IPEMA

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-06-23 to 2021-10-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

2-Propenoic acid, 2-methyl-, 3-methyl-3-buten-1-yl ester

Cas Number:

156291-88-2

Molecular formula:

C9H14O2

IUPAC Name:

2-Propenoic acid, 2-methyl-, 3-methyl-3-buten-1-yl ester

additive 1

Reference substance name:

Mequinol

EC Number:

205-769-8

EC Name:

Mequinol

Cas Number:

150-76-5

IUPAC Name:

(Polymerization inhibitor) 4-Methoxyphenol

In vitro test system

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

see under "Any other information on materials and methods incl. tables"

Vehicle / solvent control:

DMSO

Negative control:

other: DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control.

Positive control:

cinnamic aldehyde [442D]

Results and discussion

Positive control results:

Refer to the experiment 1-3 results in the pdf attachment.

In vitro / in chemico

Results

Results 1

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.66

At concentration:

500 other: µM

Cell viability:

149.8%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The corresponding cell viability determined by MTT staining was 149.8%.

Remarks:

Microscopically, a clear cytotoxic effect was observed at the three highest test item concentrations (500, 1000 and 2000 µM) and a slight cytotoxic effect at a test item concentration of 250 µM. The increase in % viability may be caused by a slight MTT reducing potential of the test item, as determined in a MTT interference test. Therefore, the slight induction above the threshold of 1.5 may be caused by the cytotoxic effect.

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

Imax [442D]

Value:

2.61

At concentration:

756.14 other: µM

Cell viability:

87.6 %.

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 3

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

1.41

At concentration:

500 other: µM

Cell viability:

87.4%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range.

Remarks:

Therefore, no EC1.5 value could be calculated. To verify the results and as the test item induced the gene activity very close to the cytotoxic level in experiment 1, a third experiment with an adapted concentration range and a narrower dose-response analysis with dilution of 1.15-fold was performed to decide if induction is at cytotoxic levels or not.

Results 4

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

289.06 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The corresponding cell viability determined by MTT staining was 149.8%.

Remarks:

Microscopically, a clear cytotoxic effect was observed at the three highest test item concentrations (500, 1000 and 2000 µM) and a slight cytotoxic effect at a test item concentration of 250 µM. The increase in % viability may be caused by a slight MTT reducing potential of the test item, as determined in a MTT interference test. Therefore, the slight induction above the threshold of 1.5 may be caused by the cytotoxic effect.

Results 5

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

EC 1.5 [442D]

Value:

313.33 µM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The lowest tested concentration with a significant luciferase induction >1.5 (1.55) was found to be 326.90 µM. The corresponding cell viability was >70% (97.9%). The calculated EC1.5 was <1000 µM (313.33 µM).

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: see table below

Any other information on results incl. tables

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

In the present study 1,3-di(3-Methyl-2-butenoxy)-2-hydroxypropane was dissolved in DMSO.

Based on a molecular weight of 228.33 g/mol a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, a max luciferase activity (I_max) induction of 2.91 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 93.9%. The lowest tested concentration with a significant luciferase induction >1.5 (1.80) was found to be 500 µM. The corresponding cell viability was >70% (121.0%). The calculated EC_1.5 was <1000 µM (256.25 µM).

In the second experiment, a max luciferase activity (I_max) induction of 5.55 was determined at a test item concentration of 2000 µM. The corresponding cell viability was 64.7%. The lowest tested concentration with a significant luciferase induction >1.5 (1.86) was found to be 500 µM. The corresponding cell viability was >70% (97.2%). The calculated EC_1.5 was <1000 µM (253.54 µM).

A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as sensitiser.

The controls confirmed the validity of the study (see Table 6).

 

Luciferase Activity - Overall Induction

Table 5: Induction of Luciferase Activity – Overall Induction

Conc.	Experiment 1			Experiment 2			Conc.	Experiment 3
[µM]	mean	sd	Sign.	mean	sd	Sign.	[µM]	mean	sd	Sign.
0.98	1.22	0.12		1.13	0.32		214.94	1.44	0.07
1.95	1.30	0.10		1.01	0.09		247.18	1.38	0.18
3.91	1.22	0.13		0.98	0.08		284.26	1.39	0.09
7.81	1.34	0.19		1.05	0.11		326.90	1.55	0.27	*
15.63	1.24	0.09		1.10	0.13		375.94	1.54	0.22	*
31.25	1.27	0.11		1.15	0.18		432.33	1.75	0.21	*
62.50	1.31	0.01		1.24	0.10		497.18	1.58	0.21	*
125.00	1.38	0.08		1.19	0.15		571.75	1.88	0.25	*
250.00	1.47	0.34		1.37	0.38		657.52	1.96	0.56	*
500.00	1.66	0.05	*	1.41	0.11		756.14	2.61	0.51	*
1000.0	0.91	0.35		0.74	0.07		869.57	1.91	1.42
2000.0	0.02	0.01		0.52	0.80		1000.0	0.74	0.34

* = significant induction according to Student’s t-test, p<0.05; grey marked: viability >70%; sign.= significant

 

Additional Parameters

Table 6: Additional Parameters

Parameter	Experiment 1	Experiment 2	Experiment 3	Mean	SD
EC1.5	289.06	-	313.33	301.19	17.17
Imax	1.66	1.41	2.61	1.89	0.63
IC30	775.28	736.75	786.88	766.30	26.24
IC50	844.29	1086.52	821.81	917.54	146.77

n.a.=not applicable; [value] excluded due to cytotoxic effects (see discussion)

Acceptance Criteria

Table 7: Acceptance Criteria

Criterion	Range	Exp. 1	pass/fail	Exp. 2	pass/fail	Exp. 3	pass/fail
CV Solvent Control [%]	< 20%	15.4	pass	9.6	pass	13.5	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	= 1	2.0	pass	2.0	pass	3.0	pass
EC1.5 PC [µM]	± 2 x SD of historical mean	20.57	pass	16.63	pass	15.16	pass
Induction PC at 64 µM	2 .00 < x < 8.00	2.05	pass	6.65	pass	5.27	pass

 

Historical Data

Table 8: Historical Data

Acceptance Criterion	Range	Mean	SD	N
CV Solvent Control	< 20%	11.6	3.4	186
No. of positive control concentration steps with significant luciferase activity induction >1.5	= 1	2.3	0.6	186
EC1.5 PC	7 < x < 34 µM	19.1	5.9	186
Induction PC at 64 µM	2.00 < x < 8.00	3.8	1.4	186

 

 

 

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study under the given conditions the test item induced the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item might be considered as sensitiser.
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:

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and
non-sensitisers.

In the first experiment, a max luciferase activity (I_max) induction of 1.66 was determined at a test item concentration of 500 µM. The corresponding cell viability determined by MTT staining was 149.8%. Microscopically, a clear cytotoxic effect was observed at the three highest test item concentrations (500, 1000 and 2000 µM) and a slight cytotoxic effect at a test item concentration of 250 µM. The increase in % viability may be caused by a slight MTT reducing potential of the test item, as determined in a MTT interference test. Therefore, the slight induction above the threshold of 1.5 may be caused by the cytotoxic effect.

In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC_1.5 value could be calculated.

To verify the results and as the test item induced the gene activity very close to the cytotoxic level in experiment 1, a third experiment with an adapted concentration range and a narrower dose-response analysis with dilution of 1.15-fold was performed to decide if induction is at cytotoxic levels or not.

In the third experiment, a max luciferase activity (I_max) induction of 2.61 was determined at a test item concentration of 756.14 µM. The corresponding cell viability was 87.6 %. The lowest tested concentration with a significant luciferase induction >1.5 (1.55) was found to be 326.90 µM. The corresponding cell viability was >70% (97.9%). The calculated EC_1.5 was <1000 µM (313.33 µM).

A dose response for luciferase activity induction was observed for experiment 1 and experiment 3.

Under the condition of this study the test item is therefore considered positive.