Sodium anisate

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

p-anisic acid

EC Number:

202-818-5

EC Name:

p-anisic acid

Cas Number:

100-09-4

Molecular formula:

C8H8O3

IUPAC Name:

4-methoxybenzoic acid

In vitro test system

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).

Results and discussion

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.

In vitro / in chemico

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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%.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.