4'-bromo-3-chloropropiophenone

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Mar 05 - Jul 13, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Remarks:

Preliminary study to determine the solubility of the test item

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Behörde für Gesundheit und Verbraucherschutz, Freie Handelsstadt Hamburg

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

4'-bromo-3-chloropropiophenone

EC Number:

250-784-5

EC Name:

4'-bromo-3-chloropropiophenone

Cas Number:

31736-73-9

Molecular formula:

C9H8BrClO

IUPAC Name:

1-(4-bromophenyl)-3-chloropropan-1-one

Test material form:

solid: bulk

In vitro test system

Details on the study design:

Test System and Origin
The test was carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures were seeded in culture medium at an appropriate density and was used for routine testing. Cells were cultured in T175 culture flasks (Greiner) in maintenance medium at 37  1°C and 5% CO2 in a humidified incubator.

Rationale for Selection of the Test System
The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitisers was reported by several studies and represents the second key event of the skin sensitisation process as described by the AOP. The ARE-Nrf2 luciferase test method makes use of an immortalised adherent cell line (e.g. KeratinoSens™) derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be upregulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances. Therefore, this test method is considered to be able to detect chemicals that cause skin sensitisation when used within Integrated Approaches to Testing and Assessment (IATA), together with other relevant complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP as well as non-testing methods, including read-across from chemical analogues. Depending on the regulatory framework, positive results generated with these methods may be used on their own to classify a chemical into UN GHS Category 1.

Culture Procedure
Cells were maintained in maintenance medium at 37°C in the presence of 5% CO2 in T175 cell culture flasks. 80 – 90% confluent cells were washed twice with 10 mL DPBS (Cat.-No.: 14190; Supplier: gibco, Life Technologies Limited, UK) containing 0.05% EDTA (Product-No.: E6758; Supplier: Sigma-Aldrich, USA), then 2 mL Trypsin-EDTA per plate (Cat.-No.: 25300; Supplier: gibco, Life Technologies Limited, UK) was added and plates were put back into the 37°C incubator. After cells had detached (usually after 5 – 10 min), they were resuspended in 10 mL maintenance medium and split at a ratio of 1:3 – 1:12 in fresh maintenance medium and grown to 80 – 90% confluency.

Preparation of the Test Items
The item solution was freshly prepared immediately prior to use.
The test item was dissolved in dimethyl sulfoxide (DMSO). A stock solution of 200 mM was prepared by pre-weighing the test item into 4 mL glass vessels.
Based on the stock solution a set of twelve master solutions in 100% DMSO were prepared. The stock solution (200 mM) of the test items were diluted eleven times using a constant dilution factor of 1:2 (100 µL stock solution + 100 µL DMSO). Then the 100x concentrated master solutions were further diluted 1:25 in test item exposure medium (10 µL + 240 µL test item exposure medium) resulting in a 4% share of the solvent.
These 4-fold concentrated test item solutions were finally diluted 1:4 when incubated with the cells (50 µL + 150 µL test item exposure medium). This resulted in the following final test item concentrations used in the assay:
0.98, 1.95, 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM
Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.

Positive Control
Designation: trans-Cinnamaldehyde >= 99%
Synonym: Cinnamic aldehyde (CA)
Supplier: Sigma-Aldrich, USA
Product No.: 239968
CAS-No.: 14371-10-9
Batch: STBJ1063
Molecular Weight: 132.16 g/mol
Expiration Date: No data available
Storage: 2 – 8°C

Cinnamic aldehyde was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test items, resulting in a final concentration range of 4 µM – 64 µM. The final concentration of DMSO was 1% (v/v) for all wells.

Negative Control
Designation: DMSO
Synonym: Dimethyl sulfoxide
Supplier: Sigma-Aldrich, USA
Product-No.: 276855
CAS-No.: 67-68-5
Lot: STBH0404
Purity (GC): > 99.99%
Expiration Date: No data available
Storage: Tightly closed, dark at room temperature (15 to 25°C)
DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control.
Six wells filled with the negative control were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.


Experimental Procedure
The incubation was performed in 96-well plates.
A cell suspension of 8 × 104 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 × 104 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability.
After seeding, cells were grown for 24h ± 1h in assay medium at 37°C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item.
All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48h ± 1h at 37°C ± 1 °C and 5% CO2.
Luciferase Activity
After 48h ± 1h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS (250 µL per well). Subsequently, 20 µL of passive lysis buffer was added into each well and the plate was incubated for 20 min at room temperature in the absence of light.
Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate was injected by the injector of the plate reader. The plate reader waited for 1s before assessing the luciferase activity for 2s. This procedure was repeated for each individual well.
Cell Viability
2.7 mL of a MTT solution (5 mg/mL in DPBS) was added to 20 mL test item exposure medium. For the cell viability assay plate, the medium was replaced with 200 µL of this fresh medium containing MTT. The plate was covered with a sealing tape and incubated for 4h at 37°C ± 1°C and 5% CO2. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37°C ± 1°C and 5% CO2 overnight. After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

The overall IC50 and IC30 were calculated as the geometric mean of the individual repetitions.
For every concentration showing >1.5-fold luciferase activity induction, statistical significance (p<0.05) was calculated using a two-tailed Student’s t-test comparing the luminescence values for the three replicated samples with the luminescence values in the solvent (negative) control wells.
The lowest concentration with >1.5-fold luciferase activity induction was the value determining the EC1.5 value. It was checked in each case whether this value was below the IC30 value, indicating that there is less than 30% reduction on cellular viability at the EC1.5 determining concentration. In addition, at least two consecutive concentrations should have >70% viability, otherwise the concentration range should have been adjusted.


Prediction Model
A KeratinoSens™ prediction was considered positive if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5-fold increased and statistically significant (p<0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity > 1.5-fold (i.e. at the EC1.5 determining concentration)
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction
If in a given repetition, all of the three first conditions are met but a clear dose-dependent increase in luciferase induction cannot be observed, then the result of that repetition should be considered inconclusive and further testing may be required. In addition, a negative result obtained with test chemicals tested at a maximal test concentration <1000 μM (or 200 μg/mL for test chemicals with no defined MW) and which do not reach cytotoxicity (<70% viability) at the maximal tested concentration should also be considered as inconclusive. A negative result for test items with a log KOW >7 should be interpreted with care due to the applicability of the test method.


Acceptance Criteria
The test met acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean (1.67 μM – 32.08 μM based on the historical data of the testing laboratory)
- the average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control is <20% in each repetition which is consisting of 6 wells.

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

4.64

Cell viability:

103.35%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

EC 1.5 [442D]

Value:

6.32

Cell viability:

92.88%

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:

In the first run, a max luciferase activity induction (Imax) of 6.03 was determined. The lowest tested concentration with a luciferase induction >1.5 (2.84) was found to be
7.8 µM, the corresponding cell viability was >70% (103.35%). The calculated EC1.5 value was 4.64 µM. A dose response for luciferase activity induction was observed.
In the second run, a max luciferase activity induction (Imax) of 9.17 was determined. The lowest tested concentration with a luciferase induction >1.5 (3.08) was found to be
15.6 µM, the corresponding cell viability was >70% (88.87%). No EC1.5 could be calculated because no clear dose response for luciferase activity induction was observed.
Because in run 2 no clear dose-dependent increase in luciferase was observed, the result was considered inconclusive and, therefore, a third run was performed.
In the third run, a max luciferase activity induction (Imax) of 4.74 was determined. The lowest tested concentration with a luciferase induction >1.5 (1.71) was found to be
7.8 µM, the corresponding cell viability was >70% (92.88%). The calculated EC1.5 value was 6.32 µM. A dose response for luciferase activity induction was observed.

Any other information on results incl. tables

Prediction Criterium	Run 1	Run 2	Run 3	Positive Sensitiser Criterium in at least two independently prepared test repetitions
Imaxis >1.5-fold increased and statistically significant (p<0.05) compared to the negative control	6.03	9.17	4.74	pass
Cell viability is >70% at the lowest concentration with an induction of luciferase activity > 1.5-fold	103.35%	88.87%	92.88%	pass
EC1.5value is <1000 µM	4.64 µM	n.d.*	6.32 µM	pass
Apparent overall dose-response for luciferase induction	yes	no	yes	pass

Applicant's summary and conclusion

Interpretation of results:

other: The result should be considered in the context of integrated approach such as IATA.

Conclusions:

In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered positive in the KeratinoSensTM assay. The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

Objective

The objective of the present study was to investigate the skin sensitising potential of the test item by assessment of its potential to induce the Keap1-Nrf2-ARE signaling pathway by quantifying the luciferase gene expression using luminescence detection.

Study Design

The KeratinoSens™ test method makes use of an immortalised adherent cell line derived from human keratinocytes stably harbouring a luciferase reporter gene under the control of the antioxidant response element of the human AKR1C2 gene. This gene is known to be upregulated by skin sensitisers. In the present study the test item was dissolved in DMSO. Based on a molecular weight of 247.52 g/mol a stock solution of 200 mM was prepared. The cells were treated with different concentrations (0.98, 1.95, 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, 500, 1000 and 2000 μM) of the test item and the luciferase gene induction was measured using light producing luciferase substrate. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from cells.

Test chemicals are considered positive in the KeratinoSens™ test method if they induce a statistically significant induction of the luciferase activity above a given threshold (≥1.5 fold), below a defined concentration which does not significantly affect cell viability (< 1000 μM) and at a concentration at which the cellular viability is above 70%. For this purpose, the maximal fold induction of the luciferase activity over solvent (negative) control (Imax) was determined. Furthermore, since cells were exposed to series of concentrations of the test chemical, the concentration needed for a statistically significant induction of luciferase activity above the threshold (EC1.5 value) was interpolated from the dose-response curve obtained from the series of tested concentrations of the test chemical. Finally, parallel cytotoxicity measurements were conducted to assess whether luciferase induction occurs at sub-cytotoxic concentrations.

Results

In the first run, a max luciferase activity induction (Imax) of 6.03 was determined. The lowest tested concentration with a luciferase induction >1.5 (2.84) was found to be

7.8 µM, the corresponding cell viability was >70% (103.35%). The calculated EC1.5 value was 4.64 µM. A dose response for luciferase activity induction was observed.

In the second run, a max luciferase activity induction (Imax) of 9.17 was determined. The lowest tested concentration with a luciferase induction >1.5 (3.08) was found to be

15.6 µM, the corresponding cell viability was >70% (88.87%). No EC1.5 could be calculated because no clear dose response for luciferase activity induction was observed.

Because in run 2 no clear dose-dependent increase in luciferase was observed, the result was considered inconclusive and, therefore, a third run was performed.

In the third run, a max luciferase activity induction (Imax) of 4.74 was determined. The lowest tested concentration with a luciferase induction >1.5 (1.71) was found to be

7.8 µM, the corresponding cell viability was >70% (92.88%). The calculated EC1.5 value was 6.32 µM. A dose response for luciferase activity induction was observed.

In 2 out of 3 runs all conditions for a positive result were clearly met and, thus, under the condition of this study the test item is considered positive in this skin sensitisation test.

 

Conclusions

In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered positive in the KeratinoSensTM assay. The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.