2,4-dinitroanisole

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27 November - 21 December, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

Skin sensitisers have been reported to induce genes that are regulated by the antioxidant response element (ARE). The KeratinoSensTM test is a method for which validation studies have been completed followed by an independent peer review conducted by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). The KeratinoSensTM test method was considered scientifically valid to be used as part of an IATA (Integrated Approach to Testing and Assessment), to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling. The method cannot be used on its own, neither to sub-categorise skin sensitisers into subcategories IA and 1B as defined by the UN GHS, for authorities implementing these two optional subcategories, nor to predict potency for safety assessment decisions. However, depending on the regulatory framework a positive result may be used on its own to classify a chemical into UN GHS category 1.

Test material

Test material information

Constituent 1

Reference substance name:

2,4-dinitroanisole

EC Number:

204-310-9

EC Name:

2,4-dinitroanisole

Cas Number:

119-27-7

Molecular formula:

C7H6N2O5

IUPAC Name:

2,4-dinitroanisole

Specific details on test material used for the study:

Supplier Sponsor
Test Item name DNAN (2,4-Dinitroanisole)
Supplier Code CAS No. 119-27-7
Supplier batch/lot number 55503625 Lot 02/13
CAS number 119-27-7
Molecular Weight 198
Purity
Expiry Date 28 April 2019
Physical state Solid
Storage Conditions Room Temperature, Dark

In vitro test system

Details on the study design:

Description of the test system:
The KeratinoSensTM cell line (test system) is an immortalized adherent cell line derived from HaCaT human keratinocytes, stably transfected with a selectable plasmid containing the luciferase gene under the transcriptional control of the Anti-oxidant Response Element (ARE) from a gene that is known to be upregulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances.

Method of administration of test item:
Per plate, single application of 12 concentrations of the test item was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1%. The top concentration was previously determined by solubility testing.

Method of administration of reference items:
Per plate, single application of 5 concentrations of the positive control was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1% and single application of culture medium with 1% DMSO was applied as the negative control (6 wells per plate). One well per plate is left empty (no cells).

Exposure times of test items and reference items:
Cells were incubated with the test or reference item for 48 ± 2h before endpoints measurements.

Number of repetitions:
Three repetitions (runs) were performed. Each repetition consisted of 3 x 96-well plates for luminescence and 2 x 96-well plates for MTT.

Overview
Preliminary testing: Determination of the top concentration by solubility testing
Day 1: Seeding cells (3 x 96-well plates for Luminescence; 2 x 96-well plate for MTT); 10,000 cells per well, 14.
Day 2: 24h after seeding the test and control items were applied and plates were incubated at 370C, 5% C02, 2 95% relative humidity for 48 ± 2h.
Day 4: Evaluation of luciferase activity by luminescence (3 plates) and cell viability by MIT testing (2 plates)

Data Analysis
XCellR8 Forms F0056 A (version 01) and B (version 04): Data Analysis for KeratinoSensTM were used to analyse data. These forms are Microsoft Excel workbooks containing formulae to process the raw data as described in SOP L0057. The spreadsheets have been validated in-house (July 2017).
The following parameters were calculated in the KeratinoSensTM test method:
the maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the test item and positive control;
the EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5- fold threshold (i.e. 50% enhanced luciferase activity);
For each concentration showing > 1.5-fold luciferase activity induction, statistical significance is calculated (e.g. by a two-tailed Student's t-test), comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). The lowest concentration with > 1.5-fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.
The percentage of viability as compared to the Negative control

Results and discussion

Positive control results:

Acceptance Criteria (Mean of three Replicates)

Criteria Result Pass or Fail
I-Positive Control (PC) (Cinnamic aldehyde) induction 21.5-fold in at least one
concentration Yes Pass
2-Average induction of PC at 32gM is [1.6-3.0] Yes (2.11) Pass
3-EC1.5value is [6-39wM] Yes (11.50) Pass
4-CV% of blank values < 20% Yes (13.06%) Pass

In vitro / in chemico

Results

Key result

Run / experiment:

other: Mean value

Parameter:

other: EC1.5

Value:

1.5

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

Table 1 Sensitisation potential of the test item - Repetition 1

Rep 1	Test Item Concentration (µM)
	0.977	1.953	3.906	7.813	15.625	31.250	62.5	125	250	500	1000	2000
Mean of fold induction	0.946	0.969	1.111	1.105	1.032	1.089	1.119	1.173	1.134	1.364	1.480	1.412
SD	0.090	0.146	0.128	0.055	0.042	0.062	0.082	0.124	0.079	0.081	0.257	0.235
Viability %	112.22	97.99	103.41	95.06	90.00	86.92	102.53	96.54	88.83	100.01	112.47	101.28
Imax	1.480 at 1000µM
EC1.5	No EC1.5 as no concentration did induce the luciferase activity above the 1.5 threshold
IC50 IC30	>2000µM as viability at 2000µM was 101.28%
	>2000µM as viability at 2000µM was 101.28%

Table 2 Sensitisation potential of the test item - Repetition 2

Rep 2		Test item concentration (µM)
		0.977	1.953	3.906	7.813	15.625	31.250	62.5	125	250	500	1000	2000
Mean of fold induction		1.027	1.150	1.118	1.110	0.969	1.191	1.245	1.237	1.358	1.861	2.422	1.067
SD		0.045	0.135	0.084	0.084	0.051	0.087	0.090	0.067	o. 165	0.078	0.247	0.134
Viability %		107.44	99.62	105.76	101.36	103.09	110.01	111.12	99.59	108.54	118.30	134.28	138.56
u' Imax		2.422 at 1000µM
EC1.5		320.67
IC50		>2000µM as viability at 2000µM was 138.56%
IC30		>2000µM as viability at 2000µM was 138.56%

Table 3 Sensitisation potential of the test item - Repetition 3

Rep 3		Test item concentration (µM)
		0.977	1.953	3.906	7.813	15.625	31.250	62.5	125	250	500	1000	2000
Mean of fold induction			0.795	0.839	0.933	0.983	0.895	0.949	0.977	1.130	0.954	1.447	0.958
SD		0.032	0.073	0.004	0.105	0.127	0.062	0.049	0.085	0.145	0.008	0.153	0.007
Viability %		96.81	99.79	102.82	107.08	98.47	101.54	106.68	96.28	101.57	112.28	129.11	165.15
Imax		1.447 at IOOOµM
EC1.5		No EC1.5 as no concentration did induce the luciferase activity above the 1.5 threshold
IC50		>2000µM as viability at 2000µM was 165.15%
IC30		>2000µM as viability at 2000µM was 165.15%

Table 4 Determination criteria for the skin sensitisation potential of DNAN

	REP1	REP2	REP3
Does at least one concentation of Test Item induce luciferase activity 21.5-fold:	No	Yes	No
Does the first concentration inducing luciferase activity above 1.5, have a viability above 70%:	N/A	Yes	N/A
Does EC1.5 value occur at a concentration <IOOOµM (or <200µg/mI)	N/A	Yes	N/A
Does the test item induce the luciferase in a dosedependent manner	N/A	Yes	N/A
Classification	Non-Sensitiser	Sensitiser	Non-Sensitiser

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this study, DNAN was classified as a non-sensitiser to human skin.

Executive summary:

The human skin sensitisation potential of DNAN was assessed using the validated in vitro method, the KeratinoSens^TMassay, adapted to fully animal-free by XCellR8, and validated in-house to determine keratinocyte activation.

After 48h exposure of cells with 12 concentrations of DNAN, Luciferase measurements and MIT viability testing were performed.

In this study, DNAN was classified as a non-sensitiser.