Registration Dossier

Administrative data

Description of key information

QSAR assessment

The prediction of the skin sensitising potential of acid orange 94 refined was performed with BIOVIA Discovery Studio (TOPKAT) 4.5, VEGA NIC 1.1.4 (SAESAR), OECD QSAR Toolbox 4.2, Toxtree 3.1.0 and DEREK Nexus 6.0.1. In addition, results from the Danish QSAR Database were also considered in this assessment. The TOPKAT model for skin sensitisation was extended by including 63 data from the Envigo database.

With two positive and two negative predictions and all considered not to be reliable or to be of low reliability, there are no arguments for a weight of evidence. The QSAR predictions are therefore not considered conclusive and In Vitro testing of acid orange 94 refined is recommended provided that the compound is within the applicability of the respective In Vitro tests.

In vitroSkin Sensitisation Test - Human Cell Line Activation Test (h-CLAT)

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of Acid Orange 94 Refined dissolved in 0.2% (v/v) DMSO 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 Acid Orange 94 Refined was previously determined by two cytotoxicity tests.

This study was performed in accordance to OECD Guidelines for the Testing of Chemicals: OECD 442E; In Vitro Skin Sensitisation: In Vitro Skin Sensitisation Assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation. Annex I: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), June 2018, in accordance with GLP.

The test item Acid Orange 94 Refined with a log Pow of -1.79 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

 

In chemicoSkin Sensitisation: Direct Peptide Reactivity Assay (DPRA),

The purpose of this study (based on the OECD guideline for the testing of chemicals, hi cli ernico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of Acid Orange 94 Refined.

Solutions of Acid Orange 94 Refined were successfully analyzed by the validated DPRA analytical method (analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. With overall mean peptide depletion (reactivity) of 76.0% in the presence of the test item, Acid Orange 94 Refined is highly reactive and hence is predicted by DPRA as positive and therefore is likely to be a potential skin sensitizer based on this assay. 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Remarks:
QSAR assessment
Type of information:
other:
Remarks:
QSAR assessment
Adequacy of study:
supporting study
Study period:
16 April 2019
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
Remarks:
QSAR assessment
Justification for type of information:
According to Article 13 of the REACH Regulation and taking into consideration the testing strategies for the 2018 registration deadline (ECHA Endpoint Specific Guidance, Chapter R 7a, 2016), the information needed for the classification or risk assessment of a substance shall be generated whenever possible by means other than vertebrate animal tests, through the use of alternative methods.
An amendment to point 8.3 of Annex VII of the REACH regulation, (Commission Regulation (EU) 2016/1688 and Commission Regulation 2017/706 of 19 April 2017) requires that alternative methods are used for assessment of skin sensitisation potential where these will generate adequate information and where the available test methods are applicable to the substance to be tested.
Principles of method if other than guideline:
According to ECHA Guidance R 7a (2016), structural considerations, physico-chemical properties, exclusion conditions specified in Annex VII section 8.3, (Q)SAR ((Quantitative) Structure-Activity Relationship) and information from structurally similar substances, In Vitro/ In Chemico data, literature data on animal studies and human data (if available) are used to elaborate at testing strategy for the substance.
GLP compliance:
no
Type of study:
other: QSAR assessment
Justification for non-LLNA method:
According to ECHA Guidance R 7a (2016), structural considerations, physico-chemical properties, exclusion conditions specified in Annex VII section 8.3, (Q)SAR ((Quantitative) Structure-Activity Relationship) and information from structurally similar substances, In Vitro/ In Chemico data, literature data on animal studies and human data (if available) are used to elaborate at testing strategy for the substance.
Specific details on test material used for the study:
Test item: Acid Orange 94 Refined
Alternative name: Disodium 5, 5’-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-aminonaphthalene-1-sulphonate]
CAS number: 70161-18-1
EC number: 274-354-1
Intended use: Industrial chemical
Appearance: Reddish brown crystals
Storage conditions: Room temperature (10 – 30C), in the dark
Lot number: 8009
Expiry/Retest date: 31 December 2019
Purity: 97%
Key result
Parameter:
other:
Remarks:
QSAR assessment
Run / experiment:
TOPKAT
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
positive indication of skin sensitisation
Remarks:
QSAR assessment
Key result
Parameter:
other:
Remarks:
QSAR assessment
Run / experiment:
CAESAR (VEGA)
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
no indication of skin sensitisation
Remarks:
QSAR assessment
Key result
Parameter:
other:
Remarks:
QSAR assessment
Run / experiment:
DEREK
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
positive indication of skin sensitisation
Remarks:
QSAR assessment
Key result
Parameter:
other:
Remarks:
QSAR assessment
Run / experiment:
Toxtree
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
not determinable because of methodological limitations
Remarks:
QSAR assessment
Key result
Parameter:
other:
Remarks:
QSAR assessment
Run / experiment:
Danish (Q)SAR Database
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
not determinable because of methodological limitations
Remarks:
QSAR assessment
Interpretation of results:
study cannot be used for classification
Conclusions:
With two positive and two negative predictions and all considered not to be reliable or to be of low reliability, there are no arguments for a weight of evidence. The QSAR predicitions are therefore not considered conclusive and In Vitro testing of acid orange 94 refined is recommended provided that the compound is within the appicability of the respective In Vitro tests.
Executive summary:

With two positive and two negative predictions and all considered not to be reliable or to be of low reliability, there are no arguments for a weight of evidence. The QSAR predicitions are therefore not considered conclusive and In Vitro testing of acid orange 94 refined is recommended provided that the compound is within the appicability of the respective In Vitro tests.

Endpoint:
skin sensitisation, other
Remarks:
Weight of Evidence assessment
Type of information:
other:
Remarks:
Weight of Evidence assessment
Adequacy of study:
supporting study
Study period:
16 April 2019
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
Remarks:
Weight of Evidence assessment
Justification for type of information:
According to Article 13 of the REACH Regulation and taking into consideration the testing strategies for the 2018 registration deadline (ECHA Endpoint Specific Guidance, Chapter R 7a, 2016), the information needed for the classification or risk assessment of a substance shall be generated whenever possible by means other than vertebrate animal tests, through the use of alternative methods.
An amendment to point 8.3 of Annex VII of the REACH regulation, (Commission Regulation (EU) 2016/1688 and Commission Regulation 2017/706 of 19 April 2017) requires that alternative methods are used for assessment of skin sensitisation potential where these will generate adequate information and where the available test methods are applicable to the substance to be tested.
Principles of method if other than guideline:
According to ECHA Guidance R 7a (2016), structural considerations, physico-chemical properties, exclusion conditions specified in Annex VII section 8.3, (Q)SAR ((Quantitative) Structure-Activity Relationship) and information from structurally similar substances, In Vitro/ In Chemico data, literature data on animal studies and human data (if available) are used to elaborate at testing strategy for the substance.
GLP compliance:
no
Type of study:
other: Weight of Evidence assessment
Justification for non-LLNA method:
According to ECHA Guidance R 7a (2016), structural considerations, physico-chemical properties, exclusion conditions specified in Annex VII section 8.3, (Q)SAR ((Quantitative) Structure-Activity Relationship) and information from structurally similar substances, In Vitro/ In Chemico data, literature data on animal studies and human data (if available) are used to elaborate at testing strategy for the substance.
Specific details on test material used for the study:
Test item: Acid Orange 94 Refined
Alternative name: Disodium 5, 5’-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-aminonaphthalene-1-sulphonate]
CAS number: 70161-18-1
EC number: 274-354-1
Intended use: Industrial chemical
Appearance: Reddish brown crystals
Storage conditions: Room temperature (10 – 30C), in the dark
Lot number: 8009
Expiry/Retest date: 31 December 2019
Purity: 97%
Parameter:
other:
Remarks:
QSAR assessment
Remarks on result:
other:
Remarks:
QSAR assessment
Interpretation of results:
study cannot be used for classification
Conclusions:
As the exclusion criteria for testing according to ECHA have not been met and (Q)SAR predications for skin sensitisation potential are not considered conclusive, In Vitro testing of acid orange 94 refined should be performed.
Executive summary:

As the exclusion criteria for testing according to ECHA have not been met and (Q)SAR predications for skin sensitisation potential are not considered conclusive, In Vitro testing of acid orange 94 refined should be performed.

Endpoint:
skin sensitisation: in vitro
Remarks:
h-CLAT
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 May 2019 to 21 May 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 442E: Skin Sensitisation: Human Cell Line Activiation Test (h-CLAT)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells
Specific details on test material used for the study:
Test item: Acid Orange 94 Refined
Alternative name: Disodium 5, 5’-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-aminonaphthalene-1-sulphonate]
CAS number: 70161-18-1
EC number: 274-354-1
Intended use: Industrial chemical
Appearance: Reddish brown crystals
Storage conditions: Room temperature (10 – 30C), in the dark
Lot number: 8009
Expiry/Retest date: 31 December 2019
Purity: 97%
Details on study design:
Experimental Design and Procedures of h-CLAT

The test item was tested in two independent runs. The tests were performed on different days. The test item was prepared separately for each run.

5.6.1 Treatment of the Cells
For the test item exposure the highest soluble test item concentration of the cytotoxicity test (without precipitations) was used instead of 1.2 × CV75, since no mean CV75 could be determined. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment. Each solution was diluted with culture medium before application of the test solution to the cells to reach a final concentration of 0.2% (v/v) DMSO in the medium.
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).

5.6.2 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.

5.6.4 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.

Preparation of the acquisition
The following acquisition plots were prepared:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of each channel (FL-1 and FL-3, respectively)
The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single population appears without contamination or excessive debris. The FL-1 and FL-3 voltage were set and compensate to appropriate position. The FL-1 voltage was set using the FITC labelled-mouse IgG1 medium-treated cells tube, as such the MFI of control cells was set in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo Mean) with the FITC labelled CD54 medium-treated cells (FACSCalibur, Becton Dickinson GmbH).
The cell viability was detected by setting an R1-gate (dead cells are gated-out by staining with 7-AAD). Therefore, the R1 gate was set approximately at the middle position between the peak of the negative fraction and the positive fraction in the FL-3 histogram using DNCB-treated cells. The negative fraction corresponds to the living cells and was kept for the subsequent analyses. For each control and all test item concentrations, the cell viability was recorded from the isotype control cell tube (stained with FITC labelled-mouse IgG1), the CD54 and CD86 cell tube, where only the isotype control cells were used for the cell viability evaluation.
The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow into the flow line.

Acquisition
Dead cells were gated-out 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:
DMSO (Dimethyl sulfoxide, - Solvent Control for the Positive Control (h-CLAT)
DNCB (2,4-dinitrochlorobenzene - Positive Control (h-CLAT)
Key result
Parameter:
other:
Remarks:
Dendritic cell activation potential
Run / experiment:
h-CLAT
Value:
> 200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
Acceptance Criteria of the first h-CLAT run for the Test Item Acid Orange 94 Refined

Cell viability of medium control and DMSO control should be more than 90%.
Medium = 94.84%
DMSO = 94.44%

In the positive control (DNCB), RFI values of both CD54 and CD86 should exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability should be > 50%.
3 μg/mL DNCB (CD 54): 208.5%
3 μg/mL DNCB (CD 86): 398.5%
4 μg/mL DNCB (CD 54): 289.8%
4 μg/mL DNCB (CD 86): 331.7%

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 should not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).
CD54: 96.7%
CD86: 94.0%

For medium and DMSO controls, the MFI ratio of CD54 and CD86 to isotype control should be > 105%. Medium CD 54: 191.9%
Medium CD 86: 276.3%
DMSO CD 54: 183.8% DMSO CD 86: 256.2%

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of Acid Orange 94 Refined dissolved in 0.2% (v/v) DMSO 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 Acid Orange 94 Refined was previously determined by two cytotoxicity tests.

Cytotoxic effects were not observed following incubation with the test item up to the highest tested concentration (334 μg/mL). Due to the lack of cytotoxicity, a CV75 value could not be calculated. Therefore, the highest soluble test item concentration 334 μg/mL was used as highest test item concentration for the h-CLAT runs.

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

93, 112, 134, 161, 193, 232, 278 and 334 μg/mL

The test item with a log Pow of -1.79 was tested in 2 independent runs. The RFI of CD54 was greater than 200% in all concentrations of both independent runs. Therefore the h-CLAT prediction is considered positive for the 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%. For details see Annex 1 and 2. All acceptance criteria met for the cytotoxicity test and the h-CLAT method.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The test item Acid Orange 94 Refined with a log Pow of -1.79 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).
Executive summary:

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of Acid Orange 94 Refined dissolved in 0.2% (v/v) DMSO 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 Acid Orange 94 Refined was previously determined by two cytotoxicity tests.

This study was performed in accordance to OECD Guidelines for the Testing of Chemicals: OECD 442E; In Vitro Skin Sensitisation: In Vitro Skin Sensitisation Assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation. Annex I: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), June 2018, in accordance with GLP.

The test item Acid Orange 94 Refined with a log Pow of -1.79 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 May 2019 to 30 May 2019
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
Specific details on test material used for the study:
Test item: Acid Orange 94 Refined
Alternative name: Disodium 5, 5’-[(1-methylethylidene)bis(4,1-phenyleneoxysulphonyl-2,1-phenyleneazo)]bis[6-aminonaphthalene-1-sulphonate]
CAS number: 70161-18-1
EC number: 274-354-1
Intended use: Industrial chemical
Appearance: Reddish brown crystals
Storage conditions: Room temperature (10 – 30C), in the dark
Lot number: 8009
Expiry/Retest date: 31 December 2019
Purity: 97%
Details on study design:
Analytical Procedure

2.4.1 Reagents

Acetonitrile (ACN): HPLC gradient grade
Trifluoroacetic acid (TFA): 99% Pure
Water: Deionised reverse osmosis
Ammonium Acetate: Analytical reagent
Sodium Phosphate, monobasic: Analytical reagent
Sodium Phosphate, dibasic: Analytical reagent
Ammonium Hydroxide: Analytical reagent
100 mM Phosphate buffer, pH 7.5: In house preparation
100 mM Ammonium Acetate buffer, pH 10.2: In house preparation
HPLC Mobile Phase A: 0.1% v/v TFA in Water, in house preparation
HPLC Mobile Phase B: 0.085% v/v TFA in ACN, in house preparation

2.4.2 Assessment of Test Item Solubility
The solubility of Acid Orange 94 Refined was assessed in acetonitrile and acetonitrile/water 50/50 v/v.

2.4.3 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).

2.4.4 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.

2.4.5 Preparation of Stability Controls and Precision Control
Stability controls (Reference Control B (n=6), a precision control of both peptides were prepared at a concentration of 0.5 mM in acetonitrile/buffer. Reference Control C (n=3) were prepared at a concentration of 0.5 mM in water/acetonitrile 50/50 v/v /buffer.

2.4.6 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 of Acid Orange 94 Refined was also prepared in acetonitrile/water 50/50 v/v.

2.4.7 Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Triplicate solutions each of the positive control and Acid Orange 94 Refined stock solutions were diluted with the Cysteine peptide stock solution to prepare solutions containing 0.5 mM Cysteine and 5 mM of Cinnamic Aldehyde or 5 mM Acid Orange 94 Refined. For the co elution control, buffer solution was used in place of the Cysteine stock solution.

2.4.8 Preparation of Positive Control and Lysine Peptide Depletion Samples and Co elution Controls
Triplicate solutions each of the positive control and Acid Orange 94 Refined stock solution were diluted with the Lysine peptide stock solution to prepare solutions containing 0.5 mM Lysine and 25 mM of Cinnamic Aldehyde or 25 mM Acid Orange 94 Refined. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

2.4.9 Incubation
The appearance of the Acid Orange 94 Refined and positive control samples in the HPLC vials was documented following preparation and then the vials were placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

2.4.10 Analysis
The concentration of both the Cysteine and Lysine peptides in the presence of Acid Orange 94 Refined and the associated positive controls was quantified by HPLC using UV detection as detailed in the chromatographic section.
Positive control results:
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 0.45-0.55 mM (CV <15%) SD <14.9%
(SD <14.9%)

Lysine r2>0.99 40.2-69.0 0.45-0.55 mM (CV <15%) SD <11.6%
(SD <11.6%)

Achieved results Cysteine r2>0.999 81.1 B: 0.501 mM (CV 1.56%, n=6)
(SD, 0.13%, n=3) C: 0.490 mM (CV 0.60%, n=3) SD 3.62 % (n=3)

Lysine r2>0.999 62.4 B: 0.525 mM (CV 1.36%, n=6)
(SD, 0.40%, n=3) C: 0.516 mM (CV 0.71%, n=3) SD 0.26% (n=3)



Key result
Parameter:
other:
Remarks:
Peptide depletion
Run / experiment:
Direct Peptide Reactivity Assay
Value:
76
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation

The depletion of peptide in the presence of Acid Orange 94 Refined was:

 Peptide

 Reference Control

 Mean peak area of peptide with test item(µV.sec)

 Mean peptide depletion by Acid Orange 94 Refined (%)

 Cysteine

Control B: 891830 (n=6)
Control C: 870820 (n=3)

 410980 (n=3)

 52.8

 Lysine

Control B: 788030 (n=6)
Control C: 774210 (n=3)

 14493 (n=3)

 98.1

Applying the following reactivity prediction depletion model (below), reactivity of Acid Orange 94 Refined is classed as “high” and the DPRA prediction is therefore positive and is therefore predicted to be a potential 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

 

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

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Solutions of Acid Orange 94 Refined were successfully analyzed by the validated DPRA analytical method (analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. With overall mean peptide depletion (reactivity) of 76.0% in the presence of the test item, Acid Orange 94 Refined is highly reactive and hence is predicted by DPRA as positive and therefore is likely to be a potential skin sensitizer based on this assay.
Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals, hi cli ernico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of Acid Orange 94 Refined.

Solutions of Acid Orange 94 Refined were successfully analyzed by the validated DPRA analytical method (analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. With overall mean peptide depletion (reactivity) of 76.0% in the presence of the test item, Acid Orange 94 Refined is highly reactive and hence is predicted by DPRA as positive and therefore is likely to be a potential skin sensitizer based on this assay. 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Justification for classification or non-classification