Benzo[rst]phenanthro[10,1,2-cde]pentaphene-9,18-dione, reaction products with 1-chlorododecane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

Dinaphtho[1,2,3-cd:1',2',3'-lm]perylene-9,18-dione, dodecyl derivs.

EC Number:

270-145-4

EC Name:

Dinaphtho[1,2,3-cd:1',2',3'-lm]perylene-9,18-dione, dodecyl derivs.

Cas Number:

68411-75-6

Molecular formula:

Unspecified

IUPAC Name:

Dinaphtho[1,2,3-cd:1',2',3'-lm]perylene-9,18-dione, dodecyl derivs.

In vitro test system

Details on the study design:

Cell line used: HaCaT cells
obtained from Givaudan and maintained following protocols outlined in the supplier’s standard operating procedures and publications (Emter et al., 2010; Natsch et al., 2011). To determine sensitization potential, keratinocytes were incubated with each test chemical for about 48 hours at approximately 37ºC. At the end of incubation, luciferase induction and cell viability was determined.

Media:
The maintenance medium for the HaCaT cell line was prepared by supplementing Dulbecco’s Modified Eagle Medium (D-MEM) media (Gibco) with 9.1% fetal bovine serum (FBS) and either with (DMEM9.1(+)) or without (DMEM9.1(-)) geneticin (antibiotic; final concentration 500 µg/ml). Treatment medium consisted of D-MEM with 1% FBS and no geneticin (DMEM1(-)). All medium prepared was stored at approximately 4°C and used within 28 days.

Preparation of the controls and treatment solutions:
DMSO was used as the solvent for the test material and the control treatment. Cinnamic aldehyde (CA, CAS # 104-55-2) was used as the positive control. CA was prepared at a concentration of 6.4 mM in DMSO and further diluted to 64 µM, 32 µM, 16 µM, 8 µM, and 4 µM in culture medium. Stock solutions of the test material were prepared fresh in DMSO, at an initial concentration of 200 mM. All stock solutions were further serial diluted in DMSO to obtain a “100X master plate” consisting of each test material at twelve consecutive two-fold dilutions (ranging from 0.098 to 200 mM). These stocks were further diluted in the assay procedure as outlined below to result in the testing of a concentration range of 0.98 to 2000 µM in the final assay.

Luciferase and Cytotoxicity assays:
Frozen HaCaT cells (approximately -150ºC) were thawed in a water bath at approximately 37ºC, resuspended in DMEM9.1(-), and were pelleted by centrifugation at 125 g for 5 minutes at room temperature. The cell pellet was resuspended in DMEM9.1(-), seeded in a flask, and maintained at about 37oC with 5% CO2. After reaching 80-90% confluency, the HaCaT cells were washed twice with Dulbecco’s Phosphate-Buffered Saline (DPBS), trypsinized, and incubated at approximately 37ºC for about 7 minutes. Detached cells were resuspended in DMEM9.1(-) and centrifuged at 125 g for 5 minutes. The resulting pellet was resuspended and hereafter maintained in DMEM9.1(+).
Cell Seeding For Testing
• Cells at 80-90% confluency were washed twice with DPBS, harvested as described above, re-suspended in DMEM9.1(-), and the cell density was adjusted to approximately 80,000 cells/ml.
• 125 µl of the cell-suspension was distributed to each well in a 96-well plate (approximately10,000 cells/well).
• Each 96 well plate consisted of EPON™ Resin CS-337 at twelve different concentrations, six negative control wells containing 1% DMSO, five wells containing the positive control CA at five different concentrations, and one well which is blank containing no cells (one well each/plate).
• In each experiment, three parallel plates for luciferase assay (solid white plate) and two plates for cytotoxicity assessment (transparent plate) were prepared as above.
• The plated cells were grown for about 24 h at approximately 37ºC and 5% CO2.

Treatment Regimen
• Following the ~24 h incubation, the medium was aspirated and replaced with 150 µl of DMEM1(-).
• The 100X stock master plate was diluted 25-fold (10 µl of test chemical solution from master plate + 240 µl of DMEM1(-)) in a fresh 96 well plate (4X master plate).
• 50 µl of solution from the resulting 4X master plate was transferred to each replicate plate already containing the keratinocytes and 150 µl of DMEM1(-) (ranging from 0.98 to 2000 µM in culture medium).
• All plates were covered with sealing tape (STR-SEAL-PLT, EXCEL Sci, Omaha, Nebraska) and incubated at approximately 37ºC for an additional ~48 h.
Luciferase Measurements
• At the end of the 48 h incubation, the supernatant from the 96 well plates was aspirated, washed once with DPBS and cells in each well was incubated with 20 µl of passive lysis buffer (Promega Corp., Madison, Wisconsin) on an orbital shaker at room temperature for
20 min.
• The plates with the cell lysate were read (relative luminescence units; RLU) in the luminometer using the following program:
i. 50 µl of the luciferase substrate was added to each well
ii. waited for 1 second and integrated the luciferase activity for 2 sec
• Luciferase induction for the chemicals was calculated using the following approach:
i. (RLUFG) – (RLUBG) = BG corrected (RLU)
ii. (BG corrected (RLU) of each chemical containing well)/( average BG corrected (RLU) of six negative control wells) = Luciferase induction
RLUFG = Foreground Relative luciferase units
RLUBG = Background Relative luciferase units (no cells blank)
Cytotoxicity Assessment
• For the cell viability assay plates, the medium was aspirated and replaced with 200 µl of DPBS and 27 µl of Thiazolyl blue tetrazolium bromide (MTT) reagent (5 mg/ml in DPBS). The plate was covered with sealing tape and was incubated at approximately 37ºC for 4 hours.
• Following incubation, the supernatant was aspirated and 200 µl of DMSO was added to each well. Following thorough mixing by repeated pipetting, the cell lysate was transferred to a new clear 96 well plate and absorbance was quantified at 600 and 630 nm. Cell viability for the cells was calculated using the following method:
i. (Abs600) – (AbsBG) = BG corrected (Abs600)
ii. (Abs630) – (AbsBG) = BG corrected (Abs630)
iii. BG corrected (Abs600) - BG corrected (Abs630) = (Abs600 - 630)
iv. ((Abs600 - 630) of each chemical containing well)/( average (Abs600 - 630) of six negative control wells) *100 = % viability
Abs600 = Foreground absorbance measured at 600 nm
Abs630 = Foreground absorbance measured at 630 nm

ii. waited for 1 second and integrated the luciferase activity for 2 sec
• Luciferase induction for the chemicals was calculated using the following approach:
i. (RLUFG) – (RLUBG) = BG corrected (RLU)
ii. (BG corrected (RLU) of each chemical containing well)/( average BG corrected (RLU) of six negative control wells) = Luciferase induction
RLUFG = Foreground Relative luciferase units
RLUBG = Background Relative luciferase units (no cells blank)
Cytotoxicity Assessment
• For the cell viability assay plates, the medium was aspirated and replaced with 200 µl of DPBS and 27 µl of Thiazolyl blue tetrazolium bromide (MTT) reagent (5 mg/ml in DPBS). The plate was covered with sealing tape and was incubated at approximately 37ºC for 4 hours.
• Following incubation, the supernatant was aspirated and 200 µl of DMSO was added to each well. Following thorough mixing by repeated pipetting, the cell lysate was transferred to a new clear 96 well plate and absorbance was quantified at 600 and 630 nm. Cell viability for the cells was calculated using the following method:
i. (Abs600) – (AbsBG) = BG corrected (Abs600)
ii. (Abs630) – (AbsBG) = BG corrected (Abs630)
iii. BG corrected (Abs600) - BG corrected (Abs630) = (Abs600 - 630)
iv. ((Abs600 - 630) of each chemical containing well)/( average (Abs600 - 630) of six negative control wells) *100 = % viability
Abs600 = Foreground absorbance measured at 600 nm
Abs630 = Foreground absorbance measured at 630 nm
AbsBG = Background absorbance (of no cells blank)

Acceptance Criteria
Cinnamic aldehyde (CA, positive control) was considered positive when the gene induction by CA was above the threshold of 1.5-fold in at least one dose level and cell viability at that dose was greater than 70%.
Maximum luciferase induction (Imax) and EC 1.5 (test material concentration at which luciferase induction was greater than 1.5 fold) was calculated for CA. The assay was acceptable only if at least one of the two following criteria were fulfilled:
• Average luciferase induction in the two replicates for CA at 64 µM was between 2 and 8.
• The EC 1.5 was between 7.5 µM and 30 µM.
If only one criterion is fulfilled, the dose-response of CA was carefully checked to decide on acceptability (Natsch et al., 2011).
The average variability in the 6 solvent control wells of each of the two parallel test plate should be below 20%. If the variability was higher, the assay was deemed unreliable and the results were discarded.
The results for these acceptable criteria were reported along with the test results. Final interpretation of the assay acceptability was based on the above criteria and expert judgment.

Test material data reporting and interpretation:
For the test chemical, Imax, EC 1.5, and cell viability were calculated as described above. A chemical was reported as positive if:
• Luciferase induction (Imax) was greater than 1.5-fold and EC 1.5 is below 1000 µM.
• At EC 1.5, the cellular viability was above 70%.
• There was an apparent overall dose-response for luciferase induction.
Final interpretation of the test material results were based on the above criteria as well as expert judgment.

Results and discussion

Positive control results:

Results for positive control and the test material were evaluated relative to the criteria specified in the OECD TG 442D. In the three independent replicates, the positive control compound, cinnamic aldehyde, exhibited a dose-dependent increase in luciferase activity with EC 1.5 values of 21.93, 14.24, and 10.93 µM, respectively. The relative cell viability at EC 1.5 was greater than 70%. In replicate 1, 2, and 3, cinnamic aldehyde exhibited a maximum luciferase induction (Imax) of 2.41-, 4.32-, and 4.44-fold, relative to the vehicle control. In addition, the average variability in the solvent control wells was below the acceptable 20% in all three replicates, thereby demonstrating appropriate assay responsiveness.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: 1

Parameter:

other: EC 1.5 (micro molar)

Value:

198.32

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 2

Key result

Run / experiment:

other: 2

Parameter:

other: EC 1.5 (micro molar)

Value:

32.09

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 3

Key result

Run / experiment:

other: 3

Parameter:

other: EC 1.5 (micromolar)

Value:

348.28

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Other effects / acceptance of results:

C.I. Solvent Red 175 Solid (solvent stripped) was tested at twelve concentrations ranging from 1 to 2000 µM in three independent assay replicates.
In replicate 1, 2, and 3 C.I. Solvent Red 175 Solid (solvent stripped) exhibited a maximum luciferase induction (Imax) of 6.33-, 5.25-, and 2.08-fold, relative to the vehicle control (Figure 1A, 1B, 1C). The EC 1.5 values in replicates 1, 2, and 3 were 198.32, 32.09, and 348.28 μM, respectively. Therefore in all three replicates, C.I. Solvent Red 175 Solid (solvent stripped) induced luciferase activity above the threshold 1.5-fold at non-cytotoxic concentrations.
Therefore, based on the findings of this study, C.I. Solvent Red 175 Solid (solvent stripped) was considered positive for skin sensitization potential in the in vitro KeratinoSens assay.

Any other information on results incl. tables

C.I. Solvent Red 175 Solid (solvent stripped)interpretation criteria			Results			Interpretation
	Rep-1	Rep-2		Rep-3	Average
Imax (relative fold)	6.33	5.25		2.08	4.55
EC 1.5 (µM)	198.32	32.09		348.28	130.38	Potential sensitizer
Cell viability at EC 1.5 (%)	>70%	>70%		>70%	>70%

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The results from this study indicate that C.I. Solvent Red 175 Solid (solvent stripped) is positive in the in vitro KeratinoSens assay and therefore is predicted to have skin sensitization potential.

Executive summary:

C.I. Solvent Red 175 Solid (solvent stripped) (Dinaphtho(1,2,3-cd:1’,2’,3’-lm) perylene-9,18-dione, lauryl derivatives)was evaluated for skin sensitization potential in anin vitroKeratinoSens assay. In this study the KeratinoSens cells were exposed to a vehicle control (1% DMSO), positive control (cinnamic aldehyde) at five concentrations (4 – 64µM), andC.I. Solvent Red 175 Solid (solvent stripped)at 12 concentrations (0.98 – 2000µM). Following 48 hours of exposure, the cell viability and luciferase activity were measured in treated and control cells. The test material was considered a sensitizer if relative luciferase activity was greater than 1.5-fold (EC 1.5 at concentration < 1000µM) and cell viability at EC 1.5 was greater than 70%. The positive control treated cells exhibited luciferase induction within the designated parameters and met all requirements for a viable assay. The relative luciferase activity ofC.I. Solvent Red 175 Solid (solvent stripped)was greater than 1.5-fold at 130.38 µM and cell viability at this concentration was greater than 70%. Therefore, under the conditions of this study,C.I. Solvent Red 175 Solidis predicted to have skin sensitization potential.