4,4'-methylenebis(cyclohexylamine)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2016-02-29 to 2016-03-10

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

The study described in this report was conducted in compliance with ECVAM (2014), DB-ALM protocol 155: KeratinoSensTM protocol and OECD guideline 442d

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Analysis of stability, homogeneity and concentration of the test substance under test conditions was not performed as part of this stuy

GLP compliance:

not specified

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-methylenebis(cyclohexylamine)

EC Number:

217-168-8

EC Name:

4,4'-methylenebis(cyclohexylamine)

Cas Number:

1761-71-3

Molecular formula:

C13H26N2

IUPAC Name:

4,4'-methylenedicyclohexanamine

Test material form:

liquid

In vitro test system

Details on the study design:

BASIS OF THE METHOD
The only feature all skin sensitizers have in common is their intrinsic electrophilicity or their potential
to be metabolically transformed to electrophilic chemicals. The signalling pathway with the repressor
protein Keap1 (Kelch-like ECH-associated protein 1) and the transcription factor Nrf2 (nuclear factor
(erythroid-derived 2)-like 2), which binds to the antioxidant / electrophile response element (ARE /
EpRE), is known to respond to electrophilic chemicals and it was found to be a valuable cellular
Givaudan in vitro toxicology laboratory Study Number: RCR 153’561
endpoint to detect skin sensitizers in vitro [8]. This result was confirmed by independent laboratories
[9-13].
EXPERIMENTAL DESCRIPTION
Test System(s):
The KeratinoSens cell line was originally derived from the human keratinocyte culture HaCaT. It
contains a stable insertion of a Luciferase gene under the control of the ARE-element of the gene
AKR1C2 [2].
Endpoint & Endpoint Detection:
Two endpoints are measured: (i) Luciferase induction after a 48 h treatment with test chemicals and
(ii) cytotoxicity as determined with the MTT assay recorded in a parallel plate with the same cell
batch and made up with the same dilutions of the test chemicals.
Endpoint Value:
For Luciferase induction the maximal fold-induction over solvent control (Imax) and the concentration
needed to reach an 1.5 fold induction (EC1.5) are calculated. For cytotoxicity the IC50 value is
extrapolated.

Basic Procedure:
Cells are grown for 24 h in 96-well plates. The medium is then replaced with medium containing a
final level of 1% of the solvent DMSO containing the test chemical. Each compound is tested at 12
concentrations in the range from 0.98 to 2000 μM. Each test plate contains six wells with the solvent
control, 1 well with no cells for background value and 5 wells with a dose response of the positive
control cinnamic aldehyde. In each repetition, three parallel replicate plates are run with this same
set-up, and a forth parallel plate is prepared for cytotoxicity determination.

Positive control
In each test Cinnamic aldehyde is included as positive control. It is tested in each test plate at five
concentrations from 4 – 64 μM.

Data Processing
Data evaluation is automatically performed by a standardized Excel template which forms part of the
SOP. The test plates are read by a plate reader, and the generated raw data are directly pasted into
this template, and all data processing is performed automatically by this Excel sheet.
For both the MTT and the luciferase data, first the background value recorded in an empty well
without added cells is subtracted.
For the MTT data the % viability is then calculated for each well in the test plate in relation to
average of the six solvent control wells.
For the luciferase data the average value of the six solvent control wells is set to 1, and for each well
in the test plate the fold induction is calculated in relation to this value.
The following parameters are then calculated from these processed raw data:
• Imax Maximal fold-gene induction of the luciferase gene over the full dose-response
up to 1000 μM
• EC 1.5, EC2, EC3 Concentration in μM for 1.5 / 2 / 3-fold gene induction
• Pos / Neg Rating of chemical according to prediction model
• reps. Positive number of independent repetitions positive / number of repetitions done
• IC50 Concentration in μM for 50% reduction of cell viability
• IC30 Concentration in μM for 30% reduction of cell viability

Results and discussion

Positive control results:

Cinnamic aldehyde was run in all three repetitions and in all three masterplates used in the study. Chemicals 1 – 7 were run in masterplate1, 8 – 14 in masterplate 2, and 15 - 20 in masterplate 3.Cinnamic aldehyde needs to be positive for a run to be accepted (i.e. induction > 1.5 fold). This was the case in all three
repetitions and in all three masterplates.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: Luciferase Induction

Parameter:

other: KeratinoSens

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 2

Run / experiment:

other: Cytotoxicity determinations

Parameter:

other: IC50 geom. mean

Value:

61.04

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Run / experiment:

other: Luciferase determinations

Parameter:

other: Imax indicating maximal fold induction up to a concentration of 1000µM

Value:

1.06

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Prediction Model
Chemicals are rated positive if the following conditions are met (see also Scheme 1):
• The Imax indicates > 1.5-fold gene induction, and this induction is statistically significant above
the solvent control in a particular repetition as determined by students T-test. The EC1.5 value
is below 1000 μM in all three repetitions or in at least 2 repetitions. (If the Imax is exactly equal to
1.5, the chemical is still rated negative and no EC1.5 value is calculated by the evaluation
sheet.)
• At the lowest concentration with a gene induction above 1.5-fold (i.e. at the EC 1.5 determining
value), the cellular viability is above 70%.
• There is an apparent overall dose-response for luciferase induction, which is similar between
the repetitions.

Any other information on results incl. tables

Cytotoxicity determinations	IC50 (ppm)
	Rep 1	Rep 2	Rep 3	Geom. Mean
Test substance	68.84	46.84	70.52	61.04

Luciferase determinations	I max
	Rep 1	Rep 2	Rep 3	Geom. Mean
Test substance	1.11	1.09	0.98	1.06

Applicant's summary and conclusion

Conclusions:

In summary, the test item revealed no sensitizing properties according to the KeratinoSens in vitro assay.

Executive summary:

The KeratinoSensTM assay is a cell-based assay with a reporter cell line to detect potential skin sensitizers by their ability to induce the Nrf2-response.

The test chemical was dissolve in DMSO and tested according to the standard operating procedure of the KeratinoSens assay at 12 concentrations in three replicates. After 48 h incubation time, luciferase induction, and cellular viability at each concentration was determined. The compound had an IC50 value of 61.04 ppm, an Imax value of 1.06 and the EC value was < 1.5. So it can be concluded that the compound is not a skin sensitizer according to the KeratinoSens in vitro assay.