p-anisic acid

Endpoint summary

Description of key information

WoE: Direct Peptide Reactivity Assay (DPRA, OEDC 442C): negative

WoE: ARE-Nrf2 Luciferase Assay (KeratinoSensTM-Assay, OECD 442D): negative

WoE: Human Cell Line Activation Test (h-CLAT, OECD 442E): positive

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:

28 Nov - 02 Dec 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Governement of the United Kindgom, Medicines & Healthcare products Regulatory Agency (MHRA)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

TEST SYSTEM
- Supplier of synthetic peptides: Anaspec
- Peptide stock solution preparation: 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 (cysteine in 100 mM phosphate buffer pH 7.5, lysine in 100 mM ammonium acetate buffer pH 10.2).
Peptide calibration standrad preparation: 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 prepared as well.

VEHICLE CONTROL
- Substance: acetonitrile

POSITIVE CONTROL
- Substance: cinnamic aldehyde
- Preparation: The positive control was prepared as 100 mM solution in acetonitrile. A 100 mM stock solution of the test item in acetonitrile/water 90/10 v/v was prepared.

CO-ELUTION CONTROL
- Co-elution controls were set up in parallel to sample preparation without respective peptide solution to verify whether a test chemical co-elutes with the cysteine or lysine peptide.

REFERENCE CONTROLS
Stability controls (Reference Control B and Reference Control C) and precision controls of both peptides were prepared at a concentration of 0.5 mM. Reference Control B samples and the precision control sample contained acetonitrile. The reference control C was prepared with propan-2-ol replacing acetonitrile.

TEST SUBSTANCE PREPARATION
The test substance was prepared as a 100 mM solution in acetonitrile.

INCUBATION CONDITIONS
- Peptide ratios: Cysteine-containing peptide: 1:10; Lysine-containing peptide: 1:50
- Temperature used during treatment / exposure: 25 °C
- Duration of treatment / exposure: minimum of 22 h

NUMBER OF REPLICATES
for each peptide triplicates were prepared for test item and control C; control B samples were prepared in 6-fold

HIGH PERFORMANCE LIQUID CHROMATOGRAPHY
- Specification of the device: Waters Alliance 2695 separation module and 2487 dual wavelength detector
- Analytical Column: Agilent Zorbax SB C18, 3.5µm, 100 x 2.1 mm
Pre-column: Phenomenex, AJO-4286
- HPLC mobile phase:
A: 0.1% trifluoracetic acid in water
B: 0.085% trifluoracetic acid in acetonitrile
- Flow: 0.35 mL/min
- Column temperature: 30 °C
- Gradient:
Time (min): 0, 10, 11, 13, 13.5, 20; MP A (%): 90, 75, 10, 10, 90, 90; MP B (%): 10, 25, 90, 90, 10, 10
- Wavelength: 220 nm
- Injection volume: 2 μL (slow draw rate)
- Retention time cysteine: approximately 11 minutes
- Retention time lysine: approximately 7 minutes

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The peptide depletion of the positive control for the cysteine peptide was 71.7%. The mean peptide depletion of the positive control for the lysine peptide was 58.8%.

Key result

Run / experiment:

other: mean of three runs

Parameter:

other: mean cysteine depletion (%)

Value:

1.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

since the cysteine depletion is ≤ 6.38 the prediction for sensitisation is considered negative

Key result

Run / experiment:

other: mean of three runs

Parameter:

other: mean lysine depletion (%)

Value:

1.01

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

since the lysine depletion is ≤ 6.38 the prediction for sensitisation is considered negative

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- The laboratory has demonstrated technical proficiency for the DPRA by successfully testing the 10 profiency chemicals outlined in the OECD 442C guideline

ACCEPTANCE CRITERIA:
The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent positve control value of the three replicates for the positive control is between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine depletion,
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine depletion,
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is in the range of 0.45 to 0.55 mM.

Both peptide runs and the test item results met the acceptance criteria of the test.

Table 2. Overall achieved depletion values

Test item	Cysteine peptide depletion (%)	Lysine peptide depletion (%)	Overall mean depletion (%)	Reactivity class	DPRA prediction
p-anisic acic	1.40	1.01	1.20	No to minimal	Negative

Table 3. Cysteine peptide depletion

Sample	Peak area (µV.sec)	Peptide concentration^1(µg/mL)	Peptide Depletion (%)	Mean Depletion (%)	SD  (%)
Positive control	236103	105.70	71.5^2	71.7	0.08
	234831	105.13	71.6^2
	235271	105.33	71.6^2
p-anisic acic	821285	369.43	1.43^3	1.40	0.43
	825232	371.21	0.953^3
	818128	368.01	1.81^3

SD Standard Deviation

¹  Samples prepared at a concentration of 376 µg/mL (0.5 mM)

²  Calculated against a mean Reference Control B area of 827610 µV.sec (n=6)

³  Calculated against a mean Reference Control C area of 833170 µV.sec (n=3)

Table 4. Lysine peptide depletion

Sample	Peak area (µV.sec)	Peptide concentration^1(µg/mL)	Peptide Depletion (%)	Mean Depletion (%)	SD  (%)
Positive control	317845	158.71	59.1^2	58.8	0.29
	322285	160.93	58.5^2
	319492	159.53	58.8^2
p-anisic acic	777172	388.56	-0.209^3	1.01	1.78
	751867	375.89	3.05^3
	774140	387.03	0.182^3

SD Standard Deviation

¹   Samples prepared at a concentration of 376 µg/mL (0.5 mM)

²   Calculated against a mean Reference Control B area of 776330 µV.sec (n=6)

³   Calculated against a mean Reference Control C area of 775550 µV.sec (n=3)

Interpretation of results:

other: no skin sensitising potential based on the key event "direct peptide binding" of skin sensitisation AOP

Conclusions:

Under the conditions of the test, it can be concluded, that the test substance is not predicted a sensitiser in the Direct Peptide Binding Assay. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation was performed.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 Aug - 07 Nov 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E (Skin Sensitization: Human Cell Line Activation Test (h-CLAT))

Version / remarks:

adopted in 2016

Deviations:

yes

Remarks:

cytotoxicity measurement and estimation of CV75 value for the dose finding assay was performed by XTT test instead of flow cytometry

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of study:

activation of dendritic cells

Details on the study design:

TEST CELL LINE
- Source: American Type Culture Collection (ATCC)
- Passage number: 9 (both XTT assays); 22 and 8 (h-CLAT runs 1 and 2, respectively)

CELL CULTURE CONDITIONS
- Type and identity of media: RPMI-1640 supplemented with 10% fetal bovine serum, 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine, 100 U/mL of penicillin and 100 µg/mL of streptomycin

CONTROLS
Negative control
- Substance: culture medium
Solvent control for positive control
- Substance: 0.2% dimethyl sulfoxide (DMSO)
Positive control
- Substance: 2 and 3 µg/mL 2,4-dinitrochlorobenzene (DNCB)

EXPOSURE CONDITIONS
- Exposure duration: 24 ± 0.5 h

TEST CONCENTRATIONS
- Justification for top dose: Cytotoxic effects were observed at 1250 µg/mL in the first XTT test and at 625 and 1250 µg/mL in the second XTT test (threshold of cytotoxicity: < 75%). The mean CV75 value of both XTT tests was calculated as 819.4 µg/mL.
- Test concentrations: 274, 329, 395, 474, 569, 683, 819 and 983 μg/mL

NUMBER OF REPLICATIONS: single measurement in two independent experiments

CYTOTOXICITY
- Method: Cytotoxicity was determined by two independent XTT tests with different cell cultures to obtain a reliable CV75; the mean of two CV75 values was used to determine the dose-range for the main experiment. At the end of the 24 h incubation period with different test item concentrations, 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 wave length 690 nm). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1). 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. The relative absorbance (= viability in [%]) as compared to the solvent control was calculated.

MEASUREMENT
- Device: FACSCalibur, Becton Dickinson GmbH
- Software: Cellquest Pro 6.0

STAINING
- Antibodies: FITC-labelled anti-CD86, FITC-labelled anti-CD54 and FITC-labelled mouse IgG1

EVALUATION CRITERIA/ PREDICTION MODEL
- For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs (OECD 442E guideline): The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%); the RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%). Otherwise, the h-CLAT prediction is considered NEGATIVE.
Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If, however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 and the other is only positive for CD54, a third run is required. If this third run is negative for both markers, the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE. An h-CLAT prediction should be considered in the framework of an IATA (OECD 442E guideline).

ACCEPTANCE CRITERIA
The test meets acceptance criteria if:
- 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 solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%)
- for both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105%
- in the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50% in at least one concentration of the two tested positive control concentrations
- for the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run
Negative results are acceptable only for test items exhibiting a cell viability of < 90% at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90% the negative result should be discarded. In such case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability > 90% (OECD 442E guideline).

Positive control results:

The positive control 2,4-dinitrochlorobenzene (DNCB) test at final concentrations of 2 and 3% led to upregulation of the cell surface markers CD54 and CD86.

Key result

Run / experiment:

other: First run

Parameter:

other: relative fluorescence intensity of CD86 (%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

since the RFI of CD86 is > 150% the prediction for sensitisation is considered positive

Key result

Run / experiment:

other: Second run

Parameter:

other: relative fluorescence intensity of CD86 (%)

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

since the RFI of CD86 is > 150% the prediction for sensitisation is considered positive

Key result

Run / experiment:

other: First run

Parameter:

other: relative fluorescence intensity of CD54 (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

since the RFI of CD54 is > 200% the prediction for sensitisation is considered positive

Key result

Run / experiment:

other: Second run

Parameter:

other: relative fluorescence intensity of CD54 (%)

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

since the RFI of CD54 is > 200% the prediction for sensitisation is considered positive

Other effects / acceptance of results:

OTHER EFFECTS:
- No test item-induced cytotoxicity was noted during the two runs of the main experiment.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- The laboratory has demonstrated technical proficiency for the h-CLAT by successfully testing the 10 profiency chemicals outlined in the OECD 442E guideline.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for DMSO control: The RFI values of the negative control of both CD86 and CD54 were 100, respectively, and thus did not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%). The cell viability was 100.0 % and thus > 90%.
- Acceptance criteria met for positive control: The RFI values of the positive control for CD86 and CD54 were > 650 and > 230, respectively, and thus exceeded the positive criteria (CD86 > 150% and CD54 > 200%) and the cell viability was > 60 % and thus > 50%.

Table 1: Results first h-CLAT run

	Concentration (µg/mL)	Antibody / ISO	MFI GeoMean(FITC)	MFI- ISO	RFI (%)	Cyto(Geo) GeoMean(7-AAD)	Mean Cyto	Viability (%)
Medium control	-	ISO	2.62			2.85	2.7	100.0
		CD54	3.26	0.64	100.0	2.83
		CD86	5.12	2.50	100.0	2.46
DMSO control	-	ISO	2.19			3.06	2.7	100.0
		CD54	3.26	1.07	100.0	2.73
		CD86	5.01	2.82	100.0	2.42
Positive control (DNCB)	2	ISO	2.92			4.82	3.7	74.9
		CD54	5.44	2.52	235.5	4.07
		CD86	17.05	14.13	501.1	2.07
	3	ISO	3.25			6.26	4.6	59.9
		CD54	6.68	3.43	320.6	4.99
		CD86	22.22	18.97	672.7	2.45
Test Item	274	ISO	2.41			3.22	2.9	93.1
		CD54	3.14	0.73	114.1	2.93
		CD86	4.57	2.16	86.4	2.59
	329	ISO	2.40			3.15	2.9	93.7
		CD54	3.20	0.80	125.0	2.96
		CD86	4.98	2.58	103.2	2.58
	395	ISO	2.43			3.13	2.9	94.7
		CD54	3.29	0.86	134.4	2.93
		CD86	4.99	2.56	102.4	2.54
	474	ISO	2.57			3.09	2.8	98.0
		CD54	3.38	0.81	126.6	2.85
		CD86	5.32	2.75	110.0	2.37
	569	ISO	2.59			3.07	2.8	96.9
		CD54	3.53	0.94	146.9	2.88
		CD86	5.57	2.98	119.2	2.45
	683	ISO	2.64			3.10	2.8	98.2
		CD54	3.60	0.96	150.0	2.81
		CD86	5.76	3.12	124.8	2.38
	819	ISO	2.88			3.36	2.9	94.5
		CD54	4.17	1.29	201.6	3.01
		CD86	7.14	4.26	170.4	2.24
	983	ISO	2.79			3.08	2.9	92.2
		CD54	3.90	1.11	173.4	2.82
		CD86	6.84	4.05	162.0	2.93

Table 1: Results second h-CLAT run

	Concentration (µg/mL)	Antibody / ISO	MFI GeoMean(FITC)	MFI- ISO	RFI (%)	Cyto(Geo) GeoMean(7-AAD)	Mean Cyto	Viability (%)
Medium control	-	ISO	2.44			3.85	3.7	100.0
		CD54	3.30	0.86	100.0	3.78
		CD86	5.05	2.61	100.0	3.61
DMSO control	-	ISO	2.57			4.15	3.7	100.0
		CD54	3.40	0.83	100.0	3.71
		CD86	5.01	2.44	100.0	3.12
Positive control (DNCB)	2	ISO	3.63			5.03	4.6	79.8
		CD54	7.18	3.55	427.7	4.70
		CD86	26.79	23.16	949.2	4.03
	3	ISO	3.92			6.12	5.7	63.9
		CD54	10.08	6.16	742.2	5.66
		CD86	19.97	16.05	657.8	5.39
Test Item	274	ISO	2.76			4.36	3.9	95.5
		CD54	3.65	0.89	103.5	3.98
		CD86	5.25	2.49	95.4	3.43
	329	ISO	2.74			4.19	3.9	96.6
		CD54	3.74	1.00	116.3	3.94
		CD86	5.60	2.86	109.6	3.50
	395	ISO	2.92			4.28	3.9	95.7
		CD54	3.86	0.94	109.3	4.05
		CD86	6.07	3.15	120.7	3.42
	474	ISO	2.91			4.21	3.8	98.6
		CD54	3.79	0.88	102.3	3.90
		CD86	5.30	2.39	91.6	3.29
	569	ISO	3.01			4.27	3.8	98.3
		CD54	3.96	0.95	110.5	3.91
		CD86	5.69	2.68	102.7	3.25
	683	ISO	3.13			4.35	3.7	101.7
		CD54	4.29	1.16	134.9	3.94
		CD86	7.14	4.01	153.6	2.76
	819	ISO	3.06			4.28	3.8	97.7
		CD54	4.12	1.06	123.3	3.98
		CD86	6.04	2.98	114.2	3.24
	983	ISO	3.51			5.28	4.6	81.8
		CD54	5.32	1.81	210.5	4.78
		CD86	9.46	5.95	228.0	3.68

Interpretation of results:

other: skin sensitising potential based on the key event “activation of dendritic cells” of skin sensitisation AOP

Conclusions:

Under the conditions of the test, it can be concluded, that the test substance is predicted a sensitiser in the human Cell Line Activation Test (h-CLAT). The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation was performed.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

26 Apr - 01 Nov 2018

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:

Trials 1-3 according to guideline adopted in 2015, trial 4 according to guideline adopted in 2018

Deviations:

no

Remarks:

The study design is based on the KeratinoSensTM assay, adapted to fully animal product-free conditions by the laboratory and validated in-house using the 10 proficiency chemicals and 11 r eference chemicals described in OECD 442D.

Principles of method if other than guideline:

Repetitions 1-3 returned one positive, one negative and one inconclusive result, which made the over all study result "inconclusive". Therefore, an additional fourth repetition was performed.

GLP compliance:

yes (incl. QA statement)

Remarks:

The Department of Health of the Government of the United Kindgom, Medicines & Healthcare products Regulatory Agency (MHRA)

Type of study:

activation of keratinocytes

Details on the study design:

Test Method:
The in vitro KeratinoSens™ assay is designed to predict and classify the skin sensitising potential of a chemical by assessment of its potential to induce the Keap1-Nrf2-ARE signalling pathway by quantifying the luciferase gene expression using luminescence detection. The induction of the Keap1 -Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitizers represents the second key event of the skin sensitisation process as described by the AOP. Therefore the KeratinoSens™ assay is considered relevant for the assessment of the skin sensitisation potential of chemicals.

Test Cell Line:
- Type: KeratinoSens™
- Source: Givaudan, Switzerland
- Passage number: 18 (for repetitions 1-3), 19 (for repetition 4)
- A licence agreement between the laboratory and the test method developer (Givaudan) was
established in 2014. KeratinoSens™ cells were supplied and propagated. The cell culture was then adapted to animal product-free conditions by the laboratory and reference chemicals described in the guideline and in the performance standards were used to confirm the reliability, accuracy, sensitiv ity and specificity values were shown to be comparable than those derived from the Validated Refere nce Method (VRM) KeratinoSens™

Cell Culture Conditions:
- Cell seeding: 10,000 cells/well
- Cell counting: trypna exclusion method using a haemocytometer

Test Concentrations:
0.977, 1.953, 3.906, 7.813, 15.625, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM

Controls:
Vehicle control
- Substance: dimethyl sulfoxide (DMSO)
- Final concentration: 1% (v/v)
Positive control
- Substance: cinnamic aldehyde
- Final concentrations: 8, 16, 32, 64 and 128 μM
EXPOSURE CONDITIONS
- Exposure duration: 48 ± 2 h
- Temperature (°C): 37
- CO2 (%): 5.0
- Relative Humidity (%): ≥ 95

Number of Replications: triplicates each in four independent experiments for each test item and each positive control concentration level, and six replicates each in four independent experiments for the negative control.

Determination of cell viability:
- Method: MTT assay
- Device: spectrophotometer
- Wavelength: 570 nm

Determination of Luminescence:
- following addition of luciferase substrate (50 μL/well) using a spectrophotometer

Positive control results:

The luciferase activity induced by the positive control at a concentration of 32 μM was between 1.6 and 3 (6.34 in experiment 1; 3.67 in experiment 2). The calculated EC1.5 was between 7 and 34 μM (24.15 μM in experiment 1; 18.10 μM in experiment 2).

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: maximum luciferase activity induction (Imax)

Value:

1.618

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Imax at 1000 µM; unit is not applicable since this parameter is defined as the maximum average fold induction of luciferase activity (Imax) value observed at any concentration of the test item

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: EC1.5 (μM)

Value:

849.105

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Unit: µM

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: maximum luciferase activity induction (Imax)

Value:

1.318

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Imax at 500 µM unit is not applicable since this parameter is defined as the maximum average fold induction of luciferase activity (Imax) value observed at any concentration of the test item

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: EC1.5 (μM)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: N/A - threshold of induction not crossed at any test item concentration.

Key result

Run / experiment:

other: Experiment 3

Parameter:

other: maximum luciferase activity induction (Imax)

Value:

1.711

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Imax at 1.953 µM unit is not applicable since this parameter is defined as the maximum average fold induction of luciferase activity (Imax) value observed at any concentration of the test item

Key result

Run / experiment:

other: Experiment 3

Parameter:

other: EC1.5 (μM)

Value:

1.514

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Unit: µM

Key result

Run / experiment:

other: Experiment 4

Parameter:

other: maximum luciferase activity induction (Imax)

Value:

0.819

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: Imax at 0.977 µM unit is not applicable since this parameter is defined as the maximum average fold induction of luciferase activity (Imax) value observed at any concentration of the test item

Key result

Run / experiment:

other: Experiment 4

Parameter:

other: EC 1.5 (µM)

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: N/A - threshold of induction not crossed at any test item concentration.

Other effects / acceptance of results:

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The laboratory has demonstrated technical proficiency using the 10 proficiency chemicals and 11 reference chemicals described in OECD 442D. Performance data for the adpatation of the test to animal-free culture coditions are appended to the report.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (mean value in experiments 1-3: 15.736% and 9.642% in experiment 4). Therefore the negative control confirmed the validity of the study.
- Acceptance criteria met for positive control: The luciferase activity induced by the positive control at a concentration of 32 μM was between 1.6 and 3.0 (mean value in experiments 1-3: 2.938 and 2.509 in experiment 4). The calculated EC1.5 was between 6 and 39 μM (mean value in experiments 1-3: 13.569 and 7.105 in experiment 4). Therefore the positive control confirmed the validity of the study.

For further details please refer to attached summary tables

Interpretation of results:

other: no skin sensitising potential based on the key event "activation of keratinocytes"

Conclusions:

Under the conditions of the test, it can be concluded, that the test item is not a sensitiser in KeratinoSensTM Assay. The result does not allow for the non-classification or classification as skin sensitiser of the test item and therefore further evaluation and/or data generation was performed.

Executive summary:

The human skin sensitisation potential of the test item was assessed using the validated in vitro method: the KeratinoSens test to determine keratinocyte activation (according to OECD 442D).

In this study, because replicates 1 and 2 provided differing results (Repl. 1 - positive, Repl. 2 - negative), and Repl. 3 was inconclusive (no clear dose response), a 4. Repl. was performed. This 4. Repl. was negative and therefore, based on 2 repetitions in agreement, the test item was classified as negative using the KeratinoSens prediction model.

Reference 4

Endpoint:

skin sensitisation, other

Remarks:

: QSAR profiling

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification

Justification for type of information:

1. SOFTWARE
Quantitative Structure-Activity Relationship model that was developed by the Laboratory of Mathematical Chemistry, Burgas, Bulgaria (http://toolbox.oasis-lmc.org).

2. MODEL
OECD QSAR Toolbox v4.1

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
See “Test material information”

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See description of profilers in attached document.

5. APPLICABILITY DOMAIN
For details regarding the models used, and their compliance with the “OECD principles for the validation, for regulatory purposes, of (Quantitative) Structure-Activity Relationship models”, please refer to the supporting documentation in the “Attached justification” section.

6. ADEQUACY OF THE RESULT
The results may be used in a weight-of-evidence approach together with other information to reach a conclusion regarding the skin sensitising potential of the test substance.

Qualifier:

according to guideline

Guideline:

other: REACH Guidance on QSARs

Deviations:

not applicable

Principles of method if other than guideline:

- Principle of test: The OECD QSAR Toolbox v4.1 is a Quantitative Structure-Activity Relationship model that was developed by the Laboratory of Mathematical Chemistry, Bulgaria (http://toolbox.oasis-lmc.org).

GLP compliance:

no

Key result

Parameter:

other: QSAR profiling

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

other: The QSAR profiling results (OECD QSAR Toolbox v4.1, ECHA database) support the absence of skin sensitising potential. The results may only be used for classification purposes together with other data in a weight-of-evidence approach.

Conclusions:

In the (Q)SAR evaluation for skin sensitisation of p-anisic acid by means of the relevant profilers present in the OECD QSAR Toolbox v4.1, only one structural alert was identified by the by profilers related to Direct Peptide Reactivity Assay (DPRA), leading to place the substance in the “Non-Conjugated carboxylic acids and esters” class. No detailed information are available for this substances, which can be however considered as non-reactive (DPRA < 9%). The results therefore support the absence of skin sensitisation effects for the evaluated substance.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The skin sensitization potential of p-anisic acid (CAS 100-09-4) has been evaluated in an WoE approach in the acknowledged, validated test battery of three in vitro studies the so-called sensitization Adverse Outcome Pathway, AOP, as defined and described in the OECD guideline, consisting of a Direct Peptide Reactivity Assay (OECD 442C), an ARE-Nrf2 luciferase test (KeratinoSensTM, OECD 442D) and a Human Cell Line Activation Test (OECD 442E). In addition a human RIPT is available and a QSAR profiling based on the OECD QSAR Toolbox v 4.1 has been performed.

In vitro skin sensitisation

A reliable in vitro Human Cell Line Activation Test (h-CLAT) was performed under GLP and according the OECD 442E (Roth, 2018). To assess the dendritic cell activation potential (the third key event of the so-called sensitization Adverse Outcome Pathway, AOP, as defined and described in the OECD guideline) p-anisic acid was stably suspended in culture medium and administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of p-anisic acid was previously determined by two XTT tests. In these tests cytotoxic effects were observed following incubation with the test item at the highest applied concentration (1250 μg/mL) in the first XTT test and at 625 μg/mL and 1250 μg/mL in the second XTT test (threshold of cytotoxicity: < 75%). The mean CV75 value of both XTT tests was calculated as 819.4 μg/mL. Accordingly, the following concentrations were tested in the main experiment (h-CLAT): 274, 329, 395, 474, 569, 683, 819 and 983 μg/mL. The test item was tested in 2 independent runs. The RFI of CD86 and CD54 was greater than 150% and 200%, respectively, in at least one concentration of both independent runs. Therefore the h-CLAT prediction is considered positive for the tested test item in this h-CLAT. In the solvent control (DMSO), 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%.

Thus under the conditions of the test, it can be concluded, that the test substance is predicted to possess a skin sensitizing potential based on the key event “activation of dendritic cells” of the skin sensitisation in the human Cell Line Activation Test (h-CLAT).

In a second study, the protein reactivity of the test substance was investigated in a Direct Peptide Reactivity Assay (DPRA) according to OECD Guideline 442C and in compliance with GLP (Fleet, 2018). The overall mean percent cysteine and lysine depletion was 1.20% and therefore the test substance is allocated to the “no to minimal reactivity” class (mean % depletion 0 - ≤ 6.38%). Accordingly, the DPRA prediction is negative, and p-anisic acidis predicted as a potential non-skin sensitizer in this test.

The activation of keratinocytes of the test item was investigated in an ARE-Nrf2 Luciferase Test in human transgenic keratinocyte cell line according to OECD 442D and in compliance with GLP (Bailey, 2019). Cells were incubated with test item concentrations of 0.977 - 2000 μg/mL in DMSO for 48 h at 37°C. After exposure, cells were lysed and antioxidant response element (ARE) dependent luciferase activity was assessed by luminescence measurement. Additionally, a MTT assay was performed to assess cytotoxicity of the test substance. In this study, because experiments 1 and 2 provided differing results (1 positive and 1 negative), and experiment 3 was inconclusive (no clear dose response), a fourthexperiment was performed. The acceptance criteria were met in all experiments. The test item induced statistically significant luciferase induction > 1.5 in experiment 1, which was dose-responsive. For experiments 2 and 4, there was no induction > 1.5. Experiment 3 had statistically significant luciferase induction > 1.5, but there was no clear dose response observed. The EC1.5 values were calculated as 849.105 μM and 1.514 μM for experiments 1 and 3 respectively. Overall, based on 2 repetitions in agreement, under the conditions of this test the test item is predicted as a potential nonskin sensitizer in this test.

Supporting human data

A reliable (Klimisch score 2) human Repeated Insult Patch Test (RIPT) was conducted (Davis, 1994) with p-anisic acid. In this test the skin sensitization potential of p-anisic acid (3% in glycerine) was tested on 56 subjects (19 males and 37 females). Testing was done in accordance with a modified Draize assay employing an 8 mm Finn Chamber (an occlusive patch). For the induction phase the products were applied to the scapular back Monday, Wednesday, and Friday of each week for 3 consecutive weeks, with a final patch on Monday of the 4th week, for a total of ten applications. Scoring of the skin sites was made at the end of each 48 hour patch period (72 hours on Weekends). The final reading was made on Wednesday of the fourth week. Following a 12-day rest period each subject received a single 48-hour occlusive challenge patch of p-anisic acid on a naive skin site on the scapular back. Scoring of the challenge sites was made after removal of the patch and again two days later. Parameters analysed / observed were erythema, edema, vesicles, bullae, toxicity/adverse effects. No productrelated systemic adverse reactions were noted during the study. Slight dermal irritation (erythema) was noted for 3 subjects on 11 occasions during the induction phase. No subject showed any indication of skin sensitization during the challenge phase. Based on the results of this study the test substance is not considered a skin sensitizer in humans.

QSAR

A QSAR profiling based on the OECD QSAR Toolbox v 4.1 was performed with p-anisic acid (QSAR, 2018). The skin sensitisation profiling was conducted with all relevant general mechanistic and endpoint specific profilers. Only one structural alert was identified by the profilers related to Direct Peptide Reactivity Assay (DPRA), leading to place the substance in the “Non-Conjugated carboxylic acids and esters” class. No detailed information are available for these substances, which can be however considered as non-reactive (DPRA < 9%). Based on these results, p-anisic acid is not expected to exhibit skin sensitising properties.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The results available from the three in vitro tests of the sensitisation AOP test battery reveal one positive (h-CLAT) and one two negative results (DPRA and KeratinoSensTM-Assay). Additionally, human RIPT data and an OECD toolbox QSAR were considered for support, and support the assumption that panisic acid is not expected a skin sensitiser. Based on these data and an Weight-of-Evidence Approach, the test item does not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.