3,3'-(methylimino)bispropiononitrile

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Guidelien 442E (In Vitro Skin Sensitisation: Activation of dendritic cells -h-CLAT

GLP compliance:

no

Remarks:

study conducted for reasons of occupational health and safety

Type of study:

other: In Vitro Skin Sensitization Turnkey Testing Strategy

Test material

Test material information

Constituent 1

Reference substance name:

3,3'-(methylimino)bispropiononitrile

EC Number:

216-305-9

EC Name:

3,3'-(methylimino)bispropiononitrile

Cas Number:

1555-58-4

Molecular formula:

C7H11N3

IUPAC Name:

3-[(2-cyanoethyl)(methyl)amino]propanenitrile

Test material form:

liquid

In vitro test system

Details on the study design:

DPRA: The test substance was incubated with synthetic peptides for 24 hours at 25 ± 2.5 °C and the remaining non-depleted peptide concentration was determined by high performance liquid chromatography (HPLC) with gradient elution and UV-detection at 220 nm. The peptides are custom-made material containing phenylalanine to facilitate UV-detection and either cysteine or lysine as the reactive center. Further, a co-elution control was assessed in order to detect possible co-elution of the test substance with the peptides. Moreover, the samples were analyzed by measuring UV absorbance at 258 nm and the area ratio 220 nm/ 258 nm was calculated as a measure of peak purity.

LuSens: The human transgenic keratinocyte cell line LuSens was treated with 8 testsubstance concentrations for 48 hours in at least two independent experiments. Three replicates of each test substance concentration were tested in each experiment. Cells were lysed and luciferase induction was evaluated by measuring luminescence signal after substrate addition (One Glo®, Promega). In parallel a MTT assay was performed to assess cytotoxicity of the test substance.

h-CLAT: The cell line THP-1 was treated with 8 test substance concentrations for 24 hours in at least two independent experiments. Duplicates of each test substance concentration were tested in each experiment. Cells were stained with FITC labeled-anti-human CD86, FITC labeled-anti-human CD54 or the corresponding isotype control FITC labeled-mouse lgG1. The relative mean fluorescence (RFI) of CD86 and CD54 was evaluated by measuring the CD86 and CD54 signal by flow cytometric analysis. In parallel cytotoxicity of the test substance was measured using the fluorescence intensity of PI.

Results and discussion

In vitro / in chemico

Results

Results 1

Remarks on result:

other: showes minimal or no chemical reactivity

Results 2

Remarks on result:

other: has a keratinocyte activating potential

Results 3

Remarks on result:

other: induces dendritic cell activation

Any other information on results incl. tables

DPRA:

The mean peptide depletion as average of cysteine- and lysine-peptide depletions is calculated and summarized in the table below.

DPRA: Mean peptide depletions of Cysteine, Lysine and both peptides.

	Cysteine-Peptide		Lsysine-Peptide		Mean of both depetions [%]
	Mean depletion [%]	SD [%]	Mean depletion [%]	SD [%]
(Methylimino)bispropiononitrile	2.69	1.28	1.27	0.42	1.98
PC: EGDMA in ACN	54.36	5.49	11.34	0.67	32.85

Based on the observed results and applying the cysteine 1: 10 / lysine 1 :50 prediction model it was concluded that 3,3'-(Methylimino)bispropiononitrile shows minimal or no chemical reactivity in the DPRA under the test conditions chosen.

LuSens:

Experiment 1: The EC 1.50 was calculated by linear regression from the results of the 275 μM and the 330 μM concentrations tobe 315 μM.

Experiment 2: The EC 1.50 was calculated by linear regression from the results of the 475 μM and the 570 μM concentrations tobe 479 μM.

In summary, after 48 hours of exposure to test substance 3,3'(Methylimino)bispropiononitrile luciferase activity in LuSens cells was induced in at least two consecutive concentrations with statistical significance affording at least 70% viability in at least two independent experiments. From this it has to be concluded that test substance 3,3'-(Methylimino)bispropiononitrile has a keratinocyte activating

potential.

h-CLAT:

In summary, after 24 hours of exposure to test substance 3,3'(Methylimino)bispropiononitrile CD86 and CD54 expression was induced in THP-1 cells affording at least 50% viability in at least two independent experiments. From this it has to be concluded that test substance 3,3'-(Methylimino)bispropiononitrile induces dendritic cell activation.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on the results summarized above, 3,3'-(Methylimino)bispropiononitrile is not peptide reactive, activates keratinocytes and activates dendritic cells. In conclusion, 3,3'-(Methylimino)bispropiononitrile is predicted to be a skin sensitizer.