[(2,4-dibromophenoxy)methyl]oxirane

Endpoint summary

Description of key information

two available in vitro/in chemico studies on skin sensitising available.

sensitising potential based on activation of human keratinocytes identified.

sensitising potential based on protein binding identified

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27. January - 14. June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

The LuSens cell line was specially designed for this test system by the BASF (Ludwigshafen,
Germany). It employs the use of a reporter gene for luciferase placed under the control
of the antioxidant response element (ARE) and hence monitors Nrf-2 transcription factor
activity. For designing this cell line, a human keratinocyte cell line (provided by RWTH,
Aachen, Germany) was transfected with the pGL4.20 [luc2/Puro] vector (Promega, Germany)
carrying the regulatory antioxidant response element (ARE) upstream of the luciferase
gene (Luc2, Promega, Germany) at the Institute of Anatomy and Cell Biology of the
RWTH, Aachen (laboratory of PD Dr. Wruck).

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:
CG272
- Expiration date of the lot/batch: 11. July 2018
- Purity test date: not stated

RADIOLABELLING INFORMATION (if applicable)
not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
room temperature
- Stability under test conditions: assumed stable
- Solubility and stability of the test substance in the solvent/vehicle:
direct applicabion
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: direct application

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
none

Details on the study design:

Cell Cultures
For mycoplasma contamination screened stocks of LuSens cells are stored in liquid nitrogen
in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guarantees
similar parameters of the experiment and reproducible characteristics of the cells.
For the Cytotoxicity Range Finder Assay cells of passage 12 were used. For the experiments
cells of passage 14 and 6 respectively, were used. After thawing the cells were cultivated
in DMEM (9 % FCS) in cell culture flasks at 37 ± 1 °C in a humidified atmosphere
with 5.0 ± 0.5 % CO2.

Cytotoxicity Range Finder Test
A Cytotoxicity Range Finder Test (CRFT) was performed in order to determine the concentration
range applicable for experiment I and II. In the CRFT cytotoxicity was determined
by measuring the cell viability with MTT.
In the CRFT the following 12 nominal concentrations of the test item were tested:
2000 μM, 1000 μM, 500 μM, 250 μM, 125 μM, 62.5 μM, 31.25 μM, 15.63 μM, 7.81 μM,
3.91 μM, 1.95 μM and 0.98 μM.
The real test item concentrations are given in chapter 19. The exposure time was 48 h and
the exposure date 31. Jan. 2017.

Performance
At the time of seeding the cells were 80 % confluent. The cells were washed twice with
PBS (without Ca2+/Mg2+) containing 0.05% EDTA. Afterwards the cells were trypsinized
until the cells detached. To stop this reaction, medium no. 2 was added. After centrifugation
(5 min at 380 * g), the supernatant was discarded and the cells were resuspended in
medium no. 2. After quantification the cell suspension was adjusted to 83 000 (± 10 %)
cells per mL. 120 μL of the cell suspension (≙ 10 000 cells) were seeded in a clear flat
bottom 96 well plate. The plate was incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified
atmosphere for 24 h.
After the incubation time the medium was removed from the cells and 150 μL medium no.
3 was added to each well. Afterwards 50 μL of the single test item concentrations as well
as controls was added to the cells in triplicates (only test item concentrations). 12 wells
were used as solvent control, 6 wells were used as growth control, 3 wells were used as
negative control, 2 wells were used as positive control and 1 well was used as blank. The
plate was sealed with breathable tapes to avoid evaporation of volatile compounds and to
avoid cross contamination between wells. Afterwards the plate was incubated for 48 h at
37 ± 1 °C in a humidified atmosphere containing 5.0 ± 0.5 % CO2.
For the viability assay the MTT working solution was prepared. The cell culture medium
was removed from all wells of the 96 well plate and 200 μL MTT working solution was
added to each well. The plates were incubated for 2 h at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in
a humidified atmosphere. Afterwards the solution was removed and 100 μL MTT-lysis
buffer was added to each well. The plate was agitated for 5 min before it was measured at
570 nm and at 690 nm at the photometer.
For calculation of the relative viability (see chapter 7.3.2) a validated Microsoft Excel® file
was used.

Dose Selection for Experiment I and II
In accordance to the OECD guideline 442D and the protocol of the BASF SE, the maximum
final test item concentration should be 2000 μM. For a test chemical which has no
defined molecular weight, the final test item concentration 400 μg/mL can also be used.
Alternative concentrations may be used upon justification (e.g. in case of cytotoxicity or
poor solubility). As solvent, DMSO will be used.
In case of cytotoxic results the highest test item concentration in experiment I and II corresponds
to the first concentration indicating viability below 70 % in the CRFT.
Since a cytotoxic reaction was observed in the CRFT the following 12 nominal concentrations
were chosen for experiment I and II: 31.3 μM, 26.0 μM, 21.7 μM, 18.1 μM, 15.1 μM,
12.6 μM, 10.5 μM, 8.7 μM, 7.3 μM, 6.1 μM, 5.0 μM, 4.2 μM.
The real test item concentrations are given in chapter 19.
In the main experiments a reduction of the viability below 70 % is considered as cytotoxic
and is not allowed to be evaluated for luciferase induction.

Experimental Parameters of Experiment I and II
7.3.1 Experimental Performance
Experiment I and II were performed in the same way. Experiment II serves only to confirm
the results of experiment I. The exposure dates were 07. Feb. 2017 and 15. Feb. 2017.
At the time of seeding the cells were 80 % confluent. The cells were washed twice with
PBS (without Ca2+/Mg2+) containing 0.05% EDTA. Afterwards the cells were trypsinized
until the cells detached. To stop this reaction, medium no. 2 was added. After centrifugation
(5 min at 380 * g), the supernatant was discarded and the cells were resuspended in
medium no. 2. After quantification the cell suspension was adjusted to 83 000 (±10 %)
cells per mL. 120 μL of the cell suspension (≙ 10 000 cells) were seeded in two clear flat
bottom 96 well plates. Both plates were incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a
humidified atmosphere for 24 h in Experiment I and 24 h in Experiment II.
The performance of the Viability test was the same as described in chapter 7.1.1 except
the concentrations of the test item.
For the performance of the luciferase induction the second plate was used. After the incubation
time the medium was removed from the cells and 150 μL medium no. 3 was added
to each well. Afterwards 50 μL of the single test item concentrations as well as controls
were added to the cells in triplicates (only for test item concentrations). 12 wells were used
as growth control. 24 wells were used as solvent control, 6 wells were used as negative
control, 5 wells were used as positive control and 1 well was used as blank. The arrangement
of the substances on the 96 well plate is demonstrated in figures 15-a and 16-a. The
plate was sealed with breathable tape to avoid evaporation of volatile compounds and to
avoid cross contamination between wells. Afterwards the plate was incubated for 48 h at
37 ± 1 °C in a humidified atmosphere containing 5.0 ± 0.5 % CO2.
For the evaluation of the luciferase expression the medium was removed from the wells
and the cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Afterwards 100 μL per
well of a Glo Lysis buffer were given to the cells and incubated for 5 min at room temperature.
During this process the plate was slightly moved. The Steady-Glo® Reagent was prepared
by mixing Steady-Glo®-Substrate with Steady-Glo®-Buffer. After lysis 100 μL Steady-
Glo® Reagent were added to each well and the plate was shaken slowly for 5 min at room
temperature. Then, 170 μL per well were transferred to a white flat bottom 96 well plate
and the luminescence was measured for 2 seconds using a luminometer.
For calculation of the luciferase induction as well as the relative viability (see chapter
7.3.2) a validated Microsoft Excel® file was used.
7.3.2 Data Evaluation
For calculation of results a validated Microsoft Excel® file was used.
The calculation of the relative Viability [%] was performed as follows:
All measured wells were corrected = OD570 Value - OD690 value
For the following calculation only the corrected values are used.
value of well of interest - blank
mean solvent control - blank
Afterwards the mean value of the single replicates was calculated.
Luciferase fold induction:
Fold induction =
(RLU value from measurement - RLU value of blank (only medium without cells)) /
(Mean RLU value solvent - RLU value of blank (only medium without cells))
RLU = Relative Light Unit
Mean fold induction (rounded to one decimal place) =
(Fold induction of replicate 1 + Fold induction of replicate 2 + Fold induction of replicate 3) /3

Positive control results:

positive control clearly induced Luciferase activity. Details see Table section

Key result

Run / experiment:

other: I

Parameter:

other: Induction of Luciferase (expressed as fold in comparision to solvent control)

Value:

1.2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: II

Parameter:

other: Induction of Luciferase (expressed as fold in comparision to solvent control)

Value:

1.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

ResultsofexperimentI

 

		InductionofLuciferase			ViabilityoftheCells
	Conc.	Induction	StandardDeviation	StandardDeviation	RelativeViability	StandardDeviation	StandardDeviation
	[µM]	fold		[%]	[%]		[%]
SolventControl	-	1.0	0.18	17.77	100.0	6.62	6.62
GrowthControl	-	1.1	0.14	13.02	144.8	6.39	4.41
NegativeControl	5000	1.1	0.16	15.07	110.5	7.56	6.84
PositiveControl	120	3.9	0.35	9.07	94.9	8.18	8.62
Testitem	4.2	1.8	0.05	2.84	107.5	3.37	3.13
Testitem	5.0	1.2	0.09	7.66	111.5	6.15	5.52
Testitem	6.1	1.4	0.14	10.00	109.9	3.70	3.36
Testitem	7.3	1.4	0.21	14.36	110.8	4.61	4.16
Testitem	8.7	1.6	0.06	3.78	116.0	5.43	4.68
Testitem	10.5	1.7	0.11	6.15	118.0	3.29	2.79
Testitem	12.6	2.1	0.06	2.76	109.8	3.66	3.33
Testitem	15.1	2.1	0.07	3.09	123.2	8.48	6.89
Testitem	18.1	2.2	0.05	2.12	108.5	5.82	5.37
Testitem	21.7	2.0	0.16	8.22	105.5	25.17	23.86
Testitem	26.0	1.7	0.07	4.28	78.2	10.45	13.36
Testitem	31.3	1.7*	0.12	6.88	47.4	18.94	39.97

 

ResultsofexperimentII

 

		InductionofLuciferase			ViabilityoftheCells
	Conc.	Induction	StandardDeviation	StandardDeviation	RelativeViability	StandardDeviation	StandardDeviation
	[µM]	fold		[%]	[%]		[%]
SolventControl	-	1.0	0.07	6.72	100	4.04	4.04
GrowthControl	-	1.0	0.09	9.27	129.5	3.30	2.55
NegativeControl	5000	1.0	0.03	3.38	109.6	3.77	3.44
PositiveControl	120	6.6	0.14	2.07	98.7	1.20	1.22
Testitem	4.2	1.4	0.07	5.00	116.5	0.82	0.71
Testitem	5.0	1.3	0.08	6.45	107.1	2.93	2.73
Testitem	6.1	1.4	0.07	4.78	108.7	3.72	3.42
Testitem	7.3	1.6	0.04	2.33	104.9	4.46	4.25
Testitem	8.7	1.7	0.03	1.56	104.7	5.25	5.02
Testitem	10.5	1.9	0.05	2.91	108.0	2.36	2.18
Testitem	12.6	1.9	0.08	4.18	109.2	3.60	3.30
Testitem	15.1	2.4	0.13	5.33	107.7	3.01	2.79
Testitem	18.1	2.6	0.21	7.96	106.7	4.11	3.85
Testitem	21.7	2.8	0.24	8.58	102.5	1.42	1.38
Testitem	26.0	3.1	0.34	10.88	86.9	9.27	10.67
Testitem	31.3	2.4*	0.43*	17.88*	54.2	9.05	16.71

* = Due tocytotoxicity values willnot beusedforthefinal evaluation

 

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, it can be stated that in this study and under the experimental conditions reported,
the test item DENACOL EX-147 possesses a sensitizing potential.

Executive summary:

This in vitro study was performed to investigate the sensitizing potential of DENACOL EX-

147, by using the LuSens cell line.

The assay was performed in a cytotoxicity range finder test (CRFT) and two independent

experiments (experiment I and II) with a treatment period of 48 h. The CRFT was performed

to detect a potential cytotoxic effect of the test item. Based on the results of this

test the concentrations for the two experiments were determined.

In the experiments, the highest nominal applied concentration (31.25 μM) was chosen with

regard to the cytotoxic reaction in the CRFT. Furthermore, a geometric series (factor 1.2)

of eleven dilutions was prepared. Precipitation of the test item was not visible in all experimental

parts.

DMSO (final concentration: 1 %) was used as solvent control and culture medium as medium

control. Furthermore, Lactic acid (5000 μM) was used as negative control and EGDMA

(120 μM) as positive control.

The evaluated experimental points and the results are summarised in chapter 8, page 18ff.

A substantial and reproducible dose dependent increase in luciferase induction above 1.5

fold in more than two non-cytotoxic test item concentrations was observed in both experiments.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05. September 2017 - 17. July 2018 (draft report

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))

GLP compliance:

yes (incl. QA statement)

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 No.of test material:
CG272
- Expiration date of the lot/batch: 11. July 2018
- Purity test date: not stated

RADIOLABELLING INFORMATION (if applicable)
not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
room temperature
- Stability under test conditions: assumed stable
- Solubility and stability of the test substance in the solvent/vehicle:
direct applicabion
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: direct application

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
none

Details on the study design:

Peptides with ≥ 95 % purity, synthesized by Genecust, Dudelange, Luxemburg, were used.
Cys-Peptide (Cysteine)
Sequence: Ac-RFAACAA-COOH (MW = 750.9 g/mol)
Batch no.:P170415-2-LR569638
Purity: 98.10%
Lys-Peptide (Lysine)
Sequence: Ac-RFAAKAA-COOH (MW = 775.9 g/mol)
Batch no.: P170415-2-LR569640
Purity: 98.85%

Positive control
Positive controls were treated identically as the test item. The following positive controls were used:
-Cinnamaldehyde (CAS 104-55-2, food grade 99.1 %, batch no. MKBT8955V) was used as 100 mM solution in acetonitrile for the Cys-peptide.
-2,3-Butanedione (CAS 431-03-8, 99.4 %, batch no. BCBS3560V) was used as 100 mM solution in acetonitrile for the Lys-peptide
As cinnamaldehyde mixed with the lysine peptide turned turbid in all experiments per-formed during the implementation phase, it was considered unsuitable as positive control. Instead, the proficiency chemical 2,3-Butanedione is used as positive control showing mid-range depletion for the lysine peptide.

Solvent controls
For both peptides, four sets of solvent controls using acetonitrile instead of test item stock solution were prepared in triplicate (sets A, B1, B2 and C, total 12 samples per peptide). Set A was analysed together with the peptide calibration standards, sets B1 and B2 were analysed at the start and end of the analysis sequence and were used as stability control for the peptide over the total analysis time. Set C was incubated and analysed together with the samples and was used for calculation of the peptide depletion of positive controls.
For the repeated experiment with the Cys-peptide the calibration standards had to be moved to the end of the sequence to ensure timely analysis of the incubated samples.

Co-elution control
Sample prepared from the respective peptide buffer and the test item, but without peptide.

Incubation
The positive control, solvent control sets C, and test item samples were incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 ± 2.5 °C for 22.2 h (Lys-peptide) and 22.1 h and 25.2 h (Cys-peptide).
All three replicates of positive control and test item from incubation 09. Oct. 2017 to 10. Oct. 2017 were turbid after incubation.

Measurements were performed using a HPLC method.

Positive control results:

Mean Peptide depletion for Lys with 2,3-Butanedione: 31.06 %, SD 1.74 %
Mean Peptide depletion for Cys with Cinnamaldehyde: 100 %, SD 0%

Key result

Run / experiment:

other: mean of three repeats

Parameter:

other: Lys-Peptide depletion (%)

Value:

13.56

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: mean of three repeats

Parameter:

other: Cys-Peptide depletion (%)

Value:

82.44

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: overall

Parameter:

other: mean of peptide depletion (%)

Remarks:

both peptides

Value:

48

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:

The test performed two times for each peptide. The determination was not evaluable for one experiment:
- Lys-peptide assay performed on 19. Sep. 2017 (no reference controls were pre-pared, no evaluation possible)
The results of this experiments are not reported but saved together with all other raw data of the study in the test facility’s GLP document archive.
The Lys-peptide assay was therefore prepared again.

The Cys-peptide experiment was performed twice, erroneously. The first experiment was valid and outside the depletion range where verification is necessary, therefore the second experiment (batch 20171009) was not necessary.

Acceptance criteria
a) The mean peptide depletion value for the positive control cinnamaldehyde should be 60.8 % - 100 % with a maximum standard deviation (SD) of < 14.9 % for the Cys-peptide.
b) The mean peptide depletion value for the positive control 2,3-butanedione should be 10 % - 45 % with a maximum standard deviation < 11.6 % for the Lys-peptide.
c) The maximum standard deviation for the test item replicates should be < 14.9 % for the percent cysteine depletion and < 11.6 % for the percent lysine depletion
8.3.4 Assessment
a) The mean peptide depletion and standard deviation of the three replicates of the positive control cinnamaldehyde were in the acceptable range of 60.8 – 100.0 % and ≤ 14.9 %, respectively, for the Cys-peptide.
b) The mean peptide depletion and standard deviation of the three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0 % and ≤ 11.6 %, respectively, for the Lys-peptide.
c) The maximum standard deviation for the test item replicates was < 14.9 % for the percent cysteine depletion for the test item.
The maximum standard deviation for the test item replicates was < 11.6 % for the percent lysine depletion for the test item.

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the described test conditions, Denacol EX-147 is “positive” with high reactivity according to the DPRA predicition.

Executive summary:

The study was performed in order to evaluate the reactivity of the test item DENACOL EX-147towards cysteine (Cys-) and lysine (Lys-) containing peptides. A test item solution in acetonitrile was incubated 24 ± 2 h at 25 °C together with cysteine and lysine peptides, respectively, and the peptide concentration after the incubation was measured using HPLC-UV.

Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured in parallel.

The peptide depletion values after incubation are shown in below table:

	Cys-peptide depletion [%]	Lys-Peptide depletion [%]	Mean peptide depletion [%]
First experiment	82.44	13.56	48.00
Verification*	86.90

* An error occurred in the evaluation of the first experiment with the Cys peptide, therefore the experiment was repeated even though it was valid. Verification was not necessary, which was noticed after its performance.

 

The DPRA predicition is “positive” with high reactivity.

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

For skin sensitisation the available information is conclusive and sufficient for classification as skin sensitisation, category 1.