D-Glucitol, 1-deoxy-1-[methyl(1-oxononyl)amino]-

Endpoint summary

Description of key information

The test item was evaluated for skin sensitisation using the DPRA (OECD 442C), KeratinoSens (OECD 442D) and h-CLAT (OECD 442E). For all three endpoints negative findings were recorded therefore the test item can be considered as a non-sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 May 2017- 19 Jun 2017

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))

Version / remarks:

February 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

January 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: (in chemico) reactivity against synthetic peptides with a thiol or amino group

Specific details on test material used for the study:

Test Item: MTDID 48740
Expiration: Dec 2017
Ptate/Purity:Solid/ 98.1%
Storage:RT
MW (g/mol): 335.44
Solvent: Water

Details on the study design:

DPRA was developed by Frank Gerberick and colleagues (2004) and was further refined in 2007 (Gerberick et al., 2007), and a full OECD guideline for the assay was released in February of 2015. In this assay, the test article was incubated concurrently in two separate buffers, one with cysteine (Ac-RFAACAA-COOH) and one with lysine (Ac-RFAAKAA-COOH). Reactive chemicals bind one or both of the peptides thereby reducing their free concentration levels. The disappearance of each peptide is measured by HPLC/UV. This method does not require any biological material such as enzymes in order for this reaction to take place. It is important to note that the cysteine peptide captures soft electrophiles, while the lysine peptide captures hard electrophiles. This makes the DPRA assay a good choice to screen for reactive chemicals which are associated with allergic contact dermatitis.

Positive control results:

Positive control, 2,3- butanedione (CAS 431-03-8), was prepared at 100 mM in acetonitrile (CAS 75- 05-8, Lot 160783).

Key result

Run / experiment:

other: cystein

Parameter:

other: mean peptide depletion [%]

Value:

0.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: lysin

Parameter:

other: mean peptide depletion [%]

Value:

2.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

In this study under the given conditions the test item showed no reactivity towards both peptides (cyten and lysin). The test item might be considered as non sensitiser.

Executive summary:

The purpose of this study was to screen three test articles 48740 for their potential to act as chemical sensitizers using the Direct Peptide Reactivity Assay (DPRA), a test used to assay reactivity of test articles with small peptides according to OECD 442C.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitation and phase separation was regarded as insignificant.

The 100 mM stock solution of the positive control (2,3 -butanedione) showed high reactivity towards the synthetic peptides.

Peptide depletion by 2,3-butandione is expected to be between 10 – 45% for the lysine (23.2%) peptide and 60 – 100% for the cysteine peptide (85.8%). The mean depletion by 2,3 -Butandione of both peptides was 54.5%.

The test Item (MTDID 48740) was found to have no/minimal reactivity with the peptides and was ranked as negative for

sensitization potential (% Cys Dep 0.5 and % lys Dep 2.1) (Mean depletion 1.3%).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

03-07-2017 to 30-09-2017

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))

Version / remarks:

February 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

January 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: (in chemico) reactivity against synthetic peptides with a thiol or amino group

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 66.12%.

Key result

Run / experiment:

other: cystein

Parameter:

other: mean peptide depletion [%]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: lysin

Parameter:

other: mean peptide depletion [%]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

In this study under the given conditions the test item showed no reactivity towards both peptides (cyten and lysin). The test item might be considered as non sensitiser.

Executive summary:

In the present study the test item was dissolved in dist. water based on the results of the pre-experiments.

Based on a molecular weightof 335 g/mol a 100 mM stock solution was prepared. The test item solutions were testedby incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h±2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Slight precipitation was observed for the samples of the positive control (excluding the co-elution control of the positive control). Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h±2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was

observed for the samples of the positive control including the co-elution control of the positive control. Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitation and phase separation was regarded as insignificant.

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 66.12%.

The 100 mM stock solution of the test item showed no reactivity towards the synthetic peptides. The mean depletion of both peptides was <= 6.38% (0.00%). Based on the prediction model 1 the test item can be considered as non-sensitiser.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23-May 2017 to 14-Feb-2018

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)

Version / remarks:

February 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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:

Cynnamic Aldehyde: Mean EC1.5: 7.77 µM
mean IC50 > 64 µM
Mean I max : Not applicable
Mean CImax (µM): not applicable

Key result

Run / experiment:

mean

Parameter:

other: EC1.5

Value:

2 000

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

7.77 µM

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

mean

Parameter:

other: IC50

Value:

2 000

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

> 64 µM

Remarks on result:

no indication of skin sensitisation

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.

substance	Mean EC 1.5 (µM)	Mean IC50 (µM)	Mean Imax	Mean CImax (µM)	Potential senitizer
Test Item	>2000	>2000	NA	NA	No
Cinnamic Aldehyde	7.77	>64	NA	NA	Yes

Interpretation of results:

other: Supperoting Study

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item might be considered as non-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 Induction of Antioxidant-Response-Element Dependent Gene Activity and Cytotoxicity (Using MTT) in the Keratinocyte ARE- Reporter Cell Line KeratinoSens assay was used to assess the skin sensitization potential of the test article. A test article was predicted to have sensitization potential if: I) The BC 1.5 value fell below 1000 iM in at least 2 of 3 repetitions; 2) At the lowest concentration with a gene induction above 1.5, cellular viability was greater than 70%; and 3) There was an apparent overall dose response which was similar between the three definitive assays. According to the current prediction model, the test article was predicted to be a non-sensitizer.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

August 2017-October 2017

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)

Version / remarks:

February 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 (6.13 in experiment 1; 4.18 in experiment 2) The calculated EC1.5 was between 7 and 34 µM (12.96 µM in experiment 1; 12.44 in experiment 2) .

Key result

Run / experiment:

other: 1

Parameter:

other: Luciferase activity

Value:

1.5

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: Luciferase activity

Value:

1.5

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:

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.

In the first experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

In the second experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

The controls confirmed the validity of the study. The luciferase activity induced by the positive control at a concentration of 64 μM was between 2 and 8 (6.13 in experiment 1; 4.18 in experiment 2). The calculated EC1.5 was between 7 and 34 μM (12.96 μM in experiment 1; 12.44 μM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (10.5% in experiment 1; 15.2% in experiment 2).

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item might be considered as non-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:

In the presentstudy the test item was dissolved in DMSO. .Based on amolecular weight of 335 g/mol astocksolution of 200 mM wasprepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilutionfactor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement. In

the first and second experiment no significant luciferase induction >1.5 and no dose response were foud in the test concentration range with viabilities >70%. Therefore no EC1.5 value could be calculated.

Under the condition of this study the test item might be considered as non-sensitiser.

Reference 5

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 May 2017- 22 June 2017

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: human Cell Line Activation Test (h-CLAT)”adopted 29 July 2016

Version / remarks:

July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

Test Article: MTDID 48740
Physical Form: Solid
Batch No. : 549612
MW: 335.44 g/mol
Storage: Room temperature

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:

Positive control: 2,4 -Dinitrochlorobenzene (DNCB) (CAS-No. 97-00-7) at 4.6 µg/mL wasa assessed as the positive control. (exposure concentration 4.6 µg/mL)
Run 1:
CD86 RFI: 293
CD54 RFI: 475
Run 2 :
CD86 RFI: 285
CD54: 620

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence CD54

Value:

118

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration 681 µg/mL

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence CD 86 [%]

Value:

114

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration 681 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative Fluorescence CD 54 [%]

Value:

80

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration 681 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence CD 86 [%]

Value:

65

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration 681 µg/mL

Other effects / acceptance of results:

Acceptance criteria:

The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

The test mets the acceptance criteria.

Test Item (µg/mL)	39.1	78.1	156.3	312.5	625.0	1250.0	2500.0	5000.0
Viability (%)	98.6	98.8	98.4	94.8	71.8	2.2	0.0	0.7

test Item	CV75 (µg/mL)
MTDID 48740	567.6

Concentration (µg/mL)	RFI for CD86	RFI for CD54	Viability above 50%
190.1	49	134	Yes
228.1	33	67	Yes
273.7	36	81	Yes
328.4	39	89	Yes
394.1	43	100	Yes
472.9	44	95	Yes
567.5	55	93	Yes
681.0	65	80	Yes

Conclusions:

In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore the test item might be considered as non-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 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.

In the present study the test item was dissolved in THP-1 culture media. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. A CV75 of 567.6 µg/mLwas derived in the dose finding assay.

Based on the CV75, the main experiment was performed covering the following concentration steps:

190.1, 228.1, 273.7, 328.4, 394.1, 472.9, 567.5 and 681.0 µg/mL

In all experiments no precipitation or turbidity of the test item was observed for all the concentration steps when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

cytotoxic effects were observed for the cells treated with the test item at concentration of 5000, 2500 and 1250 µg/mL. The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments in a viabile cells ( Viability >50%). The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments in a viabile cells ( Viability >50%)

. Therefore, the test item might be considered as
non-sensitiser.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (293% experiment 1; 285% experiment 2) and 200% for CD54 (475% experiment 1; 620% experiment 2) were clearly exceeded. The controls confirmed the validity of the study. In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore the test item might be considered as non-sensitiser.

Reference 6

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 August 2017- 01 December 2017

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: human Cell Line Activation Test (h-CLAT)”adopted 29 July 2016

Version / remarks:

July 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence CD54

Value:

121

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration 385.99 µg/mL

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence CD 86 [%]

Value:

74

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration 385.99 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative Fluorescence CD 54 [%]

Value:

104

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration 385.99 µg/mL

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence CD 86 [%]

Value:

139

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration 385.99 µg/mL

Other effects / acceptance of results:

Acceptance criteria:

The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

The test mets the acceptance criteria.

CD54 and CD 86 Expression Experiment 1

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	CD86	CD54
Medium Control	-	97.2	95.7	95.6	1349	1241	649	700	592	100	100	208	191
Solvent Control	0.20%	96.8	96.2	96.6	1580	1260	635	945	625	135	106	249	198
DNCB	4.00	87.1	84.6	84.7	3766	3271	584	3182	2687	337	430	645	560
Test item	385.99	86.2	85.7	85.0	1125	1321	606	519	715	74	121	186	218
	321.66	89.4	88.5	88.4	1454	1293	590	864	703	123	119	246	219
	268.05	92.1	91.1	90.4	1411	1283	591	820	692	117	117	239	217
	223.37	92.6	92.5	92.9	1446	1280	609	837	671	120	113	237	210
	186.14	94.7	93.6	94.2	1595	1272	675	920	597	131	101	236	188
	155.12	93.6	94.0	93.5	1517	1258	615	902	643	129	109	247	205
	129.27	94.0	94.9	94.1	1508	1258	613	895	645	128	109	246	205
	107.72	94.9	94.9	94.5	1425	1189	615	810	574	116	97	232	193

CD54 and CD 86 Expression Experiment 2

Sample	Conc. [μg/mL]	Cell Viability [%]			Mean Fluorescence Intensity			corrected Mean Fluorescence Intensity		Relative Flourescence Intensity (RFI)		Ratio Isotype IgG1 to [%]
		CD86	CD54	Isotype IgG1	CD86	CD54	Isotype IgG1	CD86	CD54	CD86	CD54	C86	CD54
Medium Control	-	96.9	96.5	96.7	1705	1505	723	982	782	100	100	236	208
Solvent Control	0.20%	96.5	96.6	96.4	1867	1411	701	1166	710	119	91	266	201
DNCB	4.0	84.3	84.3	83.0	4656	3144	751	3905	2393	335	337	620	419
Test item	385.99	78.0	78.5	78.7	2032	1482	666	1366	816	139	104	305	223
	321.66	89.6	90.2	89.4	1450	1324	650	800	674	81	86	223	204
	268.05	92.2	92.2	91.2	1731	1403	655	1076	748	110	96	264	214
	223.37	94.3	93.7	93.4	1688	1347	615	1073	732	109	94	274	219
	186.14	94.1	94.1	94.8	1625	1340	606	1019	734	104	94	268	221
	155.12	94.8	94.9	94.7	1668	1366	669	999	697	102	89	249	204
	129.27	96.0	95.4	95.6	1537	1323	655	882	668	90	85	235	202
	107.72	95.5	95.3	95.0	1625	1330	627	998	703	102	90	259	212

Conclusions:

In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore the test item might be considered as non-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 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.

In the present study the test item was dissolved in 0.9% NaCl. For the dose finding assay stock solutions with concentrations ranging from 100 mg/mL to 0.78 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis. A CV75 of321.66 ± 22.03 µg/mLwas derived in the dose finding assay.

Based on the CV75, the main experiment was performed covering the following concentration steps:

385.99, 321.66, 268.05, 223.37 186.14, 155.12, 129.27 and 107.72 µg/mL

In all experiments no precipitation or turbidity of the test item was observed for all the concentration steps when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

No cytotoxic effects were observed for the cells treated with the test item. The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item might be considered as
non-sensitiser.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (337% experiment 1; 335% experiment 2) and 200% for CD54 (430% experiment 1; 337% experiment 2) were clearly exceeded. The controls confirmed the validity of the study. In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore the test item might be considered as non-sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

The test item was evaluated for skin sensitisation using the DPRA (OECD 442C), KeratinoSens (OECD 442D) and h-CLAT (OECD 442E). For all three endpoints negative findings were recorded therefore the test item can be considered as a non-sensitiser.