Methyl 2-[[3-[4-(1,1-dimethylethyl)phenyl]-2-methylpropylidene]amino]benzoate

Endpoint summary

Description of key information

The skin sensitisation is assessed with three in vitro tests with the following results:

DPRA (OECD TG 442C): Negative

KeratinoSens (OECD TG 442D): Negative

h-CLAT (OECD TG 442E): Negative, but the result is not considered due to log Kow >3.5

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Remarks:

Direct Peptide Reactivity Assay (DPRA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

The study was conducted between 10 March 2017 and 17 March 2017

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 442C using the Skin Sensitisation: Direct Peptide Reactivity Assay method and in compliance with GLP.

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)

Specific details on test material used for the study:

Identification: Lilial ME Anthranilate
Chemical (IUPAC) Name of multiconstituent: Reaction mass of methyl 2-{[(1E)-3-(4-tert-butylphenyl)-2-methylprop-1-en-1-yl]amino}benzoate and methyl 2-{[(1Z)-3-(4-tert-butylphenyl)-2- methylprop-1-en-1-yl]amino}benzoate
CAS no multiconstituent: 1953103-61-1
Appearance: Yellow viscous liquid
Expiry/retest date: 10 May 2019
Storage conditions: Ambient temperature (15°C to 25°C), under nitrogen

Details on the study design:

Assessment of Test Item Solubility
The solubility of Lilial-Me Anthranilate in acetonitrile was assessed at a concentration of 100 mM.

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 Lilial-Me Anthranilate was prepared. Cysteine Peptide Depletion Samples (in triplicate) were prepared by dilution of the 100 mM Lilial-Me Anthranilate solution in more acetonitrile and Cysteine peptide stock solution. The final sample concentration was 5 mM of Lilial-Me Anthranilate, 0.5 mM Cysteine. For the positive control, a 100 mM solution in acetonitrile of Cinnamic Aldehyde was prepared. The final sample solution contained Cinnamic Aldehyde at a concentration of 5 mM with 0.5 mM Cysteine.The co-elution control sample contained 5 mM of Lilial-Me Anthranilate in phosphate buffer solution. An additional control sample of 5 mM of Lilial-Me Anthranilate in acetonitrile was also prepared so as to positively identify peak(s) of the test item in the co-elution control ensuring that the test item had not evaporated away during incubation and injection of the samples on the HPLC.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls
A 100 mM solution in acetonitrile of Lilial-Me Anthranilate was prepared and further diluted in HPLC vials. Lysine Peptide Depletion Samples (in triplicate) were prepared by dilution of the 100 mM Lilial-Me Anthranilate solution in Lysine peptide stock solution. The final sample concentration was 25 mM of Lilial-Me Anthranilate, 0.5 mM Lysine. For the positive control, a 100 mM solution in acetonitrile of Cinnamic Aldehyde was prepared. The final sample solution contained Cinnamic Aldehyde at a concentration of 25 mM with 0.5 mM Lysine. The co-elution control sample contained 25 mM of Lilial-Me Anthranilate in ammonium acetate buffer solution. An additional control sample of 25 mM of Lilial-Me Anthranilate in acetonitrile was also prepared so as to positively identify peak(s) of the test item in the coelution control ensuring that the test item had not evaporated away during incubation and injection of the samples on the HPLC.

Incubation
The appearance of the Lilial-Me Anthranilate, 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 Lilial-Me Anthranilate and the associated positive controls were quantified by HPLC using UV detection as detailed in the chromatographic section.

Run / experiment:

other: Cysteine

Parameter:

other: Mean Depletion (%)

Value:

-0.291

Run / experiment:

other: Lysine

Parameter:

other: Mean Depletion (%)

Value:

-7.85

Other effects / acceptance of results:

Solubility Assessment
The solubility of Lilial-Me Anthranilate in acetonitrile at a nominal concentration of 100 mM was confirmed by visual inspection.

Reactivity Assessment
All analytical acceptance criteria for each analytical run were met.
Only the cysteine result is reported by the application of the cysteine 1:10 reactivity prediction model (co eluting peaks). Based on this model, reactivity is classed as “no to minimal”, hence the DPRA prediction is negative.

Analytical acceptance criteria

	Peptide	Standard Linearity	Positive control depletion (%)	Reference control concentration and replicate precision	Test item replicate precision
Acceptance criteria	Cysteine	r2>0.99	60.8 -100 (SD <14.9 %)	0.45- 055 mM (CV < 15%)	SD < 14.9 %
	Lysine	r2>0.99	40.2 - 69.0 (SD <11.6%	0.45- 055 mM (CV < 15%)	SD < 11.6 %
Achieved results	Cysteine	r2>0.999	72.5 (SD 0.11%, n=3)	0.51 mM (CV 0.70%, n =6)	SD 0.42% (n=3)
	Lysine	r2>0.999	59.7 (SD 1.40%, n=3)	0.51 mM (CV 1.17%, n =6)	SD 1.31% (n=3)

CV Coefficient of Variation

SD Standard deviation

Depletion of peptide in the presence of Lilial-Me Anthranilate

	Mean peak area of reference control (µV.sec)	Mean peak area of LilialMe Anthranilate (µV.sec)	Mean Depletion (%)Lilial-Me Anthranilate
Cysteine	876510 (n=6)	879060 (n=3)	-0.291
Lysine	763240 (n=6)	823140 (n=3)	-7.85

Cysteine 1:10 reactivity prediction model

Mean of cysteine % depletion	Reactivity Class	DPRA Prediction
0% ≤ Cys% depletion ≤ 13.89%	No or minimal reactivity	Negative
13.89% < Cys% depletion ≤ 23.09%	Low reactivity	Positive
23.09% < Cys% depletion ≤ 98.24%	Moderate reactivity
98.24% < Cys% depletion ≤ 100%	High reactivity

Interpretation of results:

other: “no to minimal reactivity” and thus is predicted to be negative by DPRA for skin sensitization potential.

Conclusions:

Solutions of Lilial-Me Anthranilate were successfully analyzed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in the Cysteine containing synthetic peptide with all analytical acceptance criteria of the test being met. In the Lysine containing synthetic peptide, there was co-elution of the test item with the Lysine peak and following the guideline document TG 442C no conclusions on reactivity can be drawn from it.
The Cysteine peptide depletion result in the presence of Lilial-Me Anthranilate was -0.291% and from this result alone a DPRA prediction can be made. As there was no reactivity with the Cysteine peptide Lilial-Me Anthranilate is classed as “no to minimal reactivity” and thus is predicted to be negative by DPRA, this test being one indicator for skin sensitization potential.

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 Lilial-Me Anthranilate.

Solutions of Lilial-Me Anthranilate were successfully analyzed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in the Cysteine containing synthetic peptide with all analytical acceptance criteria of the test being met. In the Lysine containing synthetic peptide, there was co-elution of the test item with the Lysine peak and following the guideline document TG 442C no conclusions on reactivity can be drawn from it.

The Cysteine peptide depletion result in the presence of Lilial-Me Anthranilate was -0.291% and from this result alone a DPRA prediction can be made. As there was no reactivity with the Cysteine peptide Lilial-Me Anthranilate is classed as “no to minimal reactivity” and thus is predicted to be negative by DPRA, this test being one indicator for skin sensitization potential.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 May, 2017 - 14 July, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD).

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:

Controls:
Positive control: ethylene dimethacrylate glycol, 7.81-250 µM, tested in triplicate, final concentration DMSO of 1%
Negative control: DMSO (vehicle) (1% in exposure medium)
Blank: on each plate three blank wells were tested (no cells and no treatment) to assess background values

Number of replicates: three independent experiments, each concentration tested in triplicate for the luciferase activity measurements, one parallel replicate for MTT cell viability assay.

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 (the passage number used was 26 in experiment 1 and 28 in experiment 2) 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. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator.

Test item preparation:
No correction was made for the composition/purity of the test item.
The test item was dissolved in DMSO to a final concentration of 100 mM to form a homogeneous suspension (slight precipitation). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98 and 0.49 µM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. In experiment 2, a slightly higher DMSO stock was prepared due to a calculation error (1.2% higher). This had no effect on the study results. Test item concentrations were used within 3 hours after preparation. Precipitation was observed at 250 µM and upwards in the 96-well plates at the start and end of the treatment.

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.

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 in a humid atmosphere of 80 - 100% (actual range 65 – 99 %) at 37.0 ± 1.0°C (actual range 36.0 – 37.5°C), in the presence of 5% ± 0.5%CO2.

Luciferase acitivity 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 (e.g. by adding 10% SDS solution to each well) overnight. After shaking, the absorption is 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= (Lsample - Lblank)/(Lsolvent - Lblank)
Where:
Lsample is the luminescence reading in the test chemical well
Lblank is the luminescence reading in the blank well containing no cells and no treatment
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 (Vsample - Vblank) / (Vsolvent - 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.

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). 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.

Data intepretation:
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in 2 of 3 repetitions, otherwise the KeratinoSensTM prediction is considered negative:
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

Acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.81 to 250 µM).
• The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
• Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.

Positive control results:

Experiment 1: the positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.39 and the EC1.5 33.0 µM.
Experiment 2: the positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.74 and the EC1.5 43.3 µM.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

1.2

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: Experiment 2

Parameter:

other: Imax

Value:

1.17

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:

The test substance showed toxicity only in experiment 1 (IC30 value of 847 µM). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in any of the two experiments. The maximum luciferase activity induction (Imax) was 1.20-fold and 1.17-fold in experiment 1 and 2 respectively. The test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations of up to 1000 µM.

ACCEPTANCE OF RESULTS:
- 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 (33.0 μM and 43.3 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (3.39-fold and 2.74-fold in experiment 1 and 2, respectively).
- Acceptance criteria met for variability between replicate measurements: yes. The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% in each repetition (respectively 12% and 6.5% for experiment 1 and 2).

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

Concentration (μM)	0.49	0.98	1.95	3.91	7.8	15.6	31.25	62.5	125	250	500	1000
Exp 1 luminescence	0.98	1.03	1.07	1.05	1.20	1.16	0.86	0.57	0.35	0.20	0.19	0.16
Exp 1 Viability (%)	116.2	111.9	115.2	119.7	148.3	189.6	153.4	105.0	54.2	100.4	85.1	63.4
Exp 2 luminescence	0.94	0.64	1.06	1.05	1.13	1.17	0.94	0.65	0.47	0.32	0.16	0.18
Exp 2 Viability (%)	85.6	95.1	93.6	98.7	129.1	183.3	155.1	124.2	109.5	108.6	111.5	83.7

 

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

Concentration (μM)	7.8	16	31	63	125	250
Exp 1 luminescence	1.10	1.26	1.47	2.03^***	2.35^***	3.39^***
Exp 1 Viability (%)	105.2	106.2	114.7	124.6	129.7	127.9
Exp 2 luminescence	1.09	0.86	1.41	1.65^***	2.11^***	2.74^***
Exp 2 Viability (%)	84.8	89.6	99.5	103.9	109.4	109.5

*** p<0.001 Students t-test

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

	EC1.5 (μM)	Imax	IC30 (μM)	IC50 (μM)
Test item experiment 1	NA	1.20	847	NA
Test item experiment 2	NA	1.17	NA	NA
Pos. control experiment 1	33.0	3.39	NA	NA
Pos. control experiment 2	43.4	2.74	NA	NA

 

NA = Not applicable

Interpretation of results:

other: KeratinoSens assay was negative

Conclusions:

In a GLP-compliant guideline study, the test substance did not cause a biologically relevant induction in luciferase activity in Keratinosens assay. Based on this, the test substance is considered to give a negative result under the experimental conditions in this assay.

Executive summary:

The GLP-compliant in vitro KeratinoSens (ARE-Nrf2 luciferase reporter) assay was performed in accordance with OECD guideline 442D to assess the skin sensitising potential of the test substance. Two independent experiments were performed. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.49 – 1000 μM. Ethylene dimethacrylate glycol was used as a positive control. The acceptance criteria were met in both experiments. toxicity only in experiment 1 (IC30 value of 847µM). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in any of the two experiments. The maximum luciferase activity induction (Imax) was 1.20-fold and 1.17-fold in experiment 1 and 2, respectively. The test item is classified as negative in the KeratinoSensTM assay, since negative results (<1.5-fold induction) were observed at test concentrations of up to 1000 µM.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

The study was conducted between 02 March 2017 and 07 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is considered to be reliability 1 as it has been conducted according to OECD Test Guideline 442E using the human cell line activation test (h-CLAT) and in compliance with GLP.

Qualifier:

according to guideline

Guideline:

other: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), July 2016.

Deviations:

yes

Remarks:

The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry.

GLP compliance:

yes (incl. QA statement)

Type of study:

other: In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)

Specific details on test material used for the study:

Identification: Lilial ME Anthranilate
Appearance: Yellow viscous liquid (paste-like)
Storage Conditions: At room temperature, under N2-atmosphere

Dose calculation was not adjusted to purity.

Details on the study design:

Controls for XTT and h-CLAT: Concurrent controls were used for several Envigo CRS GmbH studies performed simultaneously.
Medium Control: Culture medium
Solvent Control for the Test Item: DMSO (final concentration for XTT cytotoxicity 0.2 - 0.5% and for h-CLAT 0.2%)
Positive Control: DNCB, Solvent: DMSO, diluted with culture medium to a final concentration of 2 and 3 µg/mL DNCB.
Solvent Control for the Positive Control (h-CLAT): DMSO (final concentration 0.2%)

TEST ITEM PREPARATION
On the day of the experiment (immediately prior to start) Lilial ME Anthranilate was dissolved in DMSO.
The maximum prepared concentration of test item was 62.5 µg/mL in 0.2% (v/v) DMSO.
For the XTT test (dose finding assay) eight concentrations of the test item were analysed. For this, dilutions were prepared by 1:2 serial dilutions from 62.5 µg/mL in 0.2% (v/v) DMSO.



Experimental Design and Procedures of XTT
Dose Finding Assay (XTT Test)
The test item concentrations investigated in the main experiment (h-CLAT) were determined with two XTT tests.
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. This method was first described 1988 by SCUDIERO et al. and improved in subsequent years by several other investigators.
Two independent cytotoxicity experiments were performed with different cell cultures to obtain a reliable CV75. The mean CV75 was calculated with the CV75 value of the first XTT test and the highest soluble tested test item concentration of the second XTT test, to determine the dose-range for the main experiment (h-CLAT).

XTT Labelling Mixture
The XTT labelling mixture consists of two components, a XTT buffer solution and the substrate solution. Both components were mixed right before application at a ratio of 1:100.

Treatment
After the cell seeding, 100 µL of the test item dilutions, the medium and solvent controls, respectively were added to the cells. All dose groups were tested in 7 replicates for each XTT test. At the end of the incubation period of 24 ± 1 hours, the cell cultures were microscopically evaluated for morphological alterations.

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).



Experimental Design and Procedures of h-CLAT
The test item was tested in two independent runs.

Treatment of the Cells
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. 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) DMSO in the culture 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, positive and DMSO control was prepared 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 x 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-AAD solution were added.
 
Flow Cytometry Acquisition
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) were 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 are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single population appears without contamination or excessive debris. The FL-1 and FL-3 voltage were set and compensate to appropriate position. The FL-1 voltage was set using the FITC labelled-mouse IgG1 medium-treated cells tube, such that the MFI of control cells were set in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo Mean) with the FITC labelled CD54 medium-treated cells (FACSCalibur, Becton Dickinson).
The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was 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 were 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 are generated due to cell membrane destruction).
 

 

Positive control results:

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

Run / experiment:

other: 2 independent runs

Parameter:

other: % relative fluorescence intensity (RFI) of CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2 independent runs

Parameter:

other: % relative fluorescence intensity (RFI) of CD54

Value:

200

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:

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the skin sensitising potential of Lilial ME Anthranilate dissolved in DMSO (final concentration 0.2% (v/v)) when administered to THP-1 cells for 24 ± 1 hours. The highest test item concentration for the main experiment (h-CLAT) of Lilial ME Anthranilate was previously determined by two XTT tests.
Cytotoxic effects (threshold of cytotoxicity: < 75%) were observed following incubation with the test item starting with the concentration of 31.25 µg/mL up to the highest tested concentration (62.5 µg/mL) in the first XTT test and at the highest concentration in the second XTT test. Precipitations were observed in the highest tested concentration of both XTT tests. Since no CV75 could be calculated for the second XTT test, the highest soluble test item concentration was used for the calculation of the mean CV75. The mean CV75 value of both XTT tests was calculated as 29 µg/mL.
The following concentrations of the test item (dissolved in DMSO with a final concentration of 0.2% (v/v)) were tested in the main experiment (h-CLAT):
10, 12, 14, 17, 20, 24, 29 and 35µg/mL
The test item with a log Pow of 4.4 was tested in 2 independent runs. The RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any dose in both independent run data. Both runs showed a NEGATIVE result However, due to a log Pow > 3.5 for the tested test item, the result of this h-CLAT should not be considered.
In the DMSO solvent control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).
Further results of the testing battery (including e.g. DPRA, ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential are not available. Therefore, consideration of the test method results within the context of an IATA (Integrated Approaches to Testing and Assessment) is not possible.

Resultsof the Dose Finding Assay (XTT Test)

Results of the first XTT test for Test Item Lilial ME Anthranilate

		Microscopic Evaluation	Photometric Evaluation
Test Group	Concen-tration [µg/mL]	Cytotoxicity	Mean Ab-sorbance*	Standard-Deviation	Chem. Blank	Mean Ab-sorbance – Chemical Blank	Absorbance in % of Solvent Control**
Medium Control	-	no	0.826	0.041	0.192	0.633	101.79
Solvent Control	-	no	0.819	0.048	0.197	0.622	100.00
Test Item	0.49	no	0.775	0.049	0.199	0.576	92.53
	0.98	no	0.814	0.032	0.201	0.613	98.56
	1.95	no	0.778	0.023	0.194	0.585	93.98
	3.91	no	0.849	0.064	0.201	0.648	104.19
	7.81	no	0.804	0.044	0.197	0.606	97.45
	15.63	no	0.733	0.024	0.202	0.531	85.27
	31.25	yes	0.647	0.030	0.207	0.440	70.74
	62.5^P	yes	0.621	0.030	0.246	0.375	60.29

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

P             precipitation

*             mean absorbance (absolute) of 7 wells

**          relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 98.25%.

The CV75 value of the first XTT test: 26.7 µg/mL

Results of the second XTT test for Test Item Lilial ME Anthranilate

		Microscopic Evaluation	Photometric Evaluation
Test Group	Concen-tration [µg/mL]	Cytotoxicity	Mean Ab-sorbance*	Standard-Deviation	Chem. Blank	Mean Ab-sorbance – Chemical Blank	Absorbance in % of Solvent Control**
Medium Control	-	no	0.963	0.048	0.200	0.763	110.42
Solvent Control	-	no	0.887	0.033	0.196	0.691	100.00
Test Item	0.49	no	0.933	0.035	0.202	0.731	105.81
	0.98	no	0.876	0.047	0.200	0.676	97.81
	1.95	no	0.881	0.022	0.199	0.681	98.61
	3.91	no	0.871	0.025	0.197	0.675	97.61
	7.81	no	0.843	0.033	0.205	0.638	92.34
	15.63	no	0.827	0.048	0.204	0.623	90.21
	31.25	no	0.770	0.038	0.208	0.562	81.36
	62.5^P	yes	0.718	0.027	0.243	0.476	68.85

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

P             precipitation

*             mean absorbance (absolute) of 7 wells

**          relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 90.57%.

The CV75 value of the second XTT test could not be determined due to precipitation observed at the highest tested test item concentration. Therefore, the highest tested soluble test itemconcentration (31.25 µg/mL) was used to calculate the mean CV75.

The mean CV75 value of both XTT tests: 29 µg/mL

Results of the h-CLAT Test

Results of the first h-CLAT run for the Test Item Lilial ME Anthranilate

	Concentration (µg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	100.0
DMSO Control	-	100.0	100.0	100.0
Positive Control (DNCB)	2.0	271.2*	247.1*	66.3
	3.0	326.0*	353.5*	61.4
Test Item	10	87.5	80.8	92.9
	12	93.3	80.2	92.4
	14	90.4	93.0	90.5
	17	94.2	83.7	90.5
	20	92.3	58.1	86.7
	24	114.4	59.9	84.9
	29	129.8	66.3	83.1
	35	151.0	83.7	78.0

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

 

Results of the second h-CLAT run for the Test Item Lilial ME Anthranilate

	Concentration (µg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	100.0
DMSO Control	-	100.0	100.0	100.0
Positive Control (DNCB)	2.0	305.7*	727.6*	74.7
	3.0	522.9*	686.6*	72.8
Test Item	10	90.5	83.5	96.4
	12	84.8	87.4	98.1
	14	96.2	78.7	97.3
	17	94.3	82.7	97.8
	20	103.8	82.7	95.4
	24	99.0	87.4	93.1
	29	121.9	88.2	90.8
	35	147.6	131.5	77.3

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

Interpretation of results:

study cannot be used for classification

Conclusions:

The test item Lilial ME Anthranilate with a log Pow of 4.4 was found to be negative up to a concentration of 35 µg/mL under the test conditions of this study. However, the negative result cannot be used in an assessment of skin sensitisation potential because the Log Pow of the test item is 4.4 and according to OECD test Guideline 442E, negative results for test chemicals with a Log Kow > 3.5 should not be considered.

Executive summary:

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the skin sensitising potential of Lilial ME Anthranilate dissolved in culture medium with 0.2% (v/v) DMSO when administered toTHP-1 cells for 24 ± 1 hours. The highest test item concentration for the main experiment(h-CLAT) of Lilial ME Anthranilate was previously determined by two XTT tests.

Cytotoxic effects (threshold of cytotoxicity: < 75%) were observed in both XTT tests. In addition, precipitations were observed in the highest tested concentration of both XTT tests. Due to the precipitation no CV75 could be calculated for the second XTT test, therefore the highest soluble test item concentration was used for the calculation of the mean CV75. The mean CV75 value of both XTT tests was calculatedas 29 µg/mL.

The following concentrations of the test item (dissolved inculture medium with 0.2% (v/v) DMSO) were tested in the main experiment (h-CLAT):

10, 12, 14, 17, 20, 24, 29 and 35µg/mL

The test item with a log Pow of 4.4 was tested in 2 independent runs. The RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any dose in both independent run data. Both runs showed a NEGATIVE result. However, due to a log Pow > 3.5 for the tested test item, the result of this h-CLAT should not be considered.

In the DMSO solvent control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%)and the cell viability was >50%.

In conclusion, the test item Lilial ME Anthranilate with a log Pow of 4.4 was found to be negative up to a concentration of 35 µg/mL under the test conditions of this study. However, the negative result cannot be used in an assessment of skin sensitisation potential because the Log Pow of the test item is 4.4 and according to OECD test Guideline 442E, negative results for test chemicals with a Log Kow > 3.5 should not be considered.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The executive summaries of the individual in vitro studies are presented.

DPRA

The skin sensitisation potential of the test substance has been tested according to OECD TG 442C: Direct Peptide Reactivity Assay. Solutions of Lilial-Me Anthranilate were successfully analyzed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in the Cysteine containing synthetic peptide with all analytical acceptance criteria of the test being met. In the Lysine containing synthetic peptide, there was co-elution of the test item with the Lysine peak and following the guideline document TG 442C no conclusions on reactivity can be drawn from it.

The Cysteine peptide depletion result in the presence of Lilial-Me Anthranilate was -0.291% and from this result alone a DPRA prediction can be made. As there was no reactivity with the Cysteine peptide Lilial-Me Anthranilate is classed as “no to minimal reactivity” and thus is predicted to be negative by DPRA, this test being one indicator for skin sensitization potential.

KeratinoSens

The GLP-compliant in vitro KeratinoSens (ARE-Nrf2 luciferase reporter) assay was performed in accordance with OECD guideline 442D to assess the skin sensitising potential of the test substance. Two independent experiments were performed. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.49 – 1000 μM. Ethylene dimethacrylate glycol was used as a positive control. The acceptance criteria were met in both experiments. toxicity only in experiment 1 (IC30 value of 847µM). No biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in any of the two experiments. The maximum luciferase activity induction (Imax) was 1.20-fold and 1.17-fold in experiment 1 and 2, respectively. The test item is classified as negative in the KeratinoSensTM assay, since negative results (<1.5-fold induction) were observed at test concentrations of up to 1000 µM.

h-CLAT:
The skin sensitisation potential of the test substance has been tested according to OECD TG 442E: in vitro Human Cell Line Activation Test (h-CLAT) method. At 10, 12, 14, 17, 20, 24, 29 and 35µg/mL the substance showed RFI of CD86 and CD54 that were not equal or greater than 150% and 200%, respectively at any dose in both independent run data. Both runs showed a NEGATIVE result. However, due to a log Pow > 3.5 for the tested test item, the result of this h-CLAT should not be considered.

Conclusion:

Lilial-Me Anthranilate does not have a skin sensitization potential based on the key information of three in vitro skin sensitization tests. This is based on the ITS proposed by Bauch et al. 2012, which presents that when 2/3 in vitro results are negative the substance is a non-skin sensitiser.

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.

Justification for classification or non-classification

Based on the presented information the substance is not a skin sensitiser according to EU CLP (EC 1272/2008 and its amendments)