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Description of key information

This assessment addressed the requirements of conducting skin sensitization assessment for 2,4-dinitroanisole according to Annex VII section 8.3 of the REACH Regulation (Regulation (EC) No 1907/2006 and Commission Regulation (EU) 2016/1688).

The assessment of physicochemical properties of 2,4-dinitroanisole indicates that substance may readily undergo dermal penetration, yet the study performed by Lent found the absorption rate to be 0.0017%. None of specified conditions in column 2 of Annex VII section 8.3 of the REACH Regulation were fulfilled to exclude the substance from the skin sensitization testing.

The skin sensitizing potential of 2,4-dinitroanisole was predicted with BIOVIA Discovery Studio (TOPKAT) 4.5, VEGA NIC 1.1.4 (CAESAR), OECD QSAR Toolbox 4.1, Toxtree 2.6.13 and DEREK Nexus 5.0.2. In addition, results from the Danish QSAR Database were also considered (Pudenz, Envigo Ref. PN66VK). The assessment in silico with two negative predictions (TOPKAT and OECD QSAR Toolbox) without alerts in Toxtree and absence of pro-haptens against three low to moderate reliable positive predictions (CAESAR of VEGA, DEREK and Danish QSAR database) suggested rather no skin sensitizing property for 2,4-dinitroanisole.

A skin sensitization study in guinea pigs using a modified Buehler design was performed with the substance for the US Air Force in 2001. Unfortunately it has not been possible to retrieve the report untitled “A Dermal Sensitization Study in Guinea Pigs with Composition B, CBR-12 and 2,4- Dinitroanisole - Modified Buehler Design (Bonnette, 2001). The substance was found not to be a skin sensitizer as cited in the Occupational Alliance for Risk Science - Workplace Environmental Exposure Levels for 2,4-dinitroanisole (2014): “Dermal sensitization was not observed when DNAN was administered to guinea pigs in a sensitization assay (OPPTS 870.2600)”. As the available information on 2,4-dinitoanisole seemed to be not consistent and of low reliability, as the next step, the in silico assessment was performed.

All three in chemico/in vitro tests OECD 442C, OECD 442D and OECD 442E were conducted on substance 2,4-dinitroanisole and showed negative results for covalent protein binding to skin proteins (key event 1), keratinocyte activation (key event 2) and dendritic cell activation (key event 3), respectively.

As weight of evidence, it may be concluded, that the substance does not require classification as a skin sensitizer, with no requirement to conduct testing in vivo.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
05 - 12 January, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
3 Keys Event on the Adverse Outcome Pathway (AOP) for Skin Sensitisation have been provided. With the QSAR statement it is not required to perdormed study on animals.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 55503625 Lot 02/13
- Expiration date of the lot/batch:
- Purity test date: Purity/correction factor 100%, use as supplied
- Molecular weight 198 g/mol
- Appearance : Pale yellow needles
- Expiry/retest date : 28 April 2019


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature (15°C to 25°C) in the dark
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:


The solubility of 2,4-Dinitroanisole was assessed at a concentration of 100 mM in a selection of solvents and solvent mixtures.
The solubility of 2,4-Dinitroanisole in acetonitrile/water 75/25 v/v at a nominal concentration of 100 mM was achieved.

Peptide :

Synthetic peptide containing Cysteine
Alternative name: Ac-RFAACAA-OH
Batch number: 1658140
Stated purity: 97.8% (by HPLC)
Molecular Weight: 751 g/mol
Supplier: AnaSpec
Storage conditions: Frozen, -10°C to -30°C

Synthetic peptide containing Lysine
Alternative name: Ac-RFAAKAA-OH
Batch number: 1658141
Stated purity: 96.8% (by HPLC).
Molecular Weight: 776 g/mol
Supplier: AnaSpec
Storage conditions: Frozen, -10°C to -30°C

Cinnamic Aldehyde (Positive control)
Batch number: MKCB9907
Stated purity: 99.1%
Molecular Weight: 132.16 g/mol
Supplier: SAFC
Storage conditions: Room temperature (15°C to 25°C)
Expiry/retest date: November 2021

Details on study design:
Preparation of Peptide Stock Solutions
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).

Preparation of Peptide Calibration Standards
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.

Preparation of Stability Controls and Precision Controls
Stability controls (Reference Control B) and precision controls (Reference control A) of both peptides were prepared at a concentration of 0.5 mM in acetonitrile. Stability controls (Reference Control C) were prepared at a concentration of 0.5 mM in acetonitrile/water 75/25 v/v.

Preparation of Positive Control Solution and Test Item Stock Solution
The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution in acetonitrile/water 75/25 v/v of 2,4-Dinitroanisole was prepared.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Acetonitrile/water 75/25 v/v solutions of 2,4-Dinitroanisole and solutions of the positive control were diluted with the Cysteine peptide to prepare solutions containing 0.5 mM Cysteine and 5 mM of either 2,4-Dinitroanisole or the positive control. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls
Acetonitrile/water 75/25 v/v solutions of 2,4-Dinitroanisole and solutions of the positive control were diluted with the Lysine peptide to prepare solutions containing 0.5 mM Lysine and 25 mM of either 2,4-Dinitroanisole or the positive control. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

Incubation
The appearance of the 2,4-Dinitroanisole and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to injection of the samples as part of analytical run. Before initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis
The concentration of both the Cysteine and Lysine peptides in the presence of 2,4-Dinitroanisole and the associated positive controls was quantified by HPLC using UV detection as detailed in the chromatographic section.

Calculations
The peak area response for the peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the equation described in the paragraph 2.4.12

Positive control results:
Cf any other information on results
Key result
Parameter:
other: Peptide depletion
Run / experiment:
Mean value percentage
Value:
ca. 1.9
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
All analytical acceptance criteria for each peptide run were met:

Peptide Standard Linearity Positive control depletion (%) Reference controls Test item
Acceptance Cysteine r2>0.99 60.8-100 (SD <14.9%) 0.45-0.55 mM (CV <15%) SD <14.9%
Criteria Lysine r2>0.99 40.2-69.0 (SD <11.6%) 0.45-0.55 mM (CV <15%) SD <11.6%

Solubility Assessment

The solubility of 2,4-Dinitroanisole in acetonitrile/water 75/25 v/v at a nominal concentration of 100 mM was achieved.

Reactivity Assessment

The DPRA prediction and the reactivity of 2,4-Dinitroanisole based on the overall mean and the individual depletion values in the Cysteine peptide and the Lysine peptide is presented in Table 1 of the study report. Peptide calibration graphs and typical sample chromatograms are presented in Figure 1, Figure 2 and Figure 3 of the study report.

All analytical acceptance criteria for each peptide run were met:

  Peptide Standard Linearity Positive control depletion (%) Reference controls Test item
Acceptance criteria Cysteine r2>0.99 60.8-100 (SD <14.9%) 0.45-0.55 mM (CV <15%) SD <14.9%
Lysine r2>0.99 40.2-69.0 (SD <11.6%) 0.45-0.55 mM (CV <15%) SD <11.6%
Achieved results Cysteine r2>0.99 71.2 (SD, 0.17%, n=3) B: 0.498 mM (CV 0.73%, n=6) C: 0.497 mM (CV 0.33%, n=3) SD 0.67% (n=3)
Lysine r2>0.99 56.6 (SD, 1.26%, n=3) B: 0.496 mM (CV 2.15%, n=6) C: 0.496 mM (CV 0.21%, n=3) SD 1.33% (n=3)

The depletion of peptide in the presence of 2,4-Dinitroanisole :

  Mean peak area of reference control (μV.sec) Mean peak area of peptide with test item (μV.sec) Mean peptide depletion by 2,4-Dinitroanisole (%)
Cysteine Control B: 845770 (n=6) 835440 (n=3) 0.994
Control C: 843830 (n=3)
Lysine Control B: 788650 (n=6) 767260 (n=3) 2.81
Control C: 789400 (n=3)

There were no co-elution peaks in either of the Lysine or Cysteine assays.

Interpretation of results:
GHS criteria not met
Conclusions:
Solutions of 2,4-Dinitroanisole were successfully analyzed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides.
Applying the following depletion model (below), the overall result of 1.90% depletion places 2,4-Dinitroanisole in the reactivity class of “no to minima”l and therefore it is predicted by DPRA to be a non-skin sensitizer.

Mean of Cysteine and Lysine% depletion Reactivity Class DPRA Prediction
0%≤ mean% depletion ≤6.38% No or minimal reactivity Negative
6.38%< mean% depletion ≤22.62% Low reactivity Positive
22.62%< mean% depletion ≤42.47% Moderate reactivity Positive
42.47%< mean% depletion ≤100% High reactivity Positive



Executive summary:

This in chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA) was performed according to the OECD guideline 442C, in GLP compliance, to assess the reactivity and sensitizing potential of 2,4-Dinitroanisole.

Solutions of 2,4-Dinitroanisole were successfully analyzed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides.

The acceptance criteria according to the guideline have been performed and validated the assay.

The overall result of 1.90% depletion places 2,4-Dinitroanisole in the reactivity class of “no to minima”l and therefore it is predicted by DPRA to be a non-skin sensitizer.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
13 December, 2017 - 15 March, 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 442E In Vitro Skin Sensitisation : human Cell Line Activation Test (h-CLAT)
Version / remarks:
Deviation No.1
The mean CV75 value of both XTT tests was calculated as 54.65 instead of 54.35 μg/mL, due to an error of the CV75 calculation for the second XTT test. In addition, the calculated mean CV75 x 1.2 value was slightly higher than the correct value (65.6 μg/mL instead of 65.2μg/mL). Therefore, all test item concentrations used in the h-CLAT runs were slightly higher.
Deviation No.2
The relative fluorescence intensity (RFI) was calculated instead of not calculated, but excluded from the evaluation, if the cell viability was less than 50% (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).
Deviations:
yes
Remarks:
These deviations to the study plan, however, did not affect the validity of the study.
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
3 Keys Event on the Adverse Outcome Pathway (AOP) for Skin Sensitisation have been provided. With the QSAR statement it is not required to perdormed study on animals.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 55503625/Lot 02/13
- Expiration date of the lot/batch: 28/04/2019
- Purity test date: 99%
- Partition coefficient (n-octanol/water): log Pow: 0.5 – 1.9, predicted with several methods
- Water solubility: 155 mg/L @ 15° C
- Appearance: Yellow solid

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions:
- Solubility and stability of the test substance in the solvent/vehicle:
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment (immediately prior to start) 2,4-Dinitroanisole was prepared in culture medium, which formed a stable suspension.
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid:

TEST SYSTEM AND SUPPORTING INFORMATION

- Reasons for the Choice of THP-1 Cells
THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202. THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitisers.

- THP-1 Cell Cultures
Stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank of Envigo CRS GmbH (aliquots of cells in freezing medium at 1 × 106 to 2 × 106 cells/mL) allowing the repeated use of the same cell culture batch in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. Thawed stock cultures are propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured twice weekly. The cell density should not exceed 1 × 106 cells/mL. The THP-1 cell suspension is incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere. Cells can be used up to two months after thawing (passage number should not exceed 30).
The passage numbers of the used THP-1 cells were 26 in both XTT assays and 16, 18 and 12 in the h-CLAT for runs 1, 2 and 3, respectively.

- Culture Medium
RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) is used to culture the cells during the assay. Medium with supplements has to be stored at 2 – 8 °C and used within one month. The culture medium has to be warmed to room temperature just before use.

- Preparation and Seeding of THP-1 Cells
On the day of the cytotoxicity experiment (XTT) directly before the application of the test item, solvent and medium control, a volume of 100 μL with a cell density of 0.9 - 1 × 106 THP-1 cells/mL was seeded in each well of a 96-well flat bottom plate.
For the main experiment (h-CLAT) 0.9 - 1 × 106 cells/well in a volume of 500 μL were seeded in a 24-well plate before the treatment.

Positive control:
Name: DNCB (2,4-dinitrochlorobenzene, CAS No.: 97-00-7)
Final concentration: 2 and 3 μg/mL
Purity ≥ 99%
Solvent: DMSO (Dimethyl sulfoxide, CAS No. 67-68-5) in culture medium, final concentration 0.2%, Purity ≥ 99%)


Details on study design:
Experimental Design and Procedures of h-CLAT :
The test item was tested in three independent runs.

- Treatment of the Cells
Due to an error of the CV75 calculation for the second XTT test, the mean CV75 and all test item concentrations used in the h-CLAT runs were slightly higher calculated. For the test item exposure the highest dose solution calculated from the XTT assay was prepared corresponding to 1.2 × mean CV75. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment.
Each volume (500 μL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).

- Staining of the Cells
The triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 μL of blocking solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 μL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).
All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures.
The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).
5.6.3 Sample Preparation for Measurement
After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-AAD solution were added.

Flow Cytometry Acquisition
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.

Acquisition
Dead cells were determined by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was calculated but excluded from the evaluation, if the cell viability was less than 50% (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).
Positive control results:
Cf results
Key result
Parameter:
other: CV75
Run / experiment:
CV75 in ug/mL
Value:
ca. 54.65
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Valid results
Key result
Parameter:
other: RFI CD86
Run / experiment:
RFI in %
Value:
>= 150
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Valid results
Key result
Parameter:
other: RFI CD54
Run / experiment:
RFI in %
Value:
>= 200
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Valid results
Other effects / acceptance of results:
The following acceptance criteria should be met when using the h-CLAT method:
• 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).

Data Analysis and Interpretation :
The RFI is used as an indicator of CD86 and CD54 expression and is calculated based on the formula described in the paragraph 5.6.5.1 of the study report.
The cell viability of the h-CLAT experiment is determined based on the formula described in the paragraph 5.6.5.1 of the study report.

Prediction model (Cf paragraph 5.6.7.1 Flow chart of the Prediction model used in the h-CLAT test method of the study report)
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 (see chapter 5.6.7.1).
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 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), 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).

Test chemicals with a Log Pow > 3.5 tend to produce false negative results. Therefore negative results with test chemicals with a Log Pow > 3.5 should not be considered (according to the OECD guideline). However, positive results obtained with test chemicals with a Log Pow > 3.5 could still be used to support the identification of the test chemical as a skin sensitiser (OECD 442E guideline).

Results of the Dose Finding Assay (XTT Test) :

Cytotoxic effects were observed following incubation with the test item starting with the concentration of 78.13 μg/mL up to the highest tested concentration (1250 μg/mL) in the first XTT test and starting with the concentration of 39.06 μg/mL up to the highest tested concentration (1250 μg/mL) in the second XTT test (threshold of cytotoxicity: < 75%).

The CV75 value of the first XTT test: 77.6 μg/mL

Due to an error of the CV75 calculation of the second XTT test, 31.7 instead of 31.1 μg/mL were used for the calculation of the mean CV75.

Therefore, the mean CV75 value of both XTT tests was calculated as 54.65 instead of 54.35 μg/mL.

In addition, the calculated mean CV75 x 1.2 value was slightly higher than the correct value (65.6 μg/mL instead of 65.2 μg/mL). Therefore, all test item concentrations used in the h-CLAT runs were slightly higher.

The following concentrations of the test item were tested in the main experiments (h-CLAT):

18.3, 22.0, 26.4, 31.6, 38.0, 45.6, 54.7 and 65.6 μg/mL

Results of the h-CLAT Test:

The test item with a predicted log Pow of 0.5 – 1.9 was tested in 3 independent runs.

Due to a cell viability below 50% at the highest concentration of the third run, this concentration is excluded from the evaluation.

The RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any tested and evaluated test item concentration in 2 out of 3 independent runs. Therefore the h-CLAT prediction is considered NEGATIVE for the tested test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%. Except the CD54 RFI value of the positive control (2.0 μg/mL DNCB) in the third h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the CD54 RFI value of the positive control (3.0 μg/mL DNCB) in the first h-CLAT run exceeded the positive criteria. For details see Annex 1 and 2.

Interpretation of results:
GHS criteria not met
Conclusions:
This human cell line activation test (h-CLAT) can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), ARE-Nrf2 luciferase test method) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.
Executive summary:

This in vitro Human Cell Line Activation Test (h-CLAT) was performed (OECD 442E guideline, GLP compliance) to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of 2,4-Dinitroanisole which formed a stable suspension in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of 2,4-Dinitroanisole was previously determined by two XTT tests.

Cytotoxic effects were observed following incubation with the test item starting with the concentration of 78.13 μg/mL up to the highest tested concentration (1250 μg/mL) in the first XTT test and starting with the concentration of 39.06 μg/mL up to the highest tested concentration (1250 μg/mL) in the second XTT test (threshold of cytotoxicity: < 75%). The mean CV75 value of both XTT tests was calculated as 54.65 instead of 54.35 μg/mL, due to an error of the CV75 calculation for the second XTT test. In addition, the calculated mean CV75 x 1.2 value was slightly higher than the correct value (65.6 μg/mL instead of 65.2 μg/mL). Therefore, all test item concentrations used in the h-CLAT runs were slightly higher.

The following concentrations of the test item were tested in the main experiments (h-CLAT):

18.3, 22.0, 26.4, 31.6, 38.0, 45.6, 54.7 and 65.6 μg/mL

The test item with a predicted log Pow of 0.5 – 1.9 was tested in 3 independent runs. Due to a cell viability below 50% at the highest concentration of the third run, this concentration is excluded from the evaluation. The RFI of CD86 and CD54 was not equal or greater than 150% and 200%, respectively at any tested and evaluated test item concentration in 2 out of 3 independent runs. Therefore the h-CLAT prediction is considered negative for the tested test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).

The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%. Except the CD54 RFI value of the positive control (2.0 μg/mL DNCB) in the third h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the CD54 RFI value of the positive control (3.0 μg/mL DNCB) in the first h-CLAT run exceeded the positive criteria.

In conclusion, the test item 2,4-Dinitroanisole with a predicted log Pow of 0.5 – 1.9 did not activate THP-1 cells up to a concentration of 54.7 μg/mL in consideration of all three runs and under the test conditions of this study. Therefore the test item is considered negative for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Endpoint:
skin sensitisation, other
Remarks:
in silico
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
April, 2018
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:
2,4-dinitroanisole is a low sensitivity explosive organic compound with an anisole core and two nitro groups attached. It is a solid compound at room temperature with a low molecular weight (198.13 g/mol), slightly soluble in water (0.276 g/L at 25 °C) with a low lipophilicity (log P = 1.58 to 1.61 at 25 °C). Based on physical and chemical properties of the substance it is expected that the substance could be well absorbed through the skin since the molecule is less than 500 Da and its log P value is between -1 and 4, yet study by Lent showed 0.0017% absorbtion rate.

2,4-dinitroanisole was not corrosive to the skin and is not spontaneously flammable solid; therefore, the compound was not exempted from the skin sensitization testing based on the listed conditions in the column 2 of Annex VII section 8.3 of the REACH Regulation. From ECHA compiled inventory of substances likely to meet the criteria of Annex III to the REACH Regulation, 2,4-dinitroanisole was suspected for skin sensitization. A skin sensitization study in guinea pigs using a modified Buehler design was performed for the US Air Force in 2001. Unfortunately, it has not been possible to retrieve the report untitled “A Dermal Sensitization Study in Guinea Pigs with Composition B, CBR-12 and 2,4- Dinitroanisole - Modified Buehler Design (Bonnette, 2001). The substance was found not to be a skin sensitizer as cited in the Occupational Alliance for Risk Science - Workplace Environmental Exposure Levels for 2,4-dinitroanisole (2014): “Dermal sensitization was not observed when DNAN was administered to guinea pigs in a sensitization assay (OPPTS 870.2600)”. As the available information on 2,4-dinitoanisole was not available of low reliability, as the next step, the in silico assessment was performed.

The skin sensitizing potential of 2,4-dinitroanisole was predicted with BIOVIA Discovery Studio (TOPKAT) 4.5, VEGA NIC 1.1.4 (CAESAR), OECD QSAR Toolbox 4.1, Toxtree 2.6.13 and DEREK Nexus 5.0.2. In addition, results from the Danish QSAR Database were also considered (Pudenz, Envigo Ref. PN66VK). The assessment in silico with two negative predictions (TOPKAT and OECD QSAR Toolbox) without alerts in Toxtree and absence of pro-haptens against three low to moderate reliable positive predictions (CAESAR of VEGA, DEREK and Danish QSAR database) suggested rather no skin sensitizing property for 2,4-dinitroanisole.

To support the assessed result and to cover the remaining key events in the cellular level, further in chemico and in vitro testing of 2,4-dinitroanisole using OECD test methods 442C, 442D and 442E was recommended. All three in chemico/in vitro tests OECD 442C (Fleet, Envigo Ref. LQ56WY), OECD 442D (Roth, Envigo Ref. PS32DN) and OECD 442E (Belot, 17ENV19) were conducted on 2,4-dinitroanisole and showed negative results for covalent protein binding to skin proteins (key event 1), keratinocyte activation (key event 2) and dendritic cell activation (key event 3), respectively. Therefore, it may be concluded that the substance does not require classification as a skin sensitizer, with no requirement to conduct testing in vivo.
Qualifier:
according to
Guideline:
other: TOPKAT and OECD QSAR Toolbox
GLP compliance:
no
Justification for non-LLNA method:
3 Keys Event on the Adverse Outcome Pathway (AOP) for Skin Sensitisation have been provided. With the QSAR statement it is not required to performed study on animals.
Key result
Parameter:
other: skin sensitisation prediction
Run / experiment:
QSAR
Remarks on result:
no indication of skin sensitisation
Remarks:
QSAR prediction therefore quantitative data not available

All three in chemico/in vitro tests OECD 442C (Fleet, Envigo Ref. LQ56WY), OECD 442D (Roth, Envigo Ref. PS32DN) and OECD 442E (Belot, 17ENV19) were conducted on substance 2,4-dinitroanisole and showed negative results for covalent protein binding to skin proteins (key event 1), keratinocyte activation (key event 2) and dendritic cell activation (key event 3), respectively. Therefore, it may be concluded that the substance does not require classification as a skin sensitizer, with no requirement to conduct testing in vivo.  

Conclusions:
As weight of evidence, it may be concluded, that the substance does not require classification as a skin sensitizer, with no requirement to conduct testing in vivo.
Executive summary:

The assessment in silico with two negative predictions (TOPKAT and OECD QSAR Toolbox) without alerts in Toxtree and absence of pro-haptens against three low to moderate reliable positive predictions (CAESAR of VEGA, DEREK and Danish QSAR database) suggested rather no skin sensitizing property for 2,4-dinitroanisole.

All three in chemico/in vitro tests OECD 442C, OECD 442D and OECD 442E were conducted on substance 2,4-dinitroanisole and showed negative results for covalent protein binding to skin proteins (key event 1), keratinocyte activation (key event 2) and dendritic cell activation (key event 3), respectively.  

As weight of evidence, it may be concluded, that the substance does not require classification as a skin sensitizer, with no requirement to conduct testing in vivo.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

As weight of evidence, it may be concluded, that the substance does not require classification as a skin sensitizer, with no requirement to conduct testing in vivo.