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

Endpoint summary

Description of key information

OECD 442 C: technically impossibe due to insolubility

OECD 442 D: negative

OECD 442 E: positive

The conflicting results observed in the OECD Guideline study 442 E and 442 D assay and the inconclucive results obtained in the OECD 442 C assay show that the in vitro batteris is not be suitable for the assessment for skin sensitisation this compound. Due to these technical problems, an in vivo LLNA was performed to get a clear result on the endpoint of skin sensitisation.

In this in vivo assay performed according to OECD 429, no increase of the SI value above 3 was detected confirming absence of skin sensitising properties in this assay.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Study period:

Jun 03 -Sep 17, 2019

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
A preliminary test to determine the solubility of the test item in the recommended solvents was performed. The test material was not soluble in any of the recommended solvents and, therefore, the DPRA test is not feasible, as the assay is not suitable for test chemicals which are not completely dissolved according to OECD 442C.

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

Type of study:

other: preliminary test on the solubility

Details on the study design:

Solubility of the test item in an appropriate solvent was assessed before performing the assay. The test item should be dissolved at a concentration of 100 mM.
The test was perfomed using the following solvents:
water,
1:1 mixture water : acetonitrile
isopropanol
acetone
1:1 mixture acetone : acetonitrile

Positive control results:

The test material was not soluble in any recommended solvent such as water, 1:1 mixture water : acetonitrile, isopropanol, acetone or 1:1 mixture
acetone : acetonitrile and, therefore, the DPRA test is not feasible with this test item.

Interpretation of results:

other: technically not possible

Conclusions:

technically not possible

Executive summary:

The test material was not soluble in any recommended solvent such as water, 1:1 mixture water : acetonitrile, isopropanol, acetone or 1:1 mixture

acetone : acetonitrile and, therefore, the DPRA test is not feasible with this test item.

Reference 2

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

Qualifier:

according to guideline

Guideline:

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

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

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.

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) .

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

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

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.

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

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.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Jan 20 - Apr 01, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 25 June 2018

Qualifier:

according to guideline

Guideline:

other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Hessisches Ministerium für Umwelt und Klimaschutz

Type of study:

activation of dendritic cells

Details on the study design:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and
non-sensitisers.

Positive control results:

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in all experiments.

The following relative fluorescense intensities and mean viabilities (values in %) have been detected:

Run Concentr. / [µg/mL] CD54 CD86 mean viability
1 4 349 527 74.2
2 4 425 367 80.1
3 4 289 311 90.0
4 4 215 244 78.6

The threshold of 150% for CD86 and 200% for CD54 were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: max relative fluorescence intensity CD86 [%]

Value:

231.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 31.3 µg/mL

Remarks:

Viability: 91.6%

Key result

Run / experiment:

other: 1

Parameter:

other: max relative fluorescence intensity CD54 [%]

Value:

143

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 631.3 µg/mL

Remarks:

Viability: 91.6%

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

239.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration:26.1 µg/mL

Remarks:

Viability: 93.16%

Key result

Run / experiment:

other: 2

Parameter:

other: max. relative fluorescence intensity CD54 [%]

Value:

178.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration:26.1 µg/mL

Remarks:

Viability: 93.16%

Other effects / acceptance of results:

Acceptance criteria:
The following acceptance criteria should be met when using the h-CLAT assay.
• The cell viabilities of medium and solvent/vehicle controls should be higher than 90%.
• In the solvent/vehicle control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%).
• For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control should be > 105%.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability should be more than 50%.
• For the test item, the cell viability should be more than 50% in at least four tested concentrations in each run.


The test mets the acceptance criteria.

Summarised Results of the hClat Assay - Relative Fluorescense Intensities (RFI) and cell viabilities

Run 1- 2

Sample	Concentration / [µg/mL]	Experiment 1			Experiment 2
		RFI to vehicle control		Mean viability	RFI to vehicle control		Mean viability
		CD54	CD86	IgG/CD54/CD86	CD54	CD86	IgG/CD54/CD86
Medium Control	0	100	100	97.44	100	100	97.18
Vehicle Control (0.2% DMSO)	0	100	100	97.44	100	100	97.60
Positive Control (DNBC)	3.0	388.6	634.1	88.93	300.0	517.4	89.23
	4.0	484.8	647.3	86.28	400.7	505.3	89.98
Test Item	8.74	117.7	175.6	97.29	112.3	108.7	96.63
	10.5	115.2	162.9	97.00	123.9	108.3	96.35
	12.6	140.5	187.3	95.20	118.1	107.2	97.08
	15.1	148.1	190.7	95.07	162.3	134.5	94.28
	18.1	129.1	201.5	95.22	154.3	135.2	95.65
	21.7	136.7	212.2	94.60	185.5	142.0	94.77
	26.1	132.9	221.5	92.53	239.1	178.8	93.16
	31.3#	143.0	231.7	91.62	205.1	175.0	91.80

RFI: relative fluorescense intensity
# : test item precipitation

Interpretation of results:

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

Conclusions:

In conclusion, the test item with a log Pow of 6.3 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of the test material.

For this purpose the test material dissolved in 0.2% (v/v) DMSO in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of the test material was previously determined by two cytotoxicity tests.
Cytotoxic effects were not observed following incubation with the test item up to the highest tested concentration (62.5 μg/mL). Due to the lack of cytotoxicity, a CV75 value could not be calculated. Due to the solubility of the test item, the highest concentration used for the h-CLAT runs was 31.3 μg/mL.
The following concentrations of the test item were tested in the main experiments (h-CLAT):
8.74; 10.5; 12.6; 15.1; 18.1; 21.7; 26.1; 31.3 μg/mL
The test item with a log Pow of 6.3 as tested in 2 independent runs.

The RFI of CD86 was greater than 150% in the first run and the RFI of CD86 and CD54 were greater than 150% and 200%, respectively, in at least one concentration of the second run. Therefore the h-CLAT prediction is considered positive for the test item in this h-CLAT.
In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was > 50%.

In conclusion, the test item with a log Pow of 6.3 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Sep 11 - Nov, 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Inbred, SPF-Quality
- Age at study initiation: 10 weeks
- Weight at study initiation: Pre-test and Main test: 21.0 to 29.1 g.
- Housing: grouped per dose
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C
- Humidity (%): 42 - 69 %
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: day 1 To: day 6

Vehicle:

methyl ethyl ketone

Concentration:

5, 10, and 35% (w/w)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
Two test item concentrations were tested; a 10% and 20% concentration.

- Compound solubility: 20 % in Propylene glycol (PG)
- Irritation: no
- Lymph node proliferation response: -


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429
- Criteria used to consider a positive response: Stimulation index > 3

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Standard statistical methods have been applied for data processing.

Positive control results:

Conc. SI
0%: 1.0
5% 2.4
10% 2.9
25% 4.5

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Test Group: 5% in PG

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

Test Group: 10% in PG

Key result

Parameter:

SI

Value:

1.3

Test group / Remarks:

Test Group: 20% in PG

Calculation of Stimulation Indices per Dose Group

Test item concentration	Group Calculation
	Mean DPM per animal (2 lymph nodes)	SD	S.I.
PG (Vehicle Control)	390	48	1.0
5 % Test Item in PG	403	68	1.0
10 % Test Item in PG	455	24	1.2
20 % Test Item in PG	506	46	1.3

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is not considered to be a skin sensitizer according based on the results of the Local Lymph Node Assay.

Executive summary:

The objective of this study was to evaluate whether the test material induces skin sensitization in mice after three epidermal exposures of the animals according to the OECD TG 429.

Test item concentrations selected for the main study were based on the results of a pre-screen test. Based on the results, the highest concentration required according to the guidelines was selected.

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 5, 10 or 20% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (PG). Three days after the last exposure, all animals were injected with 3Hmethyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 20% were 403, 455 and 506 DPM, respectively. The mean DPM/animal value for the vehicle control group was 390 DPM. The SI values calculated for the test item concentrations 5, 10 and 20% were 1.0, 1.2 and 1.3, respectively.

Since there was no indication that the test item elicits a SI = 3 when tested up to 20%, the test material was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 20%.

Based on these results, the test material The test item is not considered to be a skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).