Registration Dossier

Administrative data

Description of key information

In vitro/in chemicoskin sensitization studies were conducted on MTDID 47403. The results of the studies were:

 

-The test article was determined to have high reactivity (43.4% mean depletion) in a Direct Peptide

Reactivity Assay (DPRA) (OECD 442C).

 

-The test article was positive (EC1.5: 8.46 uM) in the ARE-Nrf2 Luciferase test method (OECD 442D).

 

-The test article was negative (CD86 <150 , CD54 <200) in the Human Cell Line Activation Test (h-CLAT) (OECD 442E).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Type of information:
other: Weight of Evidence (WoE)
Adequacy of study:
weight of evidence
Study period:
2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: See 'Remarks'
Remarks:
Weight of Evidence (WoE) conclusion based on three guideline studies conducted under GLP conditions.
Qualifier:
no guideline required
Version / remarks:
Other: Weight of Evidence summary.
Principles of method if other than guideline:
Weight of Evidence summary.
GLP compliance:
no
Type of study:
other: Weight of evidence summary.
Key result
Parameter:
other: Weight of Evidence
Remarks:
The weight of evidence indicates that the test article should be classified as a GHS Category 1A sensitizer.
Run / experiment:
Other
Value:
0
Vehicle controls validity:
not applicable
Negative controls validity:
not applicable
Positive controls validity:
not applicable
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
Based on the weight of evidence, the test article is a GHS Category 1A sensitizer.
Executive summary:

Based on the weight of evidence, the test article should be classified as a GHS Category 1A skin sensitizer. The evidence for this conclusion is as follows:

-The test article was determined to have high reactivity (43.4% mean depletion) in a Direct Peptide Reactivity Assay (DPRA) (OECD 442C).

-The test article was positive (EC1.5: 8.46 uM) in the ARE-Nrf2 Luciferase test method (OECD 442D).

-The test article was negative (CD86: , CD54: ) in the Human Cell Line Activation Test (h-CLAT) (OECD 442E).

Based on high reactivity in the DPRA and ARE-Nrf2 Luciferace test methods with reactivity values similar to the positive controls, the weight of evidence suggests that MTDID 47403 has significant skin sensitization potential and should be classified as a GHS Category 1A sensitizer.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017
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
Type of study:
direct peptide binding assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch 624730
- Expiration date of the lot/batch: 2017-12
- Purity test date: 05 May, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: No data
- Vehicle: Acetonitrile

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dissolved in acetonitrile
Details on study design:
Skin sensitisation (In chemico test system) - Details on study design:
The test article, MTDID 28640, was solubilized in acetone (48.0 mg in 1.589 mL acetone) to yield a final concentration of 100 mM and added to the peptide reactions.

DPRA was run according to Cyprotex SOP-2078. The cysteine peptide was prepared at 0.667 mM in cysteine Reaction buffer and the lysine peptide was prepared at 0.667 mM in Lysine Reaction buffer as outlined in OECD 442c. The reaction mix for cysteine peptide had a 1:10 test peptide to reference article ratio (0.5 mM cysteine to 5 mM reference article). The reaction mix for lysine peptide had a 1:50 peptide to reference article ratio (0.5 mM lysine to 25 mM reference article). Reactions for test and reference articles were run in triplicate. Vehicle control reactions were also made with acetonitrile, acetone or water containing no reference or test articles.

Reference control reactions were prepared by mixing 2.5 mL of acetonitrile with 7.5 mL of the respective buffer. Aliquots of 1 mL were added to 9 glass vials for each peptide and placed at the beginning middle and end of the run sequence. These reactions were used to ensure the consistency of peptide detection during the run. A standard curve was prepared for both peptides by adding 400 μL of acetonitrile to 1600 μL of 0.667 mM peptide to make a 0.534 mM standard. This 0.534 mM standard was serial diluted in 20% acetonitrile/buffer to make a 6-point standard curve. A zero-peptide standard was also included in the standard curve. After 24 ± 2 hours incubation DPRA samples were assayed for peptide depletion via HPLC/UV. The samples were transferred to Western Michigan University Homer Stryker M.D. School of Medicine Innovation Center for peptide assessment via HPLC with gradient elution and UV detections at 220 nm using an Agilent 1100 HPLC equipped with a Photodiode Array. Samples were run on an Agilent Zorbax SB-C-18 column under the conditions described in section 22 of the OECD guideline. Column temperature was 30°C, injection volume 5 μL, and flow rate was 0.35mL/min. Sample analysis was initiated within 24 ±2 hours of the reaction start. Samples were injected once. The first sample was injected at 13:35 on 6/2/17. The final sample was injected at 17:41 on 6/3/17. After determination of peptide remaining in the analysis, percent depletion relative to vehicle controls was calculated relative to no test article (vehicle control) samples. Peptide reactivity was reported as percent depletion and was calculated using the following formula: % Depletion = (1-(test compound area/ vehicle control area)) x 100
Positive control results:
2,3-Butanedione, the positive control, performed as expected in the assay and was ranked in the correct reactivity class according to the OECD guideline.
Key result
Parameter:
other: % Depletion DPRA
Value:
43.4
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
- Acceptance criteria met for solvent control: Yes
- Acceptance criteria met for positive control: Yes

MTDID 47403 displayed reactivity with both peptides, particularly the cysteine peptide.
Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
Based on the results of the study, MTDID 47403 is predicted to have sensitizing potential within this stage of the skin sensitization AOP.
Executive summary:

The sensitization potential of MTDID 47403 was examined using the Direct Peptide Reactivity Assay (DPRA). The test was performed under GLP conditions and followed the guidance in OECD 442C (2015). The test article, MTDID 47403, was solubilized in acetonitrile to yield a final concentration of 100 mM. The cysteine peptide was prepared at 0.667 mM in cysteine Reaction buffer and the lysine peptide was prepared at 0.667 mM in Lysine Reaction buffer as outlined in OECD 442C. The reaction mix for cysteine peptide had a 1:10 test peptide to reference article ratio (0.5 mM cysteine to 5 mM reference article). The reaction mix for lysine peptide had a 1:50 peptide to reference article ratio (0.5 mM lysine to 25 mM reference article). Reactions for test and reference articles were run in triplicate. Vehicle control reactions were also made with acetonitrile containing no reference or test articles. Aliquots of 1 mL solubilized test article were added to 9 glass vials for each peptide and placed at the beginning middle and end of the run sequence. These reactions were used to ensure the consistency of peptide detection during the run. A standard curve was prepared for both peptides by adding 400 μL of acetonitrile to 1600 μL of 0.667 mM peptide to make a 0.534 mM standard. This 0.534 mM standard was serial diluted in 20% acetonitrile/buffer to make a 6-point standard curve. A zero-peptide standard was also included in the standard curve. After 24 ± 2 hours incubation DPRA samples were assayed for peptide depletion via HPLC/UV. Samples were run on an Agilent Zorbax SB-C-18 column under the conditions described in section 22 of the OECD guideline. Column temperature was 30°C, injection volume 5 μL, and flow rate was 0.35mL/min. Sample analysis was initiated within 24 ±2 hours of the reaction start. Samples were injected once. After determination of peptide remaining in the analysis, percent depletion relative to vehicle controls was calculated relative to no test article (vehicle control) samples. Peptide reactivity was reported as percent depletion and was calculated using the following formula: % Depletion = (1-(test compound area/ vehicle control area)) x 100 MTDID 47403 showed precipitation in the lysine reaction Reactions containing precipitate were spun at 400xg for 5 minutes prior to running HPLC analysis. Because reaction with the peptide was observed (despite precipitation) the OECD guideline allows for prediction of sensitization potential. A percent depletion DPRA of 43.4% (76% Cysteine, 10.7% Lysine) was observed for MTDID 47403 indicating a high reactivity class (42.47% < Mean % Depletion ≤ 100%). Based on the results of the study, MTDID 47403 is predicted to have sensitization potential within this stage of the skin sensitization AOP.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 442E: In Vitro Skin Sensitization: Human Cell Line Activation Test (h-CLAT)
Deviations:
no
GLP compliance:
yes
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
According to OECD TG 442E (2017) in chemico and in vitro test methods have been considered scientifically valid for the evaluation of the skin sensistization hazard of chemicals. The h-CLAT is proposed as a step to address the third key event of the skin sensitization AOP, namely activation of dendritic cells (DC), by quantifying the change in expression of cell surface marker(s) associated with the process of activation of monocytes and DC following exposure to sensitizers (e.g. CD54, CD86) or changes in IL-8 expression, a cytokine associated with the activation of DC.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch 624730
- Expiration date of the lot/batch: 2017-12
- Purity test date: 05 May, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: No data
-Vehicle: DMSO

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dissolved in DMSO
Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:
THP-1 cells were analyzed for cytotoxicity following methodology from Cyprotex SOP-2099. Each test article concentration was assessed in singlet. After an initial cytotoxicity assay was completed, the CV75 was derived by calculating the test article concentration that resulted in 75% THP-1 viability
after test article exposure. Based on the results, the dose concentrations of the test article were as follows: 24.2, 29.1, 34.9, 41.9, 50.3, 60.3, 72.4, 86.9 μg/mL. For assessment of the test article, initial test article concentrations were determined according to requirements in OECD 442E and solubility data. From initial test article stock solutions of 250X in DMSO, subsequent dilutions of stock solutions were prepared by 1:2 serial dilutions in DMSO. To create a 2X working solution, stock solutions were diluted 250-fold with THP-1 culture media. The final DMSO concentration was approximately 0.2% for the h-CLAT assay. The 2X test article working solution was added to THP-1 cell suspension at a 1:1 ratio. All test article concentrations were dosed in singlet as required by OECD 442E guidance.

THP-1 cells were analyzed for expression of CD54 and CD86 surface proteins following methodology from SOP-2099 and performed across four runs. All test article concentrations were assessed in singlet for each marker and isotype control as required by OECD 422E guidance. After test article exposures of either 23 hours and 43 minutes, 23 hours and 45 minutes, 23 hours and 37 minutes, or 23 hours and 31 minutes; each THP-1 cell culture was transferred into a 12 x 75 mm tube and centrifuged. THP-1 cells were washed twice with staining buffer, centrifuged, and the supernatant aspirated. The THP-1 cells were then incubated for 16-17 minutes in 600 μL blocking buffer (0.01% Globulins Cohn Fraction in staining buffer). Three 180 μL portions of each cell suspension were then aliquoted into a 96-well round bottom plate. The plates were centrifuged, and blocking buffer aspirated. Next antibody batches were prepared for each antibody. 50 μL of each antibody batch were added to the THP-1 cells. The plates were incubated at approximately 2-8°C for 31-32 minutes. After antibody incubation, THP-1 cells were washed three times with staining buffer. THP-1 cells were resuspended in 400 μL of staining buffer and PI added at a concentration of 0.625 μg/mL before cytometric analysis. A PI working solution in PBS was prepared before use in experimental procedures. Samples were run on a validated BD FACS Canto II system. FSC, SSC, FITC, and PerCP-Cy5.5 parameters were turned on during analysis with a threshold setting of 50000 used to exclude debris.

The test article was considered to be positive if at least one of the test concentrations exhibited an RFI greater than or equal to 150 for CD86 or RFI greater than or equal to 200 for CD54. The test article was considered negative if all the test concentrations exhibited an RFI < 150 for CD86 or RFI < 200 for CD54.
The acceptance criteria were as follows:
a. The negative and vehicle controls (i.e., THP-1 Culture Media and THP-1 Culture media with 0.2% DMSO) should induce responses that are comparable with those predicted from OECD 442E. THP-1 cell viability must be >90% for the negative and vehicle control samples. For the negative and
vehicle controls the RFI (relative fluorescence intensity) values of both CD86 and CD54 should not exceed RFI 150 and RFI 200, respectively.
b. For both negative and vehicle controls the MFI ratio of both CD86 and CD54 to isotype should be >150%.
c. The positive control should induce response that are comparable with those predicted from OECD 442E. Cell viability must be over 50% for positive control samples.
d. THP-1 cell viability must be over 50% for at least four of the test article concentrations.
Positive control results:
The viability for the positive controls was above 50% across all four runs and gave a positive response for both CD86 and CD54.
Key result
Parameter:
other: RFI for CD86: 24.2 ug/mL
Run / experiment:
1a
Value:
45
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD86: 29.1 ug/mL
Run / experiment:
1a
Value:
45
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD86: 34.9 ug/mL
Run / experiment:
1a
Value:
46
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD86: 41.9 ug/mL
Run / experiment:
1a
Value:
69
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD86: 50.3 ug/mL
Run / experiment:
1a
Value:
129
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD86: 60.3 ug/mL
Run / experiment:
1a
Value:
79
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD54: 24.2 ug/mL
Run / experiment:
1a
Value:
203
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD54: 29.1 ug/mL
Run / experiment:
1a
Value:
212
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD54: 34.9 ug/mL
Run / experiment:
1a
Value:
205
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD54: 41.9 ug/mL
Run / experiment:
1a
Value:
166
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD54: 50.3 ug/mL
Run / experiment:
1a
Value:
142
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD54: 60.3 ug/mL
Run / experiment:
1a
Value:
-59
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD86: 24.2 ug/mL
Run / experiment:
2
Value:
45
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD86: 29.1 ug/mL
Run / experiment:
2
Value:
52
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD86: 34.9 ug/mL
Run / experiment:
2
Value:
51
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD86: 41.9 ug/mL
Run / experiment:
2
Value:
57
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD86: 50.3 ug/mL
Run / experiment:
2
Value:
97
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD86: 60.3 ug/mL
Run / experiment:
2
Value:
74
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD86: 72.4 ug/mL
Run / experiment:
2
Value:
50
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD86: 86.9 ug/mL
Run / experiment:
2
Value:
27
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD54: 24.2 ug/mL
Run / experiment:
2
Value:
116
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD54: 29.1 ug/mL
Run / experiment:
2
Value:
149
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD54: 34.9 ug/mL
Run / experiment:
2
Value:
140
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD54: 41.9 ug/mL
Run / experiment:
2
Value:
143
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD54: 50.3 ug/mL
Run / experiment:
2
Value:
137
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD54: 60.3 ug/mL
Run / experiment:
2
Value:
2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: RFI for CD54: 72.4 ug/mL
Run / experiment:
2
Value:
100
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Cell viability remained above 50% in run 1a at concentrations ranging from 24.2 to 60.3 μg/mL and in run 2 from 24.2 to 86.9 μg/mL for CD86 and 24.2 to 72.4 ug/mL for CD54.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the study, MTDID 47403 is considered negative for sensitization potential within this stage of the skin sensitization AOP.
Executive summary:

The sensitization potential of MTDID 47403 was examined using the human cell line activation test (h-CLAT). The test was performed under GLP conditions and followed the guidance in OECD 442E (2016). THP-1 cells were analyzed for cytotoxicity following methodology from Cyprotex SOP-2099. Each test article concentration was assessed in singlet. After an initial cytotoxicity assay was completed, the CV75 was derived by calculating the test article concentration that resulted in 75% THP-1 viability after test article exposure. Based on the results, the dose concentrations of the test article were as follows: 24.2, 29.1, 34.9, 41.9, 50.3, 60.3 μg/mL. For assessment of the test article, initial test article concentrations were determined according to requirements in OECD 442E and solubility data. From initial test article stock solutions of 250X in DMSO, subsequent dilutions of stock solutions were prepared by 1:2 serial dilutions in DMSO. To create a 2X working solution, stock solutions were diluted 250-fold with THP-1 culture media. The final DMSO concentration was approximately 0.2% for the h-CLAT assay. The 2X test article working solution was added to THP-1 cell suspension at a 1:1 ratio. All test article concentrations were dosed in singlet as required by OECD 442E guidance. THP-1 cells were analyzed for expression of CD54 and CD86 surface proteins following methodology from SOP-2099 and performed across four runs. All test article concentrations were assessed in singlet for each marker and isotype control as required by OECD 422E guidance. After test article exposures of either 23 hours and 43 minutes, 23 hours and 45 minutes, 23 hours and 37 minutes, or 23 hours and 31 minutes; each THP-1 cell culture was transferred into a 12 x 75 mm tube and centrifuged. THP-1 cells were washed twice with staining buffer, centrifuged, and the supernatant aspirated. The THP-1 cells were then incubated for 16-17 minutes in 600μL blocking buffer (0.01% Globulins Cohn Fraction in staining buffer). Three 180 μL portions of each cell suspension were then aliquoted into a 96-well round bottom plate. The plates were centrifuged, and blocking buffer aspirated. Next antibody batches were prepared for each antibody. 50 μL of each antibody batch were added to the THP-1 cells. The plates were incubated at approximately 2-8°C for 31-32 minutes. After antibody incubation, THP-1 cells were washed three times with staining buffer. THP-1 cells were resuspended in 400 μL of staining buffer and PI added at a concentration of 0.625 μg/mL before cytometric analysis. A PI working solution in PBS was prepared before use in experimental procedures. Samples were run on a validated BD FACS Canto II system. FSC, SSC, FITC, and PerCP-Cy5.5 parameters were turned on during analysis with a threshold setting of 50000 used to exclude debris.  The test article was considered to be positive if at least one of the test concentrations exhibited an RFI

greater than or equal to 150 for CD86 or RFI greater than or equal to 200 for CD54. The test article was considered negative if all the test concentrations exhibited an RFI < 150 for CD86 or RFI < 200 for CD54. The two assay runs for the test article indicated negative responses in both CD86 and CD54 levels. Therefore, neither marker was upregulated by exposure to the test article. Based on the results of the study, MTDID 47403 is considered negative for sensitization potential within this stage of the skin sensitization AOP.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
According to OECD TG 442D (2015) in chemico and in vitro test methods have been considered scientifically valid for the evaluation of the skin sensistization hazard of chemicals. The ARE-Nrf2 Luciferase Test method is proposed as a first step to address the second key event of the skin sensitization AOP, namely activation of keratinocytes, by measuring the induction of luciferase in the Keratinosens cells (a keratinocyte reporter cell line which contains an anitoxidant repsonse element [ARE]) which directly indicates activation of ARE-dependent genes.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Batch 624730
- Expiration date of the lot/batch: 2017-12
- Purity test date: 05 May, 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: No data
- Vehicle: DMSO

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dissolved in DMSO
Details on study design:
Skin sensitisation (In vitro test system) - Details on study design:
The experimental design of this study consisted of three definitive assays to determine the maximal induction (Imax), the concentration for maximal gene induction (CImax), the EC1.5 value (concentration for a statistically significant induction of 50% above solvent controls), and a mean IC50 (concentration leading to 50% viability as compared to solvent controls) for the test article, MTDID 28640. For each definitive assay, the KeratinoSens cells were cultured in quadruplicate plates for 24 hours, treated with test articles for 48 hours, and assessed for luciferase induction (3 plates) and cytotoxicity (1 plate). Each plate contained the test article with a range of 12 dosing concentrations (0.977, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000, and 2000 μM) for the article. Each plate also included 5 wells designated for the positive control (cinnamic aldehyde; 4, 8, 16, 32, and 64 μM), 6 wells designated as the DMSO solvent control, and 1 well which was left blank. After the 48-hour incubation, visual observations of the cultures were performed for the cytotoxicity plate and recorded. For the luciferase plates, each culture plate was removed from the incubator and allowed to equilibrate to room temperature for at least 30 minutes. Once at room temperature, the treatment medium was decanted from each plate and cultures were rinsed. 50 μL of Dulbecco’s Phosphate Buffered Saline without calcium and magnesium (CMF-DPBS) was added to each well followed by 50 μL of ONE-Glo Reagent. The plates remained at room temperature in the dark for at least 5 minutes before being read by the luminometer within 30 minutes of addition of the reagent. The luminescence determination of each plate was performed by a Berthold Detection Systems luminometer initiated from an IBM-PC hosting the Windows-based Simplicity software. The light intensity in each well was measured at 565 nm in the form of relative light units (RLUs). After 48 hours of incubation, the clear well plates designated for the MTT endpoint were decanted and 200 μL of the MTT (0.59 mg/mL) solution was prepared in 1% DMEM and added to each well. No rinsing was performed and the plate was incubated for approximately 4 hours. Following the 4-hour incubation, the MTT solution was decanted and 10% SLS was added to each plate. The plate was incubated at standard culture conditions overnight. After overnight incubation, each plate was placed on a shaker and shaken for at least 20 minutes at room temperature. The absorbance at 570 nm (OD570) of each well was measured with a Molecular Devices Vmax plate reader.
A test article was predicted to have sensitization potential if:
1. The EC1.5 value fell below 1000 μM in at least 2 of 3 replications;
2. At the lowest concentration with a gene induction above 1.5, cellular viability was greater than 70%;
and
3. There was apparent overall dose response which was similar between repetitions.
Positive control results:
The positive control, cinnamic aldehyde, performed as anticipated.
Key result
Parameter:
other: Mean IC50 (uM)
Run / experiment:
Mean
Value:
55.84
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: Mean Imax
Run / experiment:
Mean
Value:
4.01
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: Mean CImax (uM)
Run / experiment:
Mean
Value:
31.3
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: Mean EC 1.5 (uM)
Run / experiment:
Mean
Value:
8.46
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
Based on the results of the study, MTDID 47403 is considered positive for sensitization potential within this stage of the skin sensitization AOP.
Executive summary:

The sensitization potential of MTDID 47403 was examined using the ARE-Nrf2 Luciferase Test Method (KeratinoSens) (2015). The test was performed under GLP conditions and followed the guidance in OECD 442D. The experimental design of this study consisted of three definitive assays to determine the maximal induction (Imax), the concentration for maximal gene induction (CImax), the EC1.5 value (concentration for a statistically significant induction of 50% above solvent controls), and a mean IC50 (concentration leading to 50% viability as compared to solvent controls) for the test article, MTDID 47403. For each definitive assay, the KeratinoSens cells were cultured in quadruplicate plates for 24 hours, treated with test articles for 48 hours, and assessed for luciferase induction (3 plates) and cytotoxicity (1 plate). Each plate contained the test article with a range of 12 dosing concentrations (0.977, 1.95, 3.91, 7.81, 15.6, 31.3, 62.5, 125, 250, 500, 1000, and 2000μM) for the article. Each plate also included 5 wells designated for the positive control (cinnamic aldehyde; 4, 8, 16, 32, and 64μM), 6 wells designated as the DMSO solvent control, and 1 well which was left blank. After the 48-hour incubation, visual observations of the cultures were performed for the cytotoxicity plate and recorded. For the luciferase plates, each culture plate was removed from the incubator and allowed to equilibrate to room temperature for at least 30 minutes. Once at room temperature, the treatment medium was decanted from each plate and cultures were rinsed. 50μL of Dulbecco’s Phosphate Buffered Saline without calcium and magnesium (CMF-DPBS) was added to each well followed by 50μL of ONE-Glo Reagent. The plates remained at room temperature in the dark for at least 5 minutes before being read by the luminometer within 30 minutes of addition of the reagent. The luminescence determination of each plate was performed by a Berthold Detection Systems luminometer initiated from an IBM-PC hosting the Windows-based Simplicity software. The light intensity in each well was measured at 565 nm in the form of relative light units (RLUs). After 48 hours of incubation, the clear well plates designated for the MTT endpoint were decanted and 200μL of the MTT (0.59 mg/mL) solution was prepared in 1% DMEM and added to each well. No rinsing was performed and the plate was incubated for approximately 4 hours. Following the 4-hour incubation, the MTT solution was decanted and 10% SLS was added to each plate. The plate was incubated at standard culture conditions overnight. After overnight incubation, each plate was placed on a shaker and shaken for at least 20 minutes at room temperature. The absorbance at 570 nm (OD570) of each well was measured with a Molecular Devices Vmax plate reader. The following values were determined for the test article: mean EC1.5 = 8.46 μM, mean IC50 = 55.84 μM, mean Imax = 4.01, and mean CImax = 31.3μM. Based on these results and the current prediction model, the test article, MTDID 47403, was predicted to be a sensitizer within this stage of the skin sensitization AOP.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Justification for classification or non-classification

Based on high reactivity in the DPRA and ARE-Nrf2 Luciferace test methods with reactivity values

similar to the positive controls, the weight of evidence suggests that MTDID 47403 has significant skin

sensitization potential and should be classified as a GHS Category 1A sensitizer.