
The RFI is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration of every chemical:
RFI (%) = 100 x (MFI of test item treated cells - MFI of test item treated isotope control cells) / (MFI of solvent control cells - MFI of solvent isotope control cells), where MFI is geometric mean fluorescent intensity
The cell viability is calculated as follows:
Cell viability (%) = 100 x (Mean cytotoxicity of solvent control cells) / (Mean cytotoxicity of the test item treated cells), where Mean cytotoxicity is the mean of geometric mean (7-AAD) isotype control, geometric mean (7-AAD) CD54 and geometric mean (7-AAD) CD86.
Acceptability criteria of the h-CLAT assay:
The study is considered as valid, if the following criteria are met:
• Cell viability of medium control is adjusted to 100% and the cell viability of the DMSO control should be more than 90% in comparison to the medium control.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%.
• In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
• For medium and DMSO controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
• For the test item resulting in negative outcome, the cell viability at the 1.2 × CV75 should be less than 90%. (If the cell viability at the 1.2 × CV75 is more than 90% for a positive tested test item, the data will be acceptable. If 5 mg/mL in saline, 1 mg/mL in DMSO or the highest soluble dose will be used as the maximal test concentration instead of CV75-based dose, the data for test item are accepted independent by the cell viability.)
• The cell viability of at least 4 doses in each experiment should be ≥50%.

Evaluation of results:
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 sensitiser. Otherwise it is considered to be a non-sensitiser. 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 sensitiser. Otherwise it is considered to be a non-sensitiser.

Positive control results:

The RFI values of the positive controls (DNCB) for CD86 and CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability was >50%.

Key result

Run / experiment:

other: 1

Parameter:

other: % RFI; CD54

Value:

229.7

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

not valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

at 300 umol the value is > 200, despite positive control not valid

Key result

Run / experiment:

other: 1

Parameter:

other: % RFI; CD86

Value:

167.4

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

at 300 umol the value is > 150

Key result

Run / experiment:

other: 2

Parameter:

other: %RFI; CD 54

Value:

196.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

at 360 umol value is < 200

Key result

Run / experiment:

other: 2

Parameter:

other: % RFI; CD86

Value:

143.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

at 360 umol value is < 150

Key result

Run / experiment:

other: 3

Parameter:

other: % RFI; CD54

Value:

302

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

at 360 umol the value is > 200

Key result

Run / experiment:

other: 3

Parameter:

other: % RFI; CD86

Value:

241.4

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

At 300 umol the value is > 150

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: 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%).
- Acceptance criteria met for positive control: The RFI values of the positive controls (DNCB) for CD86 and CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability was >50%.

Results of the XTT assay:

1st test:

			Microscopic		Photometric
			Evaluation		Evaluation
Test Group	Concentration		Cytotoxicity	Mean Ab-	Standard-	Chem.		Absorbance in % of
	[µg/mL]			sorbance*	Deviation	Blanks		Solvent Control**
Medium Control	-		no	0.712	0.032	0.268		99.75
Solvent Control	-		no	0.723	0.049	0.278		100.00
	4.9		no	0.671	0.021	0.277		88.45
	9.8		no	0.704	0.034	0.28		95.36
	19.5		no	0.708	0.021	0.273		97.71
	39.1		no	0.746	0.041	0.281		104.56
Test Item
	78.1		no	0.742	0.022	0.278		104.16
	156.3		no	0.7	0.028	0.279		94.47
	312.5		no	0.698	0.025	0.273		95.41
	625		yes	0.438	0.046	0.264		39.02
Shaded test group:	cytotoxic effects occurred in the photometric evaluation (< 75% cell viability)

 * mean absorbance (absolute) of 7 wells

** relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 100.2%.

 

The CV75 value of the first XTT test: 425.6 µg/mL

2nd test:

		Microscopic Evaluation	Photometric Evaluation
Test Group	Concentration [µg/mL]	Cytotoxicity	Mean Ab-sorbance*	Standard-Deviation	Chem. Blanks	Absorbance in % of Solvent Control**
Medium Control	-	no	0.688	0.025	0.271	85.62
Solvent Control	-	no	0.760	0.039	0.273	100.00
Test Item	4.9	no	0.717	0.025	0.278	90.25
	9.8	no	0.780	0.023	0.274	103.97
	19.5	no	0.744	0.028	0.273	96.74
	39.1	no	0.760	0.017	0.278	99.18
	78.1	no	0.756	0.026	0.270	99.89
	156.3	no	0.724	0.027	0.271	92.98
	312.5^P	no	0.626	0.060	0.272	72.60
	625^P	yes	0.309	0.070	0.264	9.15

Shaded test groups:           cytotoxic effects occurred in the photometric evaluation (< 75% cell viability)

^P             precipitation

*            mean absorbance (absolute) of7wells

**          relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium controlwas 116.8%.

The CV75 value of the second XTT test: 294.1 µg/mL

The mean CV75 value of both XTT tests: 359.85µg/mL

Results of the h-CLAT test:

1st test run:

Results of the first h-CLAT run for the Test Item YLANGANATE

 

	Concentration (µg/mL)	Antibody	RFI (%)	Cell Viability (%)
Medium Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
DMSO Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
Positive Control (DNCB)	2.0	CD 54	189.1^#	75.5
		CD 86	309.0*
	3.0	CD 54	266.7*	70.0
		CD 86	451.5*
Test Item	121	CD 54	115.8	85.3
		CD 86	102.2
	145	CD 54	129.7	87.6
		CD 86	111.6
	174	CD 54	162.4	84.9
		CD 86	132.0
	208	CD 54	135.6	86.5
		CD 86	132.6
	250	CD 54	156.4	85.8
		CD 86	129.3
	300	CD 54	229.7*	68.5
		CD 86	167.4*
	360	CD 54	359.4*	38.5
		CD 86	416.0*
	432^PS	CD 54	568.3*	27.0
		CD 86	970.2*

Shaded test groups: cell viability below 50%, these test item concentrations were excluded from the evaluation

^PS   Phase separation (oily droplets), this test item concentration was excluded from the evaluation

^#CD54 RFI value of the positive control (2.0 µg/mL DNCB) did not fulfil the positive criteria
(CD54 ≥ 200%).

*   RFI value of CD86 or CD54 exceeded the positive criteria (CD86≥150% and CD54≥200%).

2nd test run:

Results of the second h-CLAT run for the Test Item YLANGANATE

 

	Concentration (µg/mL)	Antibody	RFI (%)	Cell Viability (%)
Medium Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
DMSO Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
Positive Control (DNCB)	2.0	CD 54	268.0*	80.5
		CD 86	605.9*
	3.0	CD 54	376.2*	72.3
		CD 86	669.7*
Test Item	121	CD 54	101.1	92.1
		CD 86	94.2
	145	CD 54	104.3	86.4
		CD 86	96.4
	174	CD 54	111.7	81.9
		CD 86	103.6
	208	CD 54	113.8	76.4
		CD 86	83.3
	250	CD 54	107.4	80.3
		CD 86	66.7
	300	CD 54	125.5	80.4
		CD 86	91.3
	360	CD 54	196.8	75.6
		CD 86	143.5
	432^PS	CD 54	213.8*	61.0
		CD 86	276.8*

^PS   Phase separation (oily droplets), this test item concentration was excluded from the evaluation

Results of the third h-CLAT run for the Test Item YLANGANATE

 

	Concentration (µg/mL)	Antibody	RFI (%)	Cell Viability (%)
Medium Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
DMSO Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
Positive Control (DNCB)	2.0	CD 54	545.6*	69.6
		CD 86	640.3*
	3.0	CD 54	707.0*	71.1
		CD 86	536.8*
Test Item	121	CD 54	112.0	95.0
		CD 86	109.0
	145	CD 54	108.0	94.4
		CD 86	94.0
	174	CD 54	104.0	93.8
		CD 86	108.3
	208	CD 54	110.0	95.0
		CD 86	128.6
	250	CD 54	126.0	92.9
		CD 86	140.6
	300	CD 54	150.0	76.7
		CD 86	241.4*
	360	CD 54	302.0*	66.6
		CD 86	749.6*
	432^PS	CD 54	982.0*	25.5
		CD 86	3073.7*

Shaded test group: cell viability below 50%, this test item concentration was excluded from the evaluation

^PS   Phase separation (oily droplets), this test item concentration was excluded from the evaluation.

*   RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

*   RFI value of CD86 or CD54 exceeded the positive criteria (CD86≥150% and CD54≥200%).

Interpretation of results:

other: h-CLAT is positive

Conclusions:

In a GLP-compliant guideline study, the test substance was found to be positive under the conditions of this in vitro h-CLAT test.

Executive summary:

The GLP-compliant in vitro Human Cell Line Activation Test (h-CLAT) was performed in accordance with OECD guideline 442E. The substance is dissolved in culture medium when administered to THP-1 cells for 24 ± 1 hours. The substance is in the applicability domain of this test. The validity criteria were met in the positive and negative controls. The following concentrations of the test item (solved in culture medium) were tested in the main experiment (h-CLAT), based on the results of two XTT tests: 4.9, 9.8, 19.5, 39.1, 78.1, 156.3, 312.5, 625 ug/mL. The test substance was tested in three tests. In the first test the positive control CD54 was not meeting the criteria of being positive >=200% but the results with the substance were considered positive. The results in the first test were 230 and 167, in the second test wer 197 and 143 and in the third test 302 and 241 for CD54 and CD 86, respectively. In the first test and the third test the results were > 200 and 150 or CD54 and CD86, respectively. In view of the positive results of the substance in 2/3 tests the substance is considered positive in this test.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 November 2016 - 02 December 2016

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:

February 2015

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

Vehicle: DMSO (also used as a negative control), maximal concentration of 1% in the culture medium, 18 wells/plate
Positive control: ethylene dimethacrylate glycol, 7.81 to 250 µM (final concentration DMSO of 1%)
Blank: on each plate three blank wells were tested (no cells and no treatment) to assess background values.
 
Preparation of the test item DMSO stock solution, spiking and working solutions:
No correction was made for the composition/purity of the test item.
The test item was dissolved in DMSO to a final concentration of 200 mM. The compound formed a clear colourless solution at 200 mM. From the stock 11 spike solutions in DMSO were prepared (2-fold dilution series): 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.781, 0.391, 0.195 and 0.0977 mM.
The stock and spike solution were diluted 25-fold with exposure medium resulting in concentrations of 8000, 4000, 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.813 and 3.906 µM (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 0.976 µM (final concentration DMSO of 1%). All formulations formed a clear solution.

Preparation of the positive control solution:
A 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted as described above, so that the final concentration of the positive control ranges from 7.81 to 250 µM (final concentration DMSO of 1%).

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.
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.
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. The cells were incubated overnight in the incubator.

Media:
Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.
Maintenance medium: Dulbecco’s minimal 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 supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.

Environmental conditions:
All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 60 – 100%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 36.4 –38.5°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.

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 substances 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.

Number of replicates:
Three replicates were used for the luciferase activity measurements for the test substance and positive and vehicle controls, and one parallel replicate used for the MTT cell viability assay. In total 2 independent 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.

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.

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 = (L sample - L blank) / (L solvent - L blank),
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
Lsolvent is the average luminescence reading in the wells containing cells and solvent (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 with > 1.5 fold induction
Cb is the highest concentration in μM 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 = 100 x (V sample - V blank)/(V solvent - V blank)
Where
Vsample is the MTT-absorbance reading in the test chemical well
Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment
Vsolvent is the average MTT-absorbance reading in the wells containing cells and solvent (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.

Control IC50 and IC30 are calculated by linear interpolation, and the overall IC50 and IC30 are calculated as the mean of the individual repetitions.

Statistics:
In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) 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 of the assay:
• 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 6 wells tested. If the variability is higher, results should be discarded.

Interpretation of the results:
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 (Figure 1):
1. 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 t-test)
2. The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
3. The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)
4. There is an apparent overall dose-response for luciferase induction

At least two repetitions need to be performed.
If the two repetitions are positive, final outcome is considered positive.
If the two repetitions are negative, final outcome is considered negative.
In case the first two repetitions are not concordant, a third repetition needs to be performed and a conclusion should be drawn on the basis of the mode of the outcome (i.e. 2 out of 3).
Negative results obtained with concentrations <1000 µM should be considered as inconclusive.

Positive control results:

In experiment 1, the positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.40 and the EC1.5 75.2 µM.
In experiment 2, the positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.80 and the EC1.5 27.0 µM.

Key result

Run / experiment:

other: 1

Parameter:

other: Imax

Value:

1.24

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: Imax

Value:

1.14

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 1

Parameter:

other: IC30, µM

Value:

1 785

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (12.3% and 7.7% in experiment 1 and 2, respectively).

- Acceptance criteria met for positive control: yes, 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.
The EC1.5 of the positive control was between 5 and 125 µM (75.2 µM and 27.0 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.40-fold and 2.80-fold in experiment 1 and 2, respectively).

- Acceptance criteria met for variability between replicate measurements: not applicable.

- Range of historical values if different from the ones specified in the test guideline: not applicable.

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

TEST ITEM RESULTS:
Experiment 1
The test substance showed slight toxicity. The viability of the cells was higher than 70% at all test concentrations with exception at the highest test concentration of 2000 µM. The calculated IC30 was 1785 µM. Fifty percent toxicity was not reached and thus no IC50 could be calculated.
Experiment 2
The test substance 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.

Table 1. Overview of luminescence induction and cell viability of the test substance in Experiments 1 and 2

Concentration (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.50	125	250	500	1000	2000
Exp 1 luminescence	0.84	0.84	0.87	0.91	1.09	1.01	1.03	1.15	1.24	1.24	1.16	1.13
Exp 1 viability (%)	96.1	71.5	109.7	79.5	87.2	108.6	97.1	86.8	97.9	93.4	95.1	63.1
Exp 2 luminescence	1.10	1.05	1.13	1.14	1.05	1.14	1.05	1.04	1.13	0.99	0.97	0.93
Exp 2 viability (%)	111.5	107.7	95.0	84.1	83.6	81.5	78.1	80.6	77.3	78.1	81.7	99.1

Table 2. Overview of the luminescence induction and cell viability for the positive control ethylene dimethacrylate glycol in Experiments 1 and 2

Concentration (µM)	7.81	15.6	31.3	62.5	125	250
Exp 1 luminescence	0.91	0.97	1.16	1.45	1.69***	2.40***
Exp 1 viability (%)	80.6	78.5	98.0	95.3	100.0	96.4
Exp 2 luminescence	1.34	1.40	1.61*	1.61*	1.82**	2.80***
Exp 2 viability (%)	96.6	95.5	98.0	96.2	102.9	101.4

* p<0.05; ** p<0.01; *** P<0.001 Studentsttest

Table 3. Overview of EC1.5, Imax, IC30 and IC50 values

	EC1.5 (µM)	Imax	IC30(µM)	IC50(µM)
Test substance Experiment 1	NA	1.24	1785	NA
Test substance  Experiment 2	NA	1.14	NA	NA
Pos Control Experiment 1	75.2	2.40	NA	NA
Pos Control Experiment 2	27.0	2.80	NA	NA

N.A. = Not applicable

Interpretation of results:

other: KeratinoSens assay was negative

Conclusions:

In a GLP-compliant guideline KeratinoSensTM assay the test substance is concluded to show a negative result (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

In a GLP-compliant OECD guideline 442D assay, the ability of the test substance to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway was tested in the KeratinoSens^TM assay.

Two independent experiments were performed, with each test concentration and positive and negative (vehicle) controls tested in triplicate. The test substance was tested in the concentration range of 0.976 - 2000 µM (final concentration DMSO in the culture medium of 1%). Ethylene dimethacrylate glycol in a concentration range of 7.81 to 250 µM (final concentration DMSO of 1%) was used as a positive control.

The acceptance criteria were met. The test substance showed slight toxicity in experiment 1 (no IC₅₀and an IC₃₀ of 1785 µM) and no toxicity in experiment 2 (no IC₃₀and IC₅₀ values) and no biologically relevant induction of the luciferase activity (no EC_1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (I_max) was 1.24-fold and 1.14-fold in experiment 1 and 2 respectively. The test substance is classified as negative in the KeratinoSens^TMassay, 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

For Ylanganate several sources of skin sensitisation information are available: in silico, in vitro and in vivo information.

In silico: The OECD Toolbox presents absence of protein binding.

In vitro: The substance tested was negative in the DPRA, negative in the Keratinosens but positive in the h-CLAT. In absence of protein binding (DPRA) and specific signals for skin sensitisation (Keratinosens), the potential of forming antibodies (h-CLAT) in isolation, will not present a skin sensitization potential.

In vivo: LLNA is negative up to 30%.

Overall conclusion: Ylanganate does not have a skin sensitization potential based on the key information of three in vitro skin sensitization strategy. The ITS proposed by Bauch et al. 2012) presents that when 2/3 in vitro results are negative the substance can be considered a non-skin sensitiser. In addition, there is no structural alert for skin sensitization potential and the substance is negative in the LLNA up to 30%.

Reference: Bauch, C., Kolle, S.N., Ramirez, T., Eltze, T., Fabian E., Mehling, A., Teubner, W., van Ravenzwaay, B., Landsiedel, R., 2012, Putting the parts together: Combining in vitro methods to test for skin

sensitizing potentials, Regul. Toxicol. Pharmacol., 63, 489–504.

The executive summaries are presented in the order of key in vitro information followed by key in vivo information

In vitro skin sensitization summaries

DPRA: In a GLP-compliant OECD guideline 442C study, Direct Peptide Reactivity Assay (DPRA) was used to assess the reactivity and sensitizing potential of the test substance. In the cysteine and lysine reactivity assay all analytical acceptance criteria of the test were met. The test substance caused -1.53% (CV 0.44) and -0.473% (CV 0.47) cysteine and lysine peptide depletion, respectively. These results are categorised as “no to minimal reactivity” based on the DPRA prediction model and is thus considered to be negative in the DPRA.

Keratinosens: In a GLP-compliant OECD guideline 442D assay, the ability of the test substance to activate theantioxidant/electrophile responsive element (ARE)-dependent pathway was tested in theKeratinoSens^TMassay. Two independent experiments were performed, with each test concentration and positive and negative (vehicle) controls tested in triplicate. The test substance was tested in the concentration range of0.976 - 2000 µM (final concentration DMSO in the culture medium of 1%).Ethylene dimethacrylate glycol in a concentration range of7.81 to 250 µM (final concentration DMSO of 1%) was used as a positive control. The acceptance criteria were met.The test substance showed slight toxicity in experiment 1 (no IC₅₀and an IC₃₀of 1785 µM) and no toxicity in experiment 2 (no IC₃₀and IC₅₀values) and no biologically relevant induction of the luciferase activity (no EC_1.5value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (I_max) was 1.24-fold and 1.14-fold in experiment 1 and 2 respectively. The test substance is classified as negative in the KeratinoSens^TMassay, 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.

h-CLAT: The GLP-compliant in vitro Human Cell Line Activation Test (h-CLAT) was performed in accordance with OECD guideline 442E. The substance is dissolved in culture medium when administered to THP-1 cells for 24 ± 1 hours. The substance is in the applicability domain of this test. The validity criteria were met in the positive and negative controls. The following concentrations of the test item (solved in culture medium) were tested in the main experiment (h-CLAT), based on the results of two XTT tests: 4.9, 9.8, 19.5, 39.1, 78.1, 156.3, 312.5, 625 ug/mL. The test substance was tested in three tests. In the first test the positive control CD54 was not meeting the criteria of being positive >=200% but the results with the substance were considered positive. The results in the first test were 230 and 167, in the second test were 197 and 143 and in the third test 302 and 241 for CD54 and CD 86, respectively. In the first test and the third test the results were > 200 and 150 for CD54 and CD86, respectively. In view of the positive results of the substance in 2/3 tests the substance is considered positive in this test.

In vivo information summary of LLNA

The skin sensitisation potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay" method (IFF, 2004).

Groups of 5 female CBA mice were exposed once daily for 3 consecutive days to 7.5, 15, and 30% test substance on the dorsal surface of both ears. At 7.5, 15, and 30% the substance showed SI values of 1.13, 1.59, and 1.22, respectively. No statistically significant differences in the ear measurements were observed in any of the treatment groups when compared to the vehicle group. The test item was considered not to be a sensitiser under the conditions of the test. The 30% may be considered the maximum concentration because the substance is classified for skin irritation.

In vivo supporting information from HRIPT

Two HRIPTs are performed. The test with the highest concentration is presented: at 10% and 111 subjects no sign of skin sensitization was seen.

Justification for classification or non-classification

The substance is not a skin sensitiser in view of the above results. Therefore Ylanganate does not need to be classified and labelled according to EU CLP ((EC No. 1272/2008 and its updates).