4-methyl-4'-n-propyl-[1,1-biphenyl]

Endpoint summary

Description of key information

Skin sensitisation was studied in vitro using the three key events, namely the DPRA, the Keratinosens and the hClat Assay.

In the DPRA assay, no indication for peptide depletion was shown and the study outcome was negative. In the Keratinosens assay, an increase of the luciferase activity was shown in the transgenic cells, indicating potential for skin sensitisation.

Therefore, the third key event, the hClat assay, was started to verify this result. In this OECD 442E study, there was upregulation of the expression of the relevant cell surface markers CD86 and CD54 observed, however, there was precipitation in the whole concentration range investigated. Increase in fluoerescense signals was observed independent from the concentration of the test material in the assay and no dose response relationsship was observed. Therefore, the result of this assay is considered inconclusive.

Given the fact that the results from two of the three key events give conflicting results and the third key event cannot be used due to technical incompliance, the evaluation for skin sensitisation based on in vitro data is overall inconclusive.

Consequently, the test material was studied in vivo in a local lymph node assay according to OECD 429. The substance was not a skin sensitiser in this assay. Based on these in vivo data, the substance does not need to be classified for skin sensitisation.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Jan 08 - Apr 14, 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

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight : 18.5 +/- 0.8 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 + 2°C
- Humidity (%): 24-65%
- Photoperiod (hrs dark / hrs light): 12/12

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

Vehicle:

propylene glycol

Concentration:

10, 25, and 50 (w/w)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: 50% in MEK

Range Finding Exp.
- Concentration used: 25, 50 %
- Irritation:very slight erythema of the ear skin in both animals up to Score 1


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429
- Concentration: 10, 25, and 50%
- 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% 1.68
10% 1.78
25% 8.19

Key result

Parameter:

SI

Value:

2

Test group / Remarks:

Test Group: 10%

Key result

Parameter:

SI

Value:

2

Test group / Remarks:

Test Group: 25%

Key result

Parameter:

SI

Value:

2.6

Test group / Remarks:

Test Group: 50%

Cellular proliferation data / Observations:

EC3 CALCULATION : The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

MORTALITY: No deaths occurred during the study period.

CLINICAL OBSERVATIONS: No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period.

BODY WEIGHTS: The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Calculation of Stimulation Indices per Dose Group

Test item concentration	Group Calculation
	Mean DPM per animal (2 lymph nodes)	SD	S.I.
Vehicle Control Group (acetone/olive oil (4+1, v/v))	1435.0	483.2	1.0
10 % Test Item in AOO	2866.0	1519.4	2.0
5 % Test Item in AOO	2809.0	847.3	2.0
10 % Test Item in AOO	3750.6	1778.8	2.6

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was not found to be a skin sensitiser under the test conditions of this study.

Executive summary:

Objective

In the study the test item formulated in in acetone/olive oil (4+1, v/v) was assessed for its possible skin sensitising potential.

Study Design

For this purpose a local lymph node assay was performed using test item concentrations of 10, 25, and 50 (w/w). The highest concentration tested was the highest concentration that could technically be achieved.

Results

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 2.0, 2.0, and 2.6 were determined with the test item at concentrations of 10, 25, and 50% in acetone/olive oil (4+1, v/v), respectively.

No statistically significant increase in group mean DPM values was noted in any group treated with the different concentrations of the test item if compared to the vehicle control.

Conclusion

The test item was not found to be a skin sensitiser under the test conditions of this study.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Jun 13 - Nov 13, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Freie und Hansestadt Hamburg

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 68.5%.

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion [%]

Value:

2.66

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Acceptance Criteria

The following criteria must be met for a run to be considered valid:
a) The standard calibration curve should have an r2 > 0.99.
b) The mean percent peptide depletion value of the three replicates for the positive control cinnamic aldehyde should be between 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
c) The mean peptide concentration of reference controls A should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be <15.0%.
If one or more of these criteria is not met, the run would have been repeated.

The following criteria must be met for a test item’s results to be considered valid:
a) The maximum standard deviation for the test item replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
b) The mean peptide concentration of the three reference controls C in the appropriate solvent should be 0.50 ± 0.05 mM.
If these criteria were not met, the data would have been rejected and the run have been repeated for that specific test item.

Peptide Depletion in %

1) Cystein Depletion

Sample	Peak Area	Actual Peptide [mM]	Peak Are Ref Control C	Peptide Depletion [%]	Corr. peptide Depletion [%]
1	51.0863	0.504	50.8281	-0.25	0.00
2	51.1104	0.504	51.0861	-0.29	0.00
3	51.2673	0.506	50.9661	-0.60	0.00
Mean	51.155	0.505	50.9601	-0.38	0.00
Evaluation Reactivity Class. Negative

1) Lysine Depletion

Sample	Peak Area	Actual Peptide [mM]	Peak Are Ref Control C	Peptide Depletion [%]	Corr. peptide Depletion [%]
1	38.0977	0.442	39.2617	3.98	3.98
2	38.4283	0.446	39.3871	3.14	3.14
3	39.3335	0.457	40.3771	0.86	0.86
Mean	38.620	0.448	39.6753	2.66	2.66
Evaluation Reactivity Class. Negative

Interpretation of results:

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

Conclusions:

The test item revealed a mean cysteine and lysine peptide depletion of 1.33% and, hence, the test item is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Executive summary:

The purpose of this study was to determine the sensitising potential of the test material in a Direct Peptide Reactivity Assay (DPRA). The study was performed according to OECD guideline 442C. The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following incubation with the test item. Cysteine and lysine peptide percent depletion values are then calculated and used in a prediction model, which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

The test item was dissolved at a concentration of 100 mM in acetone. No precipitate in the reaction mixture at the end of the incubation time and no coelution were observed.

The test material-treated samples revealed a cysteine peptide depletion of 0.0% and a lysine peptide depletion of 2.66% (mean peptide depletion of 1.33%) and, hence, were well below 6.38%. The test material is considered to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetone. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 68.65% for cysteine and 68.37% for lysine peptide. These values are within the required range. Therefore, the study can be regarded as valid.

Conclusion

The test item revealed a mean cysteine and lysine peptide depletion of 1.33% and, hence, the test item is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

13 Jun - 13 Nov 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 (2.12 (experiment 1); 2.90 (experiment 2) .

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

4.02

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: IC50 [µM]

Value:

98.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Luciferase determination run 1: EC 1.5

Parameter:

other: EC1.5 [µM]

Value:

1.59

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

3.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: IC50 [µM]

Value:

111.68

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Luciferase determination run 2: EC 1.5

Parameter:

other: EC1.5 [µM]

Value:

0.98

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

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	4.02*	1.59	98.8	77.92
	Repetition 2	3.60*	0.98	92.96	97.87
	Average	3.81 ± 0.29	1.28 ± 0.43	105.04 ± 9.10	87.33 ± 14.11

*= statistically significantly different as compared to the negative control

Numerical results for the positive control (cinnamic aldehyde)

Positive control: Induction values Reference							Criteria#
Cinnamic aldehyde	4 µM	8 µM	16 µM	32 µM	64 µM	EC1.5	Induction 64 µM	EC1.5
Repetition 1	1.22	1.22	1.38	1.64*	2.55*	23.40	TRUE	TRUE
Repetition 2	1.25	1.28	1.53*	2.04*	3.62*	15.12	TRUE	TRUE
Average	1.23	1.25	1.45	1.84	3.08	18.81	TRUE	TRUE

*= statistically significantly different compared to the negative control

# =the induction in the two replicates 64 µM should be between 2 and 8, the EC_1.5 value should be between 7 µM and 30 µM.

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 sensitiser.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

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 dissolved in treatment culture medium to a concentration of 62.5 µM. Test item precipitation was noted macroscopically starting at aconcentration of 125 µM.

Cinnamic aldehyde tested at five concentrations from 4 – 64 µMwas 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.The maximal average fold induction of the luciferase activity (Imax) values were 4.02 or 3.60 fold and from the dose dependent increase, EC1.5 values of 1.59 or 0.98 µM have been calculated for the first or second repetition, respectively. The corresponding cell viability was > 70% (86.74% and 108.57%) leading to IC50 values of 98.80 or 111.68 µM in repetitions 1 and 2, respectively.

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.

A dose response for luciferase activity induction was observed for each individual repetition as well as for an overall luciferase activity induction.

The test item revealed sensitising properties in the ARE-Nrf2 Luciferase test method.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Aug 22 - Nov 28, 2019

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:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

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:

125

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 83.4 µg/mL

Remarks:

Viability: 86.4%

Key result

Run / experiment:

other: 1

Parameter:

other: max relative fluorescence intensity CD54 [%]

Value:

152

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 69.5 µg/mL

Remarks:

Viability: 90.0%

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

225

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 27.9 µg/mL

Remarks:

Viability: 86.4%

Key result

Run / experiment:

other: 2

Parameter:

other: max. relative fluorescence intensity CD54 [%]

Value:

174

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 69.5 µg/mL

Remarks:

Viability: 78.6%

Key result

Run / experiment:

other: 3

Parameter:

other: max. relative fluorescence intensity CD86 [%]

Value:

187

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 33.5 µg/mL

Remarks:

Viability: 78.6%

Key result

Run / experiment:

other: 3

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

178

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 33.5 µg/mL

Remarks:

Viability: 78.6%

Key result

Run / experiment:

other: 4

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

85

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 14.2 µg/mL

Remarks:

Viability: 83.2%

Key result

Run / experiment:

other: 4

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

75

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 14.2 µg/mL

Remarks:

Viability: 83.2%

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%). RFI values of the solvent/vehicle control are calculated by using the formula described in Section 6.1.
• 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- 3

Sample	Concentration / [µg/mL]	Experiment 1			Experiment 2			Experiment 3
		RFI to vehicle control		Mean viability	RFI to vehicle control		Mean viability	RFI to vehicle control		Mean viability
		CD54	CD86	IgG/CD54/CD86	CD54	CD86	IgG/CD54/CD86	CD54	CD86	IgG/CD54/CD86
Medium Control	0	109	83	98.2	86	83	96.0	97	86	97.2
Vehicle Control (0.2% DMSO)	0	100	100	97.9	100	100	95.3	100	100	97.4
Positive Control (DNBC)	4.0	752	436	66.5	371	482	75.6	658	575	63.7
Test Item	27.9 #	136	110	89.3	151	225	86.4	172	137	85.2
	33.5 #	91	106	92.0	114	165	86.7	178	187	78.6
	40.2 #	76	95	93.3	86	128	84.7	125	148	83.7
	48.3 #	124	118	86.9	134	190	81.8	133	162	82.6
	57.9 #	127	121	87.4	149	171	82.7	153	183	78.0
	69.5 #	152	118	90.0	174	157	78.6	128	168	78.5
	83.4 #	145	125	86.4	126	161	84.3	142	171	77.0
	100.0 #	121	116	88.1	151	158	84.0	150	167	79.0

MFI: geometric mean fluorescense intensity
RFI: relative fluorescense intensity
# : test item precipitation

Interpretation of results:

study cannot be used for classification

Conclusions:

The RFI of CD86 was above 150% in the second experiment at concentrations of 27.9 µg/mL and higher and in the third experiment at concentrations of 33.5 µg test material/mL medium and higher (non-cytotoxic concentrations with mean cell viabilities of 77 to 86.7% in the second and third experiment). Precipitation was observed in the whole concentration range investigated. The data generated with this method may be not sufficient to conclude on skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

The test material was examined for sensitising properties in the h-CLAT assay, addressing the third molecular key event of the adverse outcome pathway (AOP), namely activation of dendritic cells by means of quantifying the expression of specific cell surface markers on a human monocytic leukaemia cell line (THP-1, (TIB-202™, ATCC)). The changes of surface marker expression (CD54 and CD86) were measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement was also conducted by analysing the propidium iodide (PI) uptake. The relative fluorescence intensity of surface markers compared to the solvent/vehicle control was calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers.

The maximal solubility in DMSO was determined as 50 mg/mL. Hence, the maximal feasible test concentration (after addition to medium) was 100 µg/mL.

In a preliminary experiment a dose finding assay was performed to determine the CV75 (75% cell viability), and the test material was tested at eight concentrations in the range of 0.8 - 100 µg/mL culture medium. Test item precipitation was noted macroscopically at the concentration 3.2 µg/mL medium and higher. DMSO was used as solvent control at a concentration of 0.2%. No cytotoxicity was noted and thus no CV75 could be determined (CV75 of > 100 µg/mL).

Hence, the test material was tested at 8 concentrations in the range from 27.9 to 100 µg/mL. Due to competing results of the first and second experiment in the main experiment (first negative, second positive), a third experiment had to be performed. In compliance with the guideline a prediction in the h-CLAT can only be considered as conclusive if 2 of 2 or at least 2 of 3 independent runs are concordant.

DNCB (2,4-dinitrochlorobenzene) was used as the positive control for CD86/CD54 expression measurement at a final single concentration of 4.0 µg/mL. Each experiment consisted of two independent runs for cytotoxicity and CD86/CD54 expression measurement. All quality criteria required were fulfilled.

The h-CLAT prediction was considered positive as:

The RFI of CD86 was above 150% in the second experiment at concentrations of 27.9 µg/mL and higher and in the third experiment at concentrations of 33.5 µg test material/mL medium and higher (non-cytotoxic concentrations with mean cell viabilities of 77 to 86.7% in the second and third experiment).

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

According to Commission Regulation (EU) No 286/2011 as well as GHS (Globally Harmonized Classification System) the test material has no labelling requirement for skin sensitisation.