N,N-Bis-(4-biphenyl)amine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 Jul - Dec, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Freie und Hansestadt Hamburg, Germany

Type of study:

activation of keratinocytes

Test material

Test material information

1

Reference substance name:

N-{[1,1'-biphenyl]-4-yl}-[1,1'-biphenyl]-4-amine

EC Number:

678-161-7

Cas Number:

102113-98-4

Molecular formula:

C24 H19 N

IUPAC Name:

N-{[1,1'-biphenyl]-4-yl}-[1,1'-biphenyl]-4-amine

In vitro test system

Details on the study design:

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.

Results and discussion

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (2.21 (experiment 1); 2.48 (experiment 2) .

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

IC30 [442D]

Value:

2.17 µM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

Imax [442D]

Value:

1.05

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

IC50 [442D]

Value:

3.06 µM

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

Acceptance Criteria

The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the negative (solvent) control DMSO is <20% in each repetition.

The controls fullfilled the validity criteria of the test.

Any other information on results incl. tables

Numerical results for the test item

		Luciferase determinations		Cytotoxicity determinations
	Parameter	Imax	EC1.5[µM]	IC50[µM]	IC30[µM]
Test item	Repetition 1	0.99	-	>7.81	4.62
	Repetition 2	1.11	-	3.05	1.02
	Average	1.05 ± 0.08	-	3.06	2.17 ± 2.54

Applicant's summary and conclusion

Interpretation of results:

other: The data generated with this test should be considered in the context of integrated approached such as IATA.

Conclusions:

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

Executive summary:

The test item was examined for sensitising properties in the ARE-Nrf2 luciferase test method addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes by means of quantifying the luciferase activity in the transgenic cell line KeratinoSens™. Cytotoxicity was determined with the MTT assay. The GLP compliant study was performed according to OECD TG 442D.

The test item was tested at 12 concentrations in the range from 0.98 to 2000 µM. The test item was completely dissolvedin DMSO and furhter diluted in treatment culture medium to a concentration of 2000 µM and to subsequent lower concentrations. Test item precipitationwas noted macroscopically startingat aconcentration of 3.91 µM. Cinnamic aldehyde tested at five concentrations from 4 – 64 µM was used as the positive control and the solvent (DMSO) was used as negative control. Two independent repetitions with three parallel technical replicates were run with this same set-up, and one parallel plate was prepared for cytotoxicity determination.

Due to the pronounced cytotoxicity at concentrations of 15.63 µM and higher (IC50 of 11.11 or 1.39 µM in repetitions 1 and 2, respectively) 12 lower concentrations in the range from 0.004 to 7.810 µM were employed additionally and used for the main study. The maximal average fold induction of the luciferase activity (Imax) values were 0.99 or 1.11 fold and hence, the KeratinoSensTM prediction of the test item is considered negative as the luciferase induction value was < 1.5 compared to the solvent control at any non-cytotoxic concentration (0.004 to 3.905 µM). The solvent control and the positive control cinnamic aldehyde were run in all repetitions. All quality criteria for luciferase induction and variability of the solvent control and positive control required were fulfilled.

The test item revealed no sensitising properties in the ARE-Nrf2 Luciferase test method.