reaction mass of diethyl (E)-2-methylbut-2-enedioate and diethyl (Z)-2-methylbut-2-enedioate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

15 September 2016 - 06 December 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)

Version / remarks:

2016

Deviations:

yes

Remarks:

The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry.

Principles of method if other than guideline:

In addition, the h-CLAT was performed according to the methods described in the following publications:
- Nukada Y, Ashikaga T, Miyazawa M, Hirota M, Sakaguchi H, Sasa H, Nishiyama N. (2012). Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying skin sensitization potency. Toxicol In Vitro. 2012 Oct;26(7):1150-60.
- Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa M, Ito Y, Nishiyama N, Itagaki H. (2010). A comparative evaluation of in vitro skin sensitization tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern Lab Anim. 2010 Aug;38(4):275-84.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

diethyl 2-methylidenebutanedioate

EC Number:

607-321-0

Cas Number:

2409-52-1

Molecular formula:

C9H14O4

IUPAC Name:

diethyl 2-methylidenebutanedioate

In vitro test system

Details on the study design:

Skin sensitisation (In vitro test system)
Cell line used: THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202.

Technical material and conditions:
- Culture medium: RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine and appropriate antibiotics (100 µg/mL of penicillin and 100 μg/mL of streptomycin).
- FACS buffer: PBS with 0.1% (w/v) BSA
- Antibodies: Anti - CD86 antibody (Clone: Fun-1), Anti – CD54 antibody (Clone: 6.5B5),
FITC labelled-mouse IgG1 (isotype control)

Controls used:
- Vehicle control: DMSO (final concentration 0.2%)
- Positive control: DNCB in DMSO (diluted with culture medium to a final concentration of 2 and 3 μg/mL DNCB)
- Isotype control: mouse IgG1.

Test procedure:

Dose Finding Assay, XTT Test:
Two independent cytotoxicity experiments were performed on different days to obtain a reliable CV75. The mean of two CV75 values was used to determine the dose-range for the main experiment (h-CLAT). All dose groups (eight concentrations) were tested in 7 replicates for each XTT test. The incubation period was 24 ± 0.5 hours. After incubation with the XTT labelling mixture the absorbance was measured at 450 nm.

Human Cell Line Activation Test (h-CLAT):
Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control). The expression of cell surface antigens (CD54, CD86) was analyzed by flow cytometry.

Acceptance criteria:
- Cell viability of medium control is adjusted to 100% and the cell viability of the DMSO control should be more than 90% in comparison to the medium control.
- In the positive control (DNCB), RFI values of both CD86 and CD54 should exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%.
- In the DMSO solvent control, RFI values compared to the medium control of both
CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
- For medium and DMSO controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
- For the test item resulting in negative outcome, the cell viability at the 1.2 × CV75 should be less than 90%. (If the cell viability at the 1.2 × CV75 is more than 90% for a positive tested test item, the data will be acceptable. If 5 mg/mL in saline, 1 mg/mL in DMSO or the highest soluble dose will be used as the maximal test concentration instead of CV75-based dose, the data for test item are accepted independent by the cell viability.)
- The cell viability of at least 4 doses in each experiment should be ≥50%.

Evaluation of results:
The test item is tested in 2 independent runs. The test item is considered to be a sensitizer
- if the RFI of CD86 is ≥ 150% in both independent run data or
- if the RFI of CD54 is ≥ 200% in both independent run data.
Otherwise it is considered to be a non-sensitiser.
In case of different results in both runs, a third run has to be performed. The test item is considered to be a sensitizer:
- if the RFI of CD86 is ≥ 150% at any dose in at least 2 of 3 independent run data or
- if the RFI of CD54 is ≥ 200% in at least 2 of 3 independent run data.
Otherwise it is considered to be a non-sensitiser.

Results and discussion

Positive control results:

The positive controls exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: First run - concentrations: 274, 328, 394, 473, 567 and 681 µg/mL

Parameter:

other: RFI (%) CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 2

Run / experiment:

other: First run - concentration: 817 µg/mL

Parameter:

other: RFI (%) CD54

Value:

167.1

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: excluded from the evaluation, due to observed precipitations

Results 3

Run / experiment:

other: First run - concentration: 980 µg/mL

Parameter:

other: RFI (%) CD54

Value:

295.1

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: excluded from the evaluation, due to observed precipitations

Results 4

Key result

Run / experiment:

other: Second run - concentrations: 274, 328, 394, 473, 567 and 681 µg/mL

Parameter:

other: RFI (%) CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 5

Run / experiment:

other: Second run - concentration: 817 µg/mL

Parameter:

other: RFI (%) CD86

Value:

258.6

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: excluded from the evaluation, due to observed precipitations

Results 6

Run / experiment:

other: Second run - concentration: 980 µg/mL

Parameter:

other: RFI (%) CD86

Value:

1 192.8

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: excluded from the evaluation, due to observed precipitations

Any other information on results incl. tables

Table 1: Results of the first h-CLAT run for the Test Item

	Concentration (µg/mL)	Antibody	RFI (%)	Cell Viability (%)
Medium Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
DMSO Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
Positive Control (DNCB)	2.0	CD 54	470.9*	63.9
		CD 86	371.7*
	3.0	CD 54	570.9*	67.9
		CD 86	457.2*
Test Item	274	CD 54	54.9	90.5
		CD 86	56.6
	328	CD 54	87.8	96.4
		CD 86	60.5
	394	CD 54	93.9	93.5
		CD 86	88.8
	473	CD 54	95.1	86.0
		CD 86	100.0
	567	CD 54	97.6	85.5
		CD 86	94.7
	681	CD 54	129.3	83.3
		CD 86	125.0
	817^P	CD 54	167.1	61.9
		CD 86	258.6*
	980^P	CD 54	295.1*	22.6
		CD 86	1192.8*

P: Precipitation, *RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

Table 2: Results of the second h-CLAT run for the Test Item

	Concentration (µg/mL)	Antibody	RFI (%)	Cell Viability (%)
Medium Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
DMSO Control	-	CD 54	100.0	100.0
	-	CD 86	100.0
Positive Control (DNCB)	2.0	CD 54	261.6*	70.7
		CD 86	425.1*
	3.0	CD 54	372.8*	67.0
		CD 86	383.6*
Test Item	274	CD 54	13,4.4	95.5
		CD 86	110.1
	328	CD 54	143.0	99.3
		CD 86	116.2
	394	CD 54	158.1	97.1
		CD 86	136.5
	473	CD 54	153.8	96.1
		CD 86	132.4
	567	CD 54	166.7	87.6
		CD 86	110.8
	681	CD 54	176.3	86.5
		CD 86	125.7
	817^P	CD 54	168.8	79.4
		CD 86	168.2*
	980^P	CD 54	251.6*	40.5
		CD 86	550.7*

P: Precipitation, *RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered not to be a skin sensitiser up to a test item concentration of 681 μg/mL, under the test conditions of this study.

Executive summary:

The potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h- CLAT). The test item concentration for the main experiment (h-CLAT) of the test item was previously determined by two XTT tests. The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT): 274, 328, 394, 473, 567, 681, 817 and 980μg/mL. The test substance was administered to THP-1 cells for 24 ± 0.5 hours. The RFI of CD86 was greater than 150% in the two highest tested test item concentrations and the RFI of CD54 was greater than 200% in the highest tested test item concentrations in both h-CLAT runs. The two highest tested concentrations (817 and 980μg/mL) were excluded from the evaluation in both runs, as precipitations were observed. Considering this, the test item is considered to be a non-sensitiser under the test conditions of this study. The controls confirmed the validity of the study.