Phenacetin

Endpoint summary

Description of key information

Key study (activation of keratinocytes): Key study. Test method according to the OECD 442D Guideline with GLP. Under the experimental conditions the test item may be classified as skin sensitizer using the KeratinoSensTM test method. 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 16 January 2013 to 27 January 2023

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 KeratinoSens™ test method)

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

REAGENTS AND MEDIA
Repetition 1:
-DMSO (Supplier ref. 41640, Sigma Aldrich; Batch no K0840; purity 99.9%)
-DMEM 1 g/l glucose (Supplier ref. 21885025, Fisher Bioblock; Batch no 248782)
-Non-heat inactivated foetal calf serum (Supplier ref. 10270098, Fisher Bioblock; Batch no 2337973)
-Maintenance medium: DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum, 0.05% geneticin.
-Seeding medium: DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum.
-Treatment medium: DMEM 1 g/l glucose, 1% non-heat inactivated foetal calf serum.
-Staining solution: 5 mg/ml MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) solution in PBS (phosphate buffered saline). Prepared extemporaneously and used within the day.

Repetition 2:
-DMSO (Supplier ref. 41640, Sigma Aldrich; Batch no K0840; purity 99.9%)
-DMEM 1 g/l glucose (Supplier ref. 21885025, Fisher Bioblock; Batch no 2507145)
-Non-heat inactivated foetal calf serum (Supplier ref. 11563397, Fisher Bioblock; Batch no 2337973)
-Maintenance medium: DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum, 0.05% geneticin.
-Seeding medium: DMEM 1 g/l glucose, 9.1% non-heat inactivated foetal calf serum.
-Treatment medium: DMEM 1 g/l glucose, 1% non-heat inactivated foetal calf serum.
-Staining solution: 5 mg/ml MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) solution in PBS (phosphate buffered saline). Prepared extemporaneously and used within the day.

TEST SYSTEM
-Cells: KeratinoSens™ (Givaudan)
-Culture: cells were cultured at 37ºC, 5% CO2 in maintenance medium.
-Passage number: cells were used at passage 14 in repetition 1 and passage 16 in repetition 2.

CONTROLS
-Positive control: cinnamaldehyde (CAS 104-55-2; Sigma Aldrich Ref W228613, batch no MKCJ4653; purity 98.6%)
-Negative (solvent) control: DMEM 1 g/l glucose, 1% DMSO, 1% non-heat inactivated foetal calf serum.

CELLS SEEDING.
-Culture plates: 5; 3 white cell culture plates (96 wells) for luminescence reading (induction measurement) + 2 transparent cell culture plates (96 wells) for absorbance reading (cytotoxicity).
-Cells suspension: 125 μl at 8E4 cells/ml in seeding medium were distributed in the culture plates.
-Cell density: 1E4 cells per well.
-Incubation: the seeded plates were incubated 24 hours ± 1 hour at 37ºC, 5% CO2

PREPARATION OF THE TEST ITEM AND CONTROL SUBSTANCES.
Test item stock solution: 200 mM in DMSO
Positive control stock solution: The positive control was prepared at 200 mM in DMSO then diluted to the final concentration of 6.4 mM.

1) 100 X plate: A 100-fold concentrated dilutions series was prepared in 96-well plate:
-Test item: 100 µl of DMSO were distributed from columns 1 to 11. 200 µl of the 200 mM stock solution were placed in column 12 then the series dilutions were prepared by transferring 100 µl from column 12 to column 11 and so on until the column 1. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.
-Positive control: 100 µl of DMSO were distributed in row G from columns 7 to 10. 200 µl of the 6.4 mM stock solution were placed in column 11 then the series dilutions were prepared by transferring 100 µl from column 11 to column 10 and so on until the column 7. Dilutions were mixed by repeated pipetting, at least 3 times, between each concentration.
-Negative control: 100 µl of DMSO were distributed in row G columns 1 to 6 and 12 and in the well H12.

2) 4X dilution plate: The 100 X plate was diluted 25 fold in a new plate (4 X) in treatment medium.

APPLICATION OF THE TEST ITEM AND CONTROL SUBSTANCES
In the 5 seeded plates, the medium was aspirated and replaced with 150 μl of treatment medium. Then the 4 X plate was replicated 5 times: 50 μl from the 4 X plate were placed in each of the three white plates and in the two transparent plates. The plates (1 X) were covered with an adhesive plastic foil to prevent evaporation and incubated for 48 hours ± 1 hour (37ºC, 5% CO2).

CONCENTRATIONS TESTED
-Test item: 12 concentrations according to a geometric progression of ratio 2 from 0.98 μM to 2000μM.
-Positive control: 5 concentrations according to a geometric progression of ratio 2 from 4 to 64 μM.

REPLICATES: The study was composed of two independent repetitions. For each repetition the test item and the reference items were replicated on three independent plates for the measurement of
induction and two plates for the measurement of cytotoxicity. Each repetition was performed on a different day with fresh stock solution.

LUCIFERASE ACTIVITY.
-Apparatus: Luminometer: GloMax™ (Promega)
-Validity of luminometer: a quality control analysis was performed according to the procedure described in Annex 3 of the OECD guideline 442D .
-Luciferase substrate: luciferine + ATP + lysing agent. Bright Glo™ Luciferase Assay System (Promega).
-Procedure: After 48 hours, the medium was aspirated and each well was gently washed once with 200 μl of PBS. Then 100 μl of luciferase substrate were then added in each well. The plates were incubated at least 15 minutes at room temperature to ensure cells lysis. The plates were placed in the luminometer then the luciferase activity was measured with an integration time of 2 seconds.

CITOTOXICITY ASSESSMENT.
-Apparatus: MULTISKAN EX plate reader (Thermo life sciences) - reading range 0 - 3.5 units of Absorbance -linearity range 0 - 2.200 units of Absorbance.
-Procedure: After 48 hours, the medium was aspirated and each well was gently washed once with 200 μl of PBS. Then, 225 μl of staining solution diluted at 0.6 mg/ml in treatment medium (from the
5mg/ml stock solution) were distributed in each well. The plates were covered with an adhesive plastic foil and incubated for 4 hours ± 30 minutes (37ºC, 5% CO2). After this contact time, the
staining solution was eliminated and the cells were treated with 200 μl of 10% SDS (sodium dodecylsulfate) one night in the dark (37°C, 5% CO2). After a 10 minutes homogenization, the absorbance
was measured at 540 nm.

DEVIATIONS FROM OECD GUIDELINE: No.

Negative control:

other: Treatment culture medium, 1% DMSO, 1% Non-heat inactivated foetal calf serum

Positive control:

cinnamic aldehyde [442D]

Positive control results:

Repetition 1: The maximal average induction of luciferase activity (Imax) was 4.51 at a concentration of 64 μM. The mean value EC1.5 was 10.83 μM.
Repetition 2: The maximal average induction of luciferase activity (Imax) was 3.48 at a concentration of 64 μM. The mean value EC1.5 was 11.33 μM.

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

2.99 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

1.72 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

- Visible damage on test system: No.

DEMONSTRATION OF TECHNICAL PROFICIENCY: yes, the 10 proficiency substances recommended in the OECD guideline 442D were performed, obtaining values that fall within the respective reference range for at least 8 out of the 10.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the CV of the luminescence readings for repetition 1 was 8.0% and for repetition 2 was 17.2% which are not higher than 20%.
- Acceptance criteria met for positive control: Yes, the luciferase activity induction was statistically significant above the threshold of 1.5 in at least one dose tested for each repetition. The average induction values at 64 μM were 4.51 and 3.48 in each repetition which are between 2 and 8. The E C1.5 values were 10.83 and 11.33 μM for each repetition which are within 2 SD of the historical mean of the testing facility and between 7 μM and 30 μM based on the OECD validation dataset.

Table 1. Reference items 

 

Cinnamaldeyde	4 μM	8 μM	16 μM	32 μM	64 μM	EC1.5	Imax
Rep 1	1.12	1.36	1.75	2.21	4.51	10.83	4.51
Rep 2	1.17	1.42	1.61	2.16	3.48	11.33	3.48
Mean	1.14	1.39	1.68	2.18	3.99	11.08*	3.99

*geometric mean 

Solvent control	CV % solvent control
Rep 1	8.0
Rep 2	17.2

Table 2. Test item summary results 

ID-22/09448	VIABILITY		INDUCTION
	IC50μM	IC30μM	Imax	Linear EC1.5 μM	Linear EC1.5 μM/LogμM
Rep 1	>2000	>2000	2.99	152.01	145.20
Rep 2	>2000	726.23	1.72	205.25	195.06
Mean	-	-	2.35	-	-
Geometric mean	>2000	>2000	-	176.64	168.29

Table 3. Test item mean viability percentage 

Concentration μM	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Rep 1	96.95	102.07	104.35	102.18	100.76	95.65	100.33	105.11	89.23	86.72	92.17	83.90
Rep 2	87.42	104.75	115.34	111.61	114.76	112.62	108.04	112.90	97.31	75.70	63.11	61.96
Viability	100.1	106.4	99.2	90.1	90.5	87.4	89.1	92.9	98.9	105.1	109.1	109.9

Table 4. Test item induction 

Concentration μM	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Rep 1	1.06	0.91	0.87	0.94	1.01	0.99	1.12	1.43	1.76	2.15	2.99	2.80
Rep 2	0.83	1.02	1.06	1.16	1.27	1.26	1.30	1.47	1.51	1.68	1.71	1.72
Induction	0.94	0.96	0.97	1.05	1.14	1.12	1.21	1.45	1.64	1.91	2.35	2.26
SD	0.16	0.08	0.14	0.16	0.18	0.19	0.13	0.03	0.18	0.33	0.90	0.77

 

Interpretation of results:

other: KeratinoSensTM test result is considered as part of an integrated approach to testing and ass essment (IATA) in accordance with OECD guideline 442D

Conclusions:

Under the experimental conditions the test item may be classified as potential skin sensitizer using the KeratinoSensTM test method.

Executive summary:

The KeratinoSensTM test method was performed for the test item as part of an integrated approach to testing and assessment (IATA) in accordance to OECD guideline 442D, under GLP conditions. A KeratinoSensTM cell culture was prepared, incubated for 24 hours ± 1 hour at 37ºC, 5% CO2 and distributed into 3 plates (96 wells) for the measurement of the induction of luciferase activity and 2 plates (96 wells) to assess the cytotoxicity. Test item at 12 concentrations from 0.98 μM to 2000 μM, positive control cinnamaldehyde at 5 concentrations from 4 to 64 μM and negative control 1% DMSO were placed in the seeded plates and incubated for 48 hours ± 1 hour at 37ºC, 5% CO2. 2 repetitions were performed on a different day with fresh stock solution. All validity criteria were fulfilled. Based on these findings, a positive result can be predicted for skin sensitization using the KeratinoSensTM test method.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available data, the substance is classified as sensitising according to CLP Regulation no. 1272/2008.