4-(2-acryloyloxy-ethoxy) benzophenone

Endpoint summary

Description of key information

The skin sensitization potential of AEBP was evaluated in three in vitro/in chemico studies. The results of the assays were as follows:

High reactivity in the Direct Peptide Reactivity Assay (DPRA) when tested according to OECD 442C.

Strongly positive in the Keratinosens assay when tested according to OECD 442D

Negative in the Human Cell Line Activation Test (h-CLAT) when tested according to OECD 442E.

Based on the strongly positive response that AEBP elicited in 2/3 assays, the weight of evidence suggests that it should be classified as a GHS Category 1A sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

Remarks:

No deviations that negatively impacted the results of the study ocurred.

GLP compliance:

yes

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:
3M Company, Lot 30029
- Expiration date of the lot/batch:
25 July, 2020
- Purity test date:
25 July, 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At room temperature, protected from light.
- Stability under storage conditions:
No data
- Stability under test conditions:
No data
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
Soluble and stable in acetonitrile.
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable):
No reaction observed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test article was dissolved in acetonitirile.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
: Dissolved in acetonitirile.

Details on the study design:

Skin sensitisation (In chemico test system) - Details on study design:
The Direct Peptide Reactivity Assay was used to assess the skin sensitization potential of the test article. Synthetic peptides containing cysteine or lysine were reacted with each test article for 24 ± 2 hours. After the incubation period, the extent of peptide depletion was analyzed using High Performance liquid Chromatography {HPLC) coupled with ultra-violet {UV) spectrometric detection.

Positive control results:

The positive control (Cinnamic Aldehyde) performed as expected and depleted cysteine by 71.49% and lysine by 64.02%.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

99.01 %

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

lysine depletion

Value:

61.72 %

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Mean Peptide Depletion (%) of Cysteine and Lysine

Value:

80.37

At concentration:

100 mM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

high reactivity [in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None observed.

DEMONSTRATION OF TECHNICAL PROFICIENCY: IIVS is technically proficient in running the OECD 442C.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent control: The peak area CV for the solvent controls was less than 15%.
- Acceptance criteria met for positive control: The positive control met the following criteria: The percent peptide depletion for the cysteine peptide exposed to the positive control (cinnamic aldehyde) must be> 60.8% and the SD for the replicates must be< 14.9%. For the lysine peptide, the percent depletion by the positive control must be > 40.2% and < 69.4% and the SD for the replicates must be< 11.6%.
- Acceptance criteria met for variability between replicate measurements: The SD for the test article replicates was < 14.9% for the cysteine depletion and < 11.6% for the lysine depletion.

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

Based on the results of the study, MTDID 32918 is predicted to be a sensitizer with high reactivity (99.01% cysteine depletion, 61.72% lysine depletion and 80.37% combined cysteine and lysine depletion) within this stage of the skin sensitization adverse outcome pathway (AOP).

Executive summary:

The sensitization potential of the test article 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 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. After 24 ± 2 hours incubation DPRA samples were assayed for peptide depletion via HPLC/UV. The separations module used in this assay was a Waters 2695 HPLC system. This system consisted of a solvent management system for the mobile phases and a sample management system for the test article and controls. The HPLC system was coupled to a photodiode array detector set at 220 nm. The dimensions of the column used were 2.1 mm x 100 mm x 3.5 micron. The test article depleted 99.01% cysteine and 61.72% lysine for a combined depletion (cysteine and lysine) of 80.37%. Per the DPRA prediction models, MTDID 32918 was predicted to have high reactivity in both the combined depletion model (42.48-100%) as well as the cysteine depletion model (98.25-100%). Based on the results of the study, MTDID 32918 is predicted to be a sensitizer with high reactivity (99.01% cysteine depletion, 61.72% lysine depletion and 80.37% combined cysteine and lysine depletion) within this stage of the skin sensitization adverse outcome pathway (AOP).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Test Guideline 442E

Deviations:

no

Remarks:

No deviations ocurred that negatively impacted the results of the study.

GLP compliance:

yes

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material:
3M Company, Lot 30029
- Expiration date of the lot/batch:
25 July, 2020
- Purity test date:
25 July, 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
At room temperature, protected from light.
- Stability under storage conditions:
No data
- Stability under test conditions:
No data
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium:
Soluble and stable in DMSO.
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable):
No reaction observed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
The test article was dissolved in DMSO.

FORM AS APPLIED IN THE TEST (if different from that of starting material)
: Dissolved in DMSO

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
The experimental design of this study consisted of at least one dose range assay and at least two definitive assays. The purpose of the dose range assay was to calculate the test article concentration that corresponds to 75% cell viability (CV75). The CV75 value was used to determine a range of
eight serial test article dilutions that were used for the definitive assays so as to distinguish surface marker upregulation from a cytotoxic event. The purpose of the definitive assays was to measure the upregulation of CD54 and CD86 on the cell surface after a 24 hour exposure to eight test article concentrations. Upon the completion of the 24 hour exposure, the cells were rinsed and stained with fluorescently labeled antibody for CD54 or CD86. The upregulation of the surface markers was measured with flow cytometry to determine the mean fluorescence intensity (MFI) of the cell populations.

Positive control results:

The positive control results were within the expected response range.

Key result

Group:

test chemical

Run / experiment:

other: Trial B2

Parameter:

EC200, CD54 [442E]

Value:

17.3 µg/mL

Cell viability:

>50%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

other: Trial B7

Parameter:

EC200, CD54 [442E]

Value:

20.7 µg/mL

Cell viability:

>50%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

other: Trial B2

Parameter:

EC150, CD86 [442E]

Value:

17.3 µg/mL

Cell viability:

>50%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

other: Trial B7

Parameter:

EC150, CD86 [442E]

Value:

20.7 µg/mL

Cell viability:

>50%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

CV75 [442D and 442E]

Value:

17.252 µg/mL

Cell viability:

>50%

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: None observed.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The CRO is techically proficient in running the OECD 442E.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent control: The cell viability values of the solvent control was > 90%. For the solvent control, RFI values of both CD86 and CD54 were less than the positive criteria (CD86 RFI < 150 and CD54 RFI < 200). For the medium and solvent controls, the MFI ratio of both CD86 and CD54 to isotype control was> 105%.
- Acceptance criteria met for positive control: Trials B1, B3, B4, B5 and B6 did not meet the assay acceptance criteria for the positive control and therefore those trials were not considered valid. Trials B2 and B7 were valid and were utilized to make the sensitization potential determination for the test article. The positive control acceptability criteria were as follows: For the positive control (DNCB), RFI values of both CD86 and CD54 were predicted to be positive (CD86 RFI greater than or equal to 150 and CD54 RFI greater than or equal to 200), and cell viability was> 50%.
- Acceptance criteria met for variability between replicate measurements: It was met for trials B2 and B7.
- Range of historical values if different from the ones specified in the test guideline: Per OECD 442E.

Interpretation of results:

GHS criteria not met

Conclusions:

Based on the results of the study (EC150 > 17.25 ug/mL), MTDID 32918 is not predicted to have skin sensitization potential within this stage of the skin sensitization adverse outcome pathway (AOP).

Executive summary:

The skin sensitization potential of the test article was evaluated in the Human Cell Line Activation Test (h-CLAT). The study was conducted according to OECD 442E in compliance with OECD GLP regulations. The preliminary and definitive assays consisted of a positive control, DNCB, prepared at a top stock concentration of 2 mg/mL in DMSO. The positive control was further diluted 1:250 in culture medium and then 500 uL were dosed onto 500 uL of cell suspension to make a 2-fold final dilution on the cells. The preliminary and definitive assays also required a solvent control for DMSO which was prepared as a 1:250 dilution of the stock DMSO in culture medium and dosed on the cells in the same manner as the positive control. A medium control was also prepared for the assay and consisted of 500 uL of culture medium dosed on 500 uL of cell suspension. The test article was first tested in at least one valid dose range assay to determine the CV75 value, which is the calculated concentration associated with 75% cell viability. Approximately forty eight hours prior to dosing, the THP-1 cells were seeded in T75 flasks at a density of 2.0 x 105 cells/mL. Alternately, approximately 72 hours prior to dosing, the THP-1 cells were seeded in T75 flasks at a density of 1.0 x 105 cells/mL. On the day of dosing, the cells were removed from the culture vessels, placed in conicals and centrifuged in a centrifuge set to 200-300 x G, 5 minute spin time, with a soft start/stop, and at room temperature. The supernatant was discarded and the cell pellet was re-suspended in an appropriate volume of fresh culture medium warmed to 37°C. A cell count was performed and a dilution of the cells was prepared so that 500 uL of 2.0 x 106 cells/mL could be seeded into the appropriate wells of a 24-well plate (Falcon). Once seeded, the plate(s) were returned to the incubator until dosing. The test article dilutions were prepared in the appropriate solvent. For the test article, DMSO was selected as the solvent and the top stock dilution was prepared at 500 mg/mL. The test article dilutions were then serially diluted 2-fold to make a series of eight total dilutions (including the top stock). The eight serial dilutions for the test article were further diluted 1:250 in culture medium warmed to 37 C. Five hundred microliters of each 1:250 dilution were then dosed dropwise onto the cells in the 24-well plate(s). The cells were incubated at standard culture conditions for 24±0.5 hours. At the end of the exposure period, the plate(s) were removed from the incubator and the cell suspensions from each well were placed in separate microcentrifuge tubes. The tubes were centrifuged in a centrifuge set to 200-300 x G, 5 minute spin time, with a soft start/stop, and at 4°C. The supernatant was decanted and the cell pellet was re-suspended in 1 mL of FACS buffer (1.0 mg/mL Bovine Albumin Fraction V (Calbiochem) in Dulbecco’s phosphate buffered saline (DPBS) (Gibco)). The cell suspensions were centrifuged again using the previously defined settings and the FACS buffer was decanted. This rinsing process was performed a total of three times. Upon completion of the rinsing process, the cells were re-suspended in 600 uL of FACS buffer and 200 uL were transferred from each tube to designated wells of a 96-well round-bottom plate. A 12.5 ug/mL solution of propidium iodide (PI) (Miltenyi Biotec) was prepared in FACS buffer. Living cells are impermeable to PI while dead cells are permeable to PI. The PI solution was dosed on the cells by the flow cytometer as a 1:20 dilution immediately before sampling from each well so that the final concentration of PI on the cells was 0.625 μg/mL. At least two valid definitive assays were performed to determine the sensitization potential of the test article. The cells were prepared and seeded into the 24-well dosing plates in the same manner as for the dose range assay. The test article top stocks and serial dilutions in the primary solvent (DMSO) were prepared at the concentrations determined from the CV75 value. These dilutions were further diluted 1:250 in culture medium and 500 uL of each dilution were dosed onto 500 uL of cells seeded in the 24-well plate(s) at 2.0 x 106 cells/mL. The test article dilutions were exposed to the cells for 24±0.5 hours. At the end of the exposure period, the cells were transferred to tubes and rinsed in the same fashion as performed in the dose range assay (three times with 1 mL of FACS buffer). After the completion of the three rinses, each sample of cells was exposed to 600 uL of blocking mixture (10 mg/mL Globulins Cohn Fraction II, III human (Sigma) prepared in DPBS) diluted 1:100 in FACS buffer. The cells were kept in the dark at 2-8°C for 15±1 minutes. After the exposure with the blocking mixture, the cell suspension in each tube was divided into 3 aliquots of 180 uL each into the designated wells of 96-well round-bottom plate(s). The plate(s) were centrifuged using the previously defined settings and the supernatant was discarded. The cells were then exposed to fluorescein isothiocyanate (FITC) labeled antibodies. One of the three aliquots of each cell population was designated for either CD54 antibody (6.5B5; DAKO), CD86 antibody (FUN-1; BD Pharmingen), or isotype control (DAKO). The antibodies were prepared so that 50 uL could be dosed on each appropriate cell population. The CD54 and isotype control antibodies were prepared as 6% dilutions in FACS buffer and the CD86 antibody was prepared as a 12% dilution in FACS buffer. Once the antibodies were added, the cells were incubated in the dark at 2-8°C for 30±1 minutes. After the exposure to the antibodies, 150 uL of FACS buffer were added to each well and the plate(s) were centrifuged using the previously defined settings. The supernatant was aspirated and the cells were rinsed two additional times with 200 uL of FACS buffer. Upon completion of the rinsing process the cells were resuspended in 200 uL of FACS buffer. The PI solution was prepared and added to the plate(s) in the same manner as for the dose range assay.

 

The preliminary dose range-finding assay determined the top test article concentration to be used for the definitive trials was 17.3 ug/mL in the first definitive trial and 20.7 ug/mL in the second. Due to positive control runs that did not meet the acceptability criteria, the definitive trial had to be run twice. Only data that met the acceptability criteria were presented. The test article was predicted to be a nonsensitizer in both definitive trials with EC150 (CD86) and EC200 (CD54) values of > 17.3 and > 20.7 ug/mL, respectively. Based on the results of the study (EC150 > 17.25 ug/mL), MTDID 32918 is not predicted to have skin sensitization potential within this stage of the skin sensitization adverse outcome pathway (AOP).

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch number of test material: 3M Company, Lot 30029
- Expiration date of the lot/batch: 25 July, 2020
- Purity test date: 25 July, 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from light.
- Stability under storage conditions: No data
- Stability under test conditions: No data
- Solubility and stability of the test substance in the solvent/dispersant/vehicle/test medium: Soluble and stable in DMSO.
- Reactivity of the test substance with the solvent/vehicle /test medium (if applicable): No reaction observed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test article was dissolved in DMSO.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : Dissolved in DMSO

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
The Induction of Antioxidant-Response-Element Dependent Gene Activity and Cytotoxicity (Using MTI) in the Keratinocyte ARE-Reporter Cell Line KeratinoSens skin sensitization assay is a high-throughput cell based in vitro test to screen for the skin sensitization potential of chemicals. The KeratinoSen cells (Givaudan, Switzerland) were propagated as a reporter cell line. The KeratinoSens cells are transfected HaCaT keratinocytes that include a 56-base-pair insertion containing the ARE sequence from the AKR1C2 gene, a SV40 promoter, the luciferase gene in the vector pGL4 from Promega, and a stable insertion allowing for cells to be selected in the presence of Geneticin (G418). The signaling pathway with the repressor protein Keap1 and the transcription factor Nrf2, which binds to the antioxidant I electrophile response element (ARE I EpRE), was shown to be a valuable cellular endpoint to detect skin sensitizers in vitro. The induction of luciferase directly indicates the activation of ARE dependent genes. Additionally, the cytotoxicity of a test article was assessed using MTI (3-[4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide). Cytotoxicity was determined by measuring the relative conversion of MTI in test article-treated cultures compared to the solvent control.

Positive control results:

The positive control, Cinnamic Aldehyde, was tested at 5 concentrations ranging from 4 to 64 uM. The IC50 and IC30 were both greater than 64 uM and the EC1.5 was 7.96. Maximal Induction (Imax) was 16.92 at 64 uM.

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC 1.5 [442D]

Value:

2.16 µM

Cell viability:

The mean IC50 and IC30 values were 17.2 uM and 14.78 uM, respectively. As a result, the cell viability was above 70% at the EC1.5 concentration.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

Imax [442D]

Value:

39.7 µM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

other: Concentration for Maximal Gene Induction (CImax)

Value:

23.45 µM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

IC50 [442D]

Value:

17.2 µM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

IC30 [442D]

Value:

14.78 µM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No visible damage noted.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The contract research organization that conducted the study is technically proficient in OECD 442D

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for solvent control: The standard deviation of the viability of the solvent controls was less than 20%.
- Acceptance criteria met for positive control: Cinnamic aldehyde resulted in an EC1.5 value less than 64 uM an dproduced a 2 to 8 fold induction at 64 uM.
- Acceptance criteria met for variability between replicate measurements: The first of three trials failed so the remaiing two assays were utilized to assess the sensitizaton potential of the test article.
- Range of historical values if different from the ones specified in the test guideline: Per OECD 442D.

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

Based on the results of the study, MTDID 32918 was positive in the Keratinosens assay with an EC1.5 value of 2.16 uM and is predicted to have skin sensitization potential within this stage of the skin sensitization adverse outcome pathway (AOP).

Executive summary:

The skin sensitization potential of MTDID 32918 was evaluated in the Keratinosens assay. The study was conducted according to OECD 442D in compliance with OECD GLP. The test article was dissolved in DMSO and each plate tested a range of 12 dosing concentrations for each test article ranging from 0.977 to 2000 uM. Each plate also included 5 wells designated for the positive control (tested over a range of 5 dosing concentrations), 6 wells designated as the DMSO solvent control, and 1 well which was left blank. After approximately 24 hours of incubation, the Assay Medium was removed from the cells. The plates were decanted and gently blotted on sterile paper towels. One hundred and fifty microliters of fresh pre-warmed 1% DMEM were added to all wells, including the blank. The plates were returned to the incubator until the dosing was initiated. For each test article, twelve decreasing doses were selected for the assay (test articles were diluted to a final concentration of 200 mM in DMSO). For the positive control, 5 decreasing doses were prepared. For each experiment, the positive control (5 doses), and the solvent control, a 100X DMSO master plate was made, followed by a 4X Master Plate. When added to the 150 μL of 1% DMEM already in each well, the addition of the 50 μL 4X dose brought the final dose on the plates to 1X. The resulting 4X Master Plate was then used to dose the replicate assay plates (transparent or white-walled) containing cells. A final 1X concentration was achieved for each dose by removing 50 μL from each well of the 4X Master Plate and adding the dose to the corresponding well in the 1X plates (already containing 150 μL of 1% DMEM in each well) . The blank well received 50 μL of 1% DMEM. When all of the plates were dosed, the plates were sealed with a plate sealer to avoid evaporation of volatile compounds and to avoid cross-contamination between wells. The plates were then incubated at standard culture conditions for 48±1 hours. After approximately 48 hours of post-treatment incubation, visual observations of the cultures were performed for the cytotoxicity plate and recorded. After 48 ±1 hours of exposure, each white-walled 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. The cultures were rinsed with 250 μL of CMF-DPBS (room temperature), the CMF-DPBS rinsate was decanted from the wells, and the plates were gently blotted onto paper towels. Fifty microliters of CMF-DPBS was added to each well followed by fifty microliters of ONE-Glo Reagent. The plates remained at room temperature in the dark for at least 5 minutes before being read by the luminometer. The plates were read within 30 minutes of addition of the ONEGlo 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). A 0.59 mg/mL MTT solution was prepared in 1% DMEM and used within 2 hours. After 48 ±1 hours, the clear 96-well plates designated for the MTT endpoint were decanted and gently blotted on paper towels. No rinsing was performed. Two hundred μL of 1% DMEM containing 0.59 mg/mL MTT was added to each well. The plate was incubated with a plate seal at standard culture conditions for approximately 4 hrs. After approximately 4 hours, the MTT solution was decanted, the plate was blotted, and 200 μL of 10% SLS was added to each well. The plate was covered with a plate seal and incubated at standard culture conditions overnight. After the overnight incubation, each plate was placed on a plate 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 solvent and positive controls performed as expected, indicating that the test system was valid. MTDID 32918 induced a 1.5-fold increase in luciferase activity (EC1.5) at 2.16 uM. Additionally, MTDID decreased cell viability by 50% (IC50) at 17.20 uM and 30% (IC30) at 14.78 uM. Maximal Induction (Imax) was 39.70 at a concentration (CImax) of 23.45 uM. Based on the results of the study, MTDID 32918 was positive in the Keratinosens assay with an EC1.5 value of 2.16 uM and is predicted to have skin sensitization potential within this stage of the skin sensitization adverse outcome pathway (AOP).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Justification for classification or non-classification

Based on the strongly positive response that AEBP elicited in 2/3 assays, the weight of evidence suggests that it should be classified as a GHS Category 1A sensitizer.