Methyl benzoylformate

Endpoint summary

Description of key information

All key studies are conducted to recognised testing guidelines, or draft testing guidelines based upon recognised testing methods. All key studies have GLP certification.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Although the study predates the adoption of the OECD 442 C guideline (4 Feb 2015) it was conducted to the methods described in the following literature sources, and used as the basis for the OECD 442 C guideline:
• Gerberick GF, Vassallo JD, Bailey RE, Chaney JG, Morrall SW, Lepoittevin JP. Development of a Peptide Reactivity Assay for Screening Contact Allergens. Toxicological Sciences 81,332-343, 2004.
• Gerberick GF, Vassallo JD, Foertsch LM, Price BB, Chaney JG, Lepoittenvin JP. Quantificationn of Chemical Peptide Reactivity for Screening Contact Allergens: A Classification Tree Model Approach. Toxicological Sciences 97(2), 417-427, 2007.
• Bauch C, Kolle SN, Fabian E, Pachel C, Ramirez T, Wiench B, Wruck CJ, van Ravenzwaay B, Landsiedel R. Intralaboratory validation of four in vitro assays for the prediction of the skin sensitizing potential of chemicals. Toxicology in Vitro 25, 1162 – 1168, 2011.
• Maxwell G, Aeby P, Ashikaga T, Bessou-Touya S, Diembeck W, Gerberick F, Kern P, Marrec-Fairley M, Ovigne JM, Sakaguchi H, Schroeder K, Tailhardat M, Teissier S, Winkler P. Skin sensitisation: the Colipa strategy for developing and evaluating nonanimal test methods for risk assessment. ALTEX 28(1): 50-5, 2011.
The study report contains no deviations from the OECD 442 C test method.

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

The test substance was incubated with synthetic peptides for ca. 24 hours at room temperature and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The test substance was dissolved at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides. Further, a co-elution control was performed in order to detect possible interference of the test substance with the peptides. The samples consisted of the test substance, vehicle and the respective peptide buffer but without peptide. Moreover the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 / 258 was calculated as a measure of peak purity.

Test system:
Synthetic peptides:
- Cysteine- (C-) containing peptide: Ac-RFAACAA-COOH (MW=751.9 g/mol)
- Lysine- (K-) containing peptide: Ac-RFAAKAA-COOH (MW=776.2 g/mol)
The peptides are custom material (Supplier: RS Synthesis, Louisville KY, USA) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.

HPLC:
- Liquid chromatograph: Agilent HP 1100 with DAD
- Software: Dionex Chromeleon
- Column: Phenomenex Luna 3µ C18 (2), 100 mm x 2 mm with guard column "Security Guard“ C18, 4 mm x 2 mm
- Analytical balance: Accuracy 0.1 mg
- Pipettes / positive displacement pipette: For pipetting liquids of different viscosity up to 5 mL and graduated pipette with pipettor or graduated cylinder for higher volumes.
- pH meter: Readability +/- 0.1 pH units.
For adjusting pH-values of buffers.

- HPLC mobile phase A: 0.1% (v/v) trifluoracetic acid (99+%) in de-ionized water, HPLC grade
- HPLC mobile phase B: 0.085% (v/v) trifluoracetic acid (99+%) in acetonitrile, HPLC grade

- Reagents for preparing the buffers:
Sodium phosphate, monobasic monohydrate, CAS-no. 10049-21-5 (e.g. Sigma-Aldrich S9638)
Sodium phosphate, dibasic heptahydrate, CAS-no. 7782-85-6 (e.g. Sigma-Aldrich S9390)
Ammonium acetate, CAS-no. 631-61-8 (e.g. Sigma-Aldrich 32301)
Ammonium hydroxide, 28% – 30%, CAS-no. 1336-21-6 (e.g. Sigma-Aldrich 320145)

Controls:
- Negative control (NC): vehicle control = acetonitrile
- Positive control (PC): Ethylene glycol dimethacrylate (EGDMA; CAS-no. 97-90-5) (prepared as a 50 mM solution in acetonitrile)
- Co-elution control: Sample prepared of the respective peptide buffer and the test substance but without peptide.

Test-substance preparation:
The test substance solutions were prepared within 4 hours of performing the assay (preparation of samples).
- Test-substance preparation: The test substance was prepared as a 100 mM solution in acetonitrile. After short stirring the test substance was soluble
in the vehicle.
- Vehicle: acetonitrile
- Reason for the vehicle: The test substance was soluble in acetonitrile.

Measurement of peptide concentrations:
The analyses of the samples were performed via HPLC under the following conditions:
- Column: Phenomenex Luna 3µ C18 (2), 100 mm x 2 mm with guard column "Security Guard“ C18, 4 mm x 2 mm
- Eluent:
A: 0.1% (v/v) trifluoracetic acid in de-ionized water
B: 0.085% (v/v) trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Gradient:
time [min] %B
0 10
10 25
11 90
13 90
13.5 10
25 10
- Wavelength: 220 nm and 258 nm
- Injection volume: 2 µL

Positive control results:

See "Any other information on results incl. tables".

Parameter:

other: Mean C-peptide depletion [%]

Value:

3.18

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Parameter:

other: Mean K-peptide depletion [%]

Value:

19.34

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The test was determined to be valid. See below for full result tables and historical controls.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: See below for full result tables and historical controls.

RESULTS WITH CYSTEINE-PEPTIDE

Reaction with cysteine-peptide	peak area [mAU*s] at 220 nm			peptide concentration [mM]			mean	SD
	sample 1	sample 2	sample 3	sample 1	sample 2	sample 3
Negative control - Acetonitrile	744.3	753.2	741.0	0.491	0.497	0.489	0.492	0.004
Test material	717.0	726.5	723.7	0.473	0.479	0.477	0.476	0.003
Positive control - Ethylene glycol dimethacrylate	356.0	320.8	2655	0.235	0.212	0.176	0.208	0.03

Reaction with cysteine-peptide	peptide depletion [%]			mean	SD
	sample 1	sample 2	sample 3
Negative control - Acetonitrile	0.24	-0.94	0.7	0.0	0.84
Test material	3.9	2.64	3.0	3.18	0.65
Positive control - Ethylene glycol dimethacrylate	52.18	56.88	64.28	57.78	6.1

Reaction with cysteine-peptide	peak area [mAU*s] at 258 nm			area ratio 220/258
	sample 1	sample 2	sample 3	sample 1	sample 2	sample 3
Negative control - Acetonitrile	20.9	21.4	20.8	35.5	35.2	35.7
Test material	20.9	20.9	19.5	34.4	34.8	37.1
Positive control - Ethylene glycol dimethacrylate	9.5	8.6	6.7	37.5	37.5	39.4

RESULTS WITH LYSINE-PEPTIDE

Reaction with cysteine-peptide	peak area [mAU*s] at 220 nm			peptide concentration [mM]			mean	SD
	sample 1	sample 2	sample 3	sample 1	sample 2	sample 3
Negative control - Acetonitrile	722.0	728.5	721.8	0.503	0.507	0.502	0.504	0.003
Test material	583.8	588.9	576.1	0.407	0.41	0.403	0.407	0.004
Positive control - Ethylene glycol dimethacrylate	624.1	620.0	610.5	0.435	432	0.425	0.431	0.005

Reaction with cysteine-peptide	peptide depletion [%]			mean	SD
	sample 1	sample 2	sample 3
Negative control - Acetonitrile	0.29	-0.6	0.32	0	0.52
Test material	19.31	18.62	20.09	19.34	0.74
Positive control - Ethylene glycol dimethacrylate	13.76	14.33	15.64	14.58	0.96

Reaction with cysteine-peptide	peak area [mAU*s] at 258 nm			area ratio 220/258
	sample 1	sample 2	sample 3	sample 1	sample 2	sample 3
Negative control - Acetonitrile	21.0	21.3	21.1	34.3	34.2	34.2
Test material	17.0	17.0	16.5	34.4	34.6	35.0
Positive control - Ethylene glycol dimethacrylate	18.4	17.7	17.7	33.9	35	34.5

HISTORICAL CONTROL DATA

Historical Range of NC

Acetonitrile

Historical Period	mean peak area [mAU*s]	mean peptide concentration [mM]	SD of peptide concentration
Jan - Feb 2013 (no of tests performed: 29)
Cysteine-peptide	779.0	0.487	0.0201
Lysine-peptide	701.0	0.503	0.031

Historical Range of PC

Ethylene Glycol dimethacrylate 98% (50 mM in ACN)

Historical Period	mean peak area [mAU*s]	mean peptide concentration [mM]	SD of peptide concentration	mean peptide depletion [%]
Feb 2012 - Feb 2013 (no of tests performed: 25)
Cysteine-peptide	312	0.194	0.057	60
Lysine-peptide	625	0.448	0.026	11

Interpretation of results:

GHS criteria not met

Conclusions:

The mean depletion % to the test material were determined to be C-peptide 3.18% and K-peptide 19.34%. The test material was determined to be of low reactivity under the conditions of the test.

Executive summary:

The reactivity of the test material towards synthetic cysteine (C)- or lysine (K)-containing peptides was evaluated in the Direct Peptide Reactivity Assay (DPRA). For this purpose the test substance was incubated with synthetic peptides for ca. 24 hours at

room temperature and the remaining non-depleted peptide concentrations were determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm.

The test substance was dissolved at a 100 mM concentration in acetonitrile. Three samples of the test substance were incubated with each peptide in ratios of 1:10 (for C-peptide) or 1:50 (for K-peptide). Additionally triplicates of the concurrent vehicle control (= NC) were incubated with the peptides.

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

The following results were obtained in the DPRA:

The test substance was solved in acetonitrile. The samples of the test substance with the peptides were solutions. Visual observation after the 24-hour incubation time did not reveal precipitates in all samples of the test substance with both peptides.

The mean C-peptide depletion, caused by the test substance was determined to be 3.18%.

The mean K-peptide depletion, caused by the test substance was determined to be 19.34%.

Thus, the mean peptide depletion was calculated to be 11.26%.

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

Based on the observed results and applying the prediction model proposed in Gerberick et. al (2007) and cited in chapter 3.10 it was concluded that the test material shows a low chemical reactivity in the DPRA under the test conditions chosen.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013

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)

Deviations:

no

Principles of method if other than guideline:

The study was performed before the adoption of the OECD 442 D guideline; however, the study was performed according to the methods described in the following publication, which was used abs the basis for the OECD guiideline:
• Bauch C, Kolle SN, 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(3):489-504.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

The test substance was incubated with a luciferase reporter cell line (LuSens cells) for ca. 48 hours at 37°C and antioxidant response element (ARE) dependent luciferase activity was measured in a luminometer. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined thereafter by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve. In the main test luciferase activity was measured after 48 hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 5 valid experiments were performed.

CONTROLS
- Negative control (NC): 450 µg/mL, DL-Lactic acid (LA), CAS no.: 50-21-5
- Positive control (PC): 18 µg/mL, Ethylene glycol dimethacrylate (EGDMA), CAS-no. 97-90-5
- Vehicle control: 1% DMSO in culture medium 3
- Blank control: Medium 3 without cells
- Basal control: Medium 3 with cells

- Luciferace reagent: 10 g SDS, 99.6 mL DMSO, 0.4 mL glacial acetic acid SteadyGloLuciferase Assay, Promega
- MTT Solution: Thiazolyl Blue Tetrazolium Bromide 5 mg/mL with PBS without Mg2+/Ca2+
- Negative control (NC): 450 μg/mL, DL-Lactic acid (LA)
- Positive control (PC): 18 μg/mL, Ethylene glycol dimethacrylate
- Vehicle control: 1% DMSO in culture medium 3

TEST-SUBSTANCE PREPARATION
The test substance was weighed and topped up with the chosen vehicle (DMSO) to achieve the required 100x concentration of the highest concentration (stock solution). Further concentrations were prepared as 100x concentrations by serial 1:1.2 dilution1 according to the planned doses (master plate) and will be further diluted (1:25) in culture medium 3 to obtain 4x concentrations (dilution plate). The test-substance preparations were prepared within 4 hours before application by stirring. #
- Reason for the vehicle: The test substance was soluble in DMSO.
- Form of application: Visual inspection of each dilution step was performed

ANALYSES
Because the test-substance preparations were applied shortly after preparation, no analysis of the test substance in the vehicle is required.

EXPERIMENTAL PROCEDURE
- Preparation of the cells
LuSens cells from the working cell bank were thawed and cultured using culture medium 1, under standard culture conditions (37°C, ca. 5% CO2, ≥ 90% humidity) for at least passage ≥5 but not longer than 15 passages prior to testing. Before substance incubation, cells were seeded in 96-well microtiter plates (120 µL of 0.83 x 105 cells/mL cell suspensions), using culture medium 2 for incubation for 24 hours. As a rule, two independent experiments were performed. In each experiment, three replicates of each treatment were tested. In case of contradictory results further experiments were conducted. Further experiments were also conducted if no cytotoxicity was obtained in the case of a negative result.

- Test-substance preparation and application of MTT and Luciferase Assay
After cell adaption for 24 hours cell culture medium 2 was aspirated and replaced with 150 µL medium 3. The test substance was prepared as described in section 3.5. Each preparation of the dilution plate was then applied in a ratio of 1:4 (50 µL) to the cells (final DMSO concentration in the test medium = 1%). After test substance application the plates were sealed with semi-permeable plate sealers in order to prevent evaporation of the test substance. The plates were placed into the incubator under standard culture conditions for the exposure period of 48 hours. For the luciferase assay a white plate (luminescence compatible plate) was used. In addition a clear plate was treated in parallel for the determination of cell viability.

- Visual inspections
A visual inspection for test-substance precipitates was performed for each test-substance concentration directly after application. In addition, each well was inspected under a microscope after the exposure period of 48 hours in order to detect test-substance precipitates.

- Luciferase assay
After visual inspection of the cells, the supernatant was aspirated from the white assay plate and discarded. The cells were washed twice with 300 µL PBS (with Ca2+/Mg2+). Subsequently 200µL of Steady-Glo-preparation (= 100 µL Steady-Glo- Mix and 100 µL PBS (without Ca2+/Mg2+)) per well was added and cells were shaken on a plate shaker for 10 min at room temperature in darkness. After the incubation the luminescence was measured in the luminometer.

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

ACCEPTANCE CRITERIA
A tested concentration was not further evaluated when relative viability was less than 70%.
• The cell viability of untreated cells must yield at least 90%.
• The positive control EGDMA should be >2.5 fold induction and LA <1.5 and viability ≥70%.
• The average standard of the variability in the vehicle control wells for each plate should be below 20%.
If any of the acceptance criteria mentioned above was not met, repetition of the test was considered. A study was considered acceptable if the positive and negative and vehicle control data lied within the range of the historical data.

EVALUATION OF RESULTS
A test substance was concluded to exhibit a keratinocyte activating potential when the luciferase activity exceeded 1.5 fold induction with respect to the vehicle control and at concentrations that do not reduce a viability below 70% in at least two independent experiments.

HISTORICAL CONTROL DATA
Historical control values of negative and positive controls, collected over an appropriate time period, are presented below. These data demonstrate the reproducibility of results and robustness of the procedures.

Positive control results:

See "Any other information on results incl. tables".

Run / experiment:

other: 1 - 3

Parameter:

other: ARE-dependent luciferase induction

Value:

1.5

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 4 - 5

Parameter:

other: ARE-dependent luciferase induction

Value:

15

Vehicle controls validity:

not specified

Negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The test was determined to be valid. See below for full result tables and historical controls.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: See below

PRELIMINARY CYTOTOXICITY STUDY

Concentration (active ingredient) µg/ml	Concentration (test substance) µg/ml	mean viability of 3 replicates	rel. viability [%]
VC	VC	0.44	100
0.5	0.52	411	93.5
1	12	0.383	87
5	5.2	0.393	89.2
10	10.5	0.411	93.5
50	52.4	0.4	90.8
100	104.8	0.4	90.8
500*	524.1*	0.369	83.9
1000*	1048.2*	0.395	89.7
2000*	2096.4*	0.395	89.8

MEAN VALUES AND STANDARD DEVIATIONS OF LUCIFERASE INDUCTION AND REL. VIABILITY.

Concentration (test substance) µg/ml	Concentration (active ingredient) µg/ml	1st experiment
		fold induction		rel. viability [%]
		mean	SD	mean	SD
842.5*	803.7*	0.76	0.28	79.3	11.7
1011*	964.5*	0.83	0.21	81.9	4.9
1213.2*	1157.4*	1.02	0.33	59.2	24.9
1455.8*	1388.9*	0.68	0.3	70.6	25.4
1747*	166.6*	0.55	0.73	14.9	20.2
2096.4*	2000*	-0.11	0.34	9.9	11.7
VC		1	0.28	100	4.1
EGDMA (18 µg/ml)		4.12	0.6	96.4	1.8
LA (450 µg/ml)		1.23	0.15	111.7	5.6

Concentration (test substance) µg/ml	Concentration (active ingredient) µg/ml	2nd experiment
		fold induction		rel. viability [%]
		mean	SD	mean	SD
702.1*	669.8*	0.62	0.08	99.7	2.6
842.5*	803.7*	0.64	0.16	101.1	4.2
1011*	964.5*	0.9	0.05	102.5	5.5
1213.2*	1157.4*	1.16	0.31	92.6	4.9
1455.8*	1288.9*	1.39	0.14	51.5	2.1
1747*	1666.6*	1.29	0.12	45.2	26.1
VC		1	0.15	100	2.3
EGDMA (18 µg/ml)		4.5	0.19	101	2.3
LA (450 µg/ml)		0.94	0.14	104.4	1.6

Concentration (test substance) µg/ml	Concentration (active ingredient) µg/ml	3rd experiment
		fold induction		rel. viability [%]
		mean	SD	mean	SD
702.1*	669.8*	0.62	0.18	94.6	2.8
842.5*	803.7*	0.82	0.37	94.2	3.8
1011*	964.5*	0.97	0.39	92.6	0.7
1213.2*	1157.4*	1.18	0.23	85.8	5.6
1455.8*	1288.9*	1.14	0.47	36.1	47.2
1747*	1666.6*	0.97	0.87	35.4	51.3
VC		1	0.21	100	2.7
EGDMA (18 µg/ml)		5.62	0.24	101.1	1
LA (450 µg/ml)		1.1	0.25	104.7	5.3

Concentration (test substance) µg/ml	Concentration (active ingredient) µg/ml	4th experiment
		fold induction		rel. viability [%]
		mean	SD	mean	SD
842.5*	803.7*	0.58	0.1	99.5	3.5
1011*	964.5*	1.03	0.1	93	0.4
1213.2*	1157.4*	1.59	0.19	98.1	6.8
1455.8*	1388.9*	0.31	0.58	40.2	10.1
1601.4*	1527.7*	0.82	0.8	7.7	9
1747*	1666.6*	1.06	0.64	6.9	11
VC		1	0.2	100	5
EGDMA (18 µg/ml)		5.45	0.83	113.8	2.5
LA (450 µg/ml)		0.96	0.13	106.1	2.6

Concentration (test substance) µg/ml	Concentration (active ingredient) µg/ml	5th experiment
		fold induction		rel. viability [%]
		mean	SD	mean	SD
842.5*	803.7*	0.8	0.26	99.1	2.1
1011*	964.5*	0.97	0.15	95.3	3.4
1213.2*	1157.4*	1.14	0.1	96.1	1.1
1455.8*	1388.9*	0.9	0.14	96.6	3.3
1601.4*	1527.7*	1.5	0.12	85.3	6.2
1747*	1666.6*	1.56	0.03	57.7	23.4
VC		1	0.27	100	5.5
EGDMA (18 µg/ml)		5.44	0.3	110.1	3
LA (450 µg/ml)		0.99	0.37	112.1	5.9

* precipitation after 48 hours

HISTORICAL CONTROLS

Negative Control (Lactic acid 450 µg/ml)	Luminescence (RLU)	SD [%] Luminescence	Fold Ind.	rel. viability [%]
Min	14.8	4.9	0.52	96.8
Max	64.2	32.4	1.41	120.8
Mean	32.3	14.5	1	106.1
SD	12.4		0.17	4.1
n		131

Positive Control (EGDMA 18 µg/ml)	Luminescence (RLU)	SD [%] Luminescence	Fold Ind.	rel. viability [%]
Min	80.3	2.6	3.59	86.6
Max	317.3	37	14.82	123.4
Mean	126.5	9.4	6.09	107.3
SD	36.4		1.58	7.1
n		131

Vehicle Control (1% DMSO)	Luminescence (RLU)	SD [%] Luminescence	Fold Ind.	rel. viability [%]
Min	16.2	7.7	1	100
Max	58.2	20.1	1	100
Mean	32.2	14.2	1	100
SD	10.7		0	0
n		131

Basal expression	Luminescence (RLU)	SD [%] Luminescence	Fold Ind.	rel. viability [%]
Min	13.7	4.8	0.44	113
Max	50	74.3	1.56	160.3
Mean	28.1	14.9	0.81	134.5
SD	8.6		0.17	9.8
n		131

Interpretation of results:

study cannot be used for classification

Conclusions:

The keratinocyte activating potential of Darocur MBF cannot be conclusively evaluated. In the 1st to 3rd experiment no ARE-dependent luciferase activity induction above 1.5-fold at sufficient non-toxic doses was observed. However, in the 4th and 5th experiment luciferase activities just above 1.5-fold at test substance concentrations that did not reduce cell viability below 70% was noticed. In addition precipitation was observed in all concentrations.

Executive summary:

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

In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 9 concentrations of the test substance and cytotoxicity was determined thereafter by MTT assay. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve. In the main test luciferase activity was measured after 48 hour exposure. In parallel a MTT assay was performed to assess cytotoxicity of the test substance. A total of 5 valid experiments were performed. The following results were observed:

Solubility in DMSO:

At concentrations used in the main test the test substance (100 x stock solutions) was soluble in DMSO and soluble in 1% DMSO in medium 3 (final concentrations). After 48 hours precipitates were noticed in all concentrations (main test).

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

other: DRAFT OECD in vitro skin sensitisation: U-937 skin sensitisation test (U-SENS)

Version / remarks:

Draft

Deviations:

no

Principles of method if other than guideline:

The study was performed according to the methods described in the following publications, which were used as the basis for the testing guideline.
• Bauch C, Kolle SN, Fabian E, Pachel C, Ramirez T, Wiench B, Wruck CJ, van Ravenzwaay B, Landsiedel R. (2011) Intralaboratory validation of four in vitro assays for the prediction of the skin sensitizing potential of chemicals. Toxicology in Vitro 25, 1162 – 1168.
• Bauch C, Kolle SN, 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(3):489- 504.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Details on the study design:

The test substance was incubated with human pro-monocytic cell line U937 for ca. 48 hours at 37°C and membrane marker expression measured by flow cytometry. In order to determine the concentrations suitable for the main experiment a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Cells were exposed to 9 concentrations of the test substance preparation as provided by the sponsor and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve. In the main test after 48 hour exposure U937 cells were stained with FITC labeled antihuman- CD86 antibody and propidium iodide, the fluorescence intensity was analyzed using flow cytometry. A total of 3 valid experiments were performed.

A test substance is predicted to activate dendritic cells when CD86 cell surface expression exceeds the threshold of 1.2 with respect to the vehicle control and at concentrations that do not reduce viability below 70% in at least two independent experiments.

Historical control values of negative and positive controls, gathered over an appropriate time period, are presented in section 4.2. These data demonstrate the reproducibility of results and robustness of the procedures. They are used to derive suitable acceptance criteria (see section 3.9) for the test system.

TEST-SUBSTANCE PREPARATION
The test substance was weighed and topped up with the chosen vehicle (DMSO) to achieve the required 400x concentration of the highest concentration (stock solution). Further concentrations were prepared in the vehicle as 400x concentration by serial dilution according to the planned doses and were further diluted (1:200) in culture medium to obtain 2x concentrations. The test-substance preparations were prepared within 4 hours before application by stirring.

- Reason for the vehicle: The test substance is soluble in DMSO.
- Form of application: Visual inspection of each dilution step was performed
- Analysis: Because the test-substance preparations were applied shortly after preparation, no analysis of the test substance in the vehicle is required.

EXPERIMENTAL PROCEDURE
- Preparation of the cells:
U937 cells from the working cell bank were thawed and cultured in suspension using complete RPMI 1640 medium with 25 mM HEPES buffer and 2 mM L-glutamine supplemented with 10% fetal bovine serum and 100 U/mL penicillin and 100 µg/mL streptomycin under standard culture conditions (37°C, ca. 5% CO2, ≥ 90% humidity) until for 5 passages but not longer than passage 13 prior to testing. Before substance incubation, cells were seeded in 96-well microtiter plates (100 µL of 0.5x106 cells/mL cell suspensions). As a rule, two independent experiments were performed. In each experiment, duplicates of each treatment were tested. In case of contradictory results further experiments were conducted. Further experiments were also conducted if no cytotoxicity was obtained in the case of a negative result.

- Test-substance application
Treatment was performed by adding 100 µL of test-substance preparation to the cells, thus diluting the 2x concentrated test-substance preparations to their final concentration and the cells to 0.25x106 cells/mL. After test substance application the plates were sealed with semi-permeable plate sealers in order to prevent evaporation of the test substance. The plates were placed into the incubator under standard culture conditions for the exposure period of 48 hours.

- Visual inspections
A visual inspection for test-substance precipitates was performed for each test-substance concentration prior to application. In addition, each well was inspected under a microscope after the exposure period of 48 hours in order to detect test-substance precipitates.

- Cell staining and flow cytometric analysis
After visual inspection the cells were transferred into V-shaped 96-well microtiter plates and centrifuged. Supernatants were discarded. The cells were washed once with PBS with 5% FBS at 4°C. Cells were resuspended in 100 µL PBS (with 5% FBS) and labeled for 30 min at 4°C in the dark with 5 µL IgG-FITC (isotype control) or 5 µL anti-CD86-FITC antibody. Following incubation, cells were washed twice with PBS (with 5% FBS) and once with PBS and were then resuspended in 200 µL PBS. For cell viability analysis, cells were stained with PI (1.25 µg/mL final concentration in PBS) for 5 min at 4°C in darkness. Fluorescence intensity was analyzed using flow cytometry.

Species:

other: U937

Strain:

other: U937

Details on test animals and environmental conditions:

U937, The cell line was obtained from the German Resource Center for Biological Material (DSMZ, Germany, catalog no.: ACC 5).

Negative control (NC): 200 μg/mL, DL-Lactic acid (LA), CAS no.: 50-21-5

Positive control (PC): 70 μg/mL Ethylene diamine (EDA), CAS no.: 97-90-5

Vehicle control: 0.25% DMSO in culture medium

Isotype control: In order to help distinguish non-specific (“background”) staining from specific antibody staining each test-substance concentration
and control is additionally incubated with IgG1 FITC (CD86),

Concentration / amount:

Exp I and II: 125, 250, 500, 1000 and 2000 ug/ml
Exp III: 500, 600, 700, 800, 900, 1000 ug/ml

Concentration / amount:

Exp I and II: 125, 250, 500, 1000 and 2000 ug/ml
Exp III: 500, 600, 700, 800, 900, 1000 ug/ml

Positive control results:

See "Any other information on results incl. tables".

Run / experiment:

other: 1

Parameter:

other: CD86 induction

Value:

1

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: CD86 induction

Value:

0.99

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: CD86 induction

Value:

0.94

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: CD86 induction

Value:

1.18

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: CD86 induction

Value:

1.66

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: CD86 induction

Value:

0.07

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: CD86 induction

Value:

1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: CD86 induction

Value:

0.88

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: CD86 induction

Value:

0.85

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: CD86 induction

Value:

1.12

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: CD86 induction

Value:

3.58

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: CD86 induction

Value:

-0.87

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:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The test was determined to be valid. See below for full result tables and historical controls.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes
- Range of historical values if different from the ones specified in the test guideline: See below

PRELIMINARY CYTOTOXICITY ASSESSMENT

	Concentration (active ingredient) µg/ml	%PI negative cells replicate 1	%PI negative cells replicate 2	%PI negative cells replicate 3	%PI negative cells mean	rel. Viability mean
VC	VC	98.8	99	98.9	98.9	100
0.5	0.48	99.2	97.9	98.9	98.6	99.7
1	0.95	98.9	99.2	98.4	98.8	99.9
5	4.77	96.7	99.2	98.9	98.3	99.3
10	9.5	98.5	98.9	98.7	98.7	99.8
50	48	98.5	98.7	99.1	98.7	99.8
100	95	99.1	98.8	98.6	98.8	99.9
500*	477	96	95.2	98.4	96.5	97.6
1000*	954	ND	61.5	98.8	80.2	81
2000*	1908	88.1	94	97.3	93.1	94.1

* precipitation after 48 hours

ND not determined

EXPERIMENT 1

Concentration (test substance) [µg/ml]	Concentration (active ingredient) [µg/ml]	CD 86 induction	rel. viability
VC	VC	1	100
125	119	0.99	100.6
250	239	0.94	100
500	477	1.18	98.5
1000	954	1.66	65.6
2000	1908	0.07	6.4
VC		1	100
LA 200 µg/ml		1.16	100.6
EDA 70 µg/ml		2.21	95.9

EXPERIMENT 2

Concentration (test substance) [µg/ml]	Concentration (active ingredient) [µg/ml]	CD 86 induction	rel. viability
VC	VC	1	100
125	119	0.88	99.7
250	239	0.85	99.5
500	477	1.12	95.9
1000	954	3.58	48.7
2000	1908	-0.87	9
VC		1	100
LA 200 µg/ml		0.98	99.9
EDA 70 µg/ml		2.11	90.7

EXPERIMENT 3

Concentration (test substance) [µg/ml]	Concentration (active ingredient) [µg/ml]	CD 86 induction	rel. viability
VC	VC	1	100
500	477	0.88	99.5
600	572	1.13	98.9
700	668	1.25	98.2
800	763	1.21	97.3
900	859	1.38	96.8
1000	954	1.35	96.5
VC		1	100
LA 200 µg/ml		0.97	100
EDA 70 µg/ml		2.06	97.1

HISTORICAL CONTROLS

Negative Control (Lactic acid 200 µg/ml)	CD86 MFI	CD86 fold induction	%PI negative cells	rel. viability
Min	4.52	0.56	96.8	98.1
Max	21.7	1.2	99.6	101.4
Mean	13.7	1.01	98.9	100.1
SD	3.7	0.12	0.6	0.4
n		91

Positive Control (EDA 70 µg/ml)	CD86 MFI	CD86 fold induction	%PI negative cells	rel. viability
Min	16.8	1.5	61.6	63.7
Max	51.6	4.72	98.9	100.5
Mean	33.2	2.42	88.9	89.9
SD	8.3	0.54	8.5	8.5
n		91

Vehicle Control (Medium)	CD86 MFI	CD86 fold induction	%PI negative cells	rel. viability
Min	5.3		96.8
Max	19.9		99.5
Mean	12.9	1	98.8	100
SD	3.7		0.6
n		46

Vehicle Control (DMSO)	CD86 MFI	CD86 fold induction	%PI negative cells	rel. viability
Min	8.2		97
Max	19.9		99.5
Mean	14.4	1	98.8	100
SD	3.1		0.7
n		40

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

CD86 cell surface expression was shown to exceed 1.2 with respost to the vehicle control in 2 separate experiments. Relative viability was not reduced below 70% in these instances. The test material was determined to activate dendric cells under the conditions of the test.

Executive summary:

The potential of the test substance to induce the cell membrane marker CD86 expression was evaluated in the Myeloid U937 Skin Sensitization Test (MUSST). For this purpose the test substance was incubated with human pro-monocytic cell line U937 for ca. 48 hours at 37°C and membrane marker expression measured by flow cytometry.

In order to determine the concentrations suitable for the main experiment a pre-test (experimental conduct in accordance with GLP but without a GLP status) was performed. Cells were exposed to 9 concentrations of the test substance preparation as provided by the sponsor and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (= estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve.

In the main test after 48 hour exposure U937 cells were stained with FITC labeled anti-human-CD86 antibody and propidium iodide, the fluorescence intensity was analyzed using flow cytometry. A total of 3 valid experiments were performed. The following results were

observed:

At concentrations used in the main test the test substance (400 x stock solutions) was soluble in DMSO and an emulsion in culture medium (2 x stock solutions) in all concentrations. For details see section “Solubility of the test substance preparations” in the Appendix.

After 48 hours precipitates were observed at 250 µg/mL onwards.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A combination of several in vitro and in chemico methods addressing key steps of the adverse outcome pathway (AOP) for skin sensitization as defined by OECD, has been conducted to assess the skin sensitizing potential of the test item

• protein reactivity (DPRA) (OECD 442 C) (negative)

• activation of keratinocytes (LuSens) (OECD 442 D) (inconclusive)

• activation of dendritic cells (MUSST) (draft OECD guideline) (positive)

The interpretation of the results is based upon the relevant OECD or draft OECD guideline.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is considered to be classified for skin sensitization under Regulation (EC) No. 1272/2008.