2,2'-[1,3-phenylenebis(oxy)]bisethanol

Endpoint summary

Description of key information

Two in vitro testing (OECD 442 D and OECD 442 E ) were performed and also one in Chemico study (OECD 442C).

Two of the three studies were positive for skin sensitization (OECD 442E and OECD 442D).  Therefore, the material has to be classified as a Cat. 1 skin sensitizer based on those in vitro and in Chemico results .

Subsequently, an LLNA study (OECD 429) was performed and the test item was determined to not be a skin sensitizer under the conditions of this study.

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:

Experimental start date: 09 October 2018 and Experimental completion date: 01 November 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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

Cell Culture:
The cells used in this assay were the transgenic cell line KeratinoSens™ with a stable insertion of the luciferase construct supplied by Givaudan (Dubendorf, Switzerland). The cells were routinely grown and subcultured in maintenance medium at 37°C ± 2°C in a humidified atmosphere containing 5% CO2 in air.

Cell Culture from Frozen Stocks
Vials of KeratinoSens™ cells, stored frozen in cryotubes at -196°C under liquid nitrogen

Cell Passage
Actively growing cell stocks were maintained and expanded by subculturing (passage). When the cells had reached 80 – 90% confluence, the medium from each flask was removed and harvested using trypsin-EDTA solution. Cultures were incubated at 37 ± 2°C in a humidified atmosphere containing 5% CO2 in air until complete detachment and disaggregation of the cell monolayer had occurred. The cells were then resuspended in medium to neutralise the trypsin (cells from several flasks may have been pooled at this point). The cells were resuspended and distributed into flasks containing fresh maintenance medium. This passage procedure was repeated to provide a sufficient number of cells for a test, and were passaged at least twice before using the cells in a test. The passages of KeratinoSens™ cells were limited to 25 passages.

Preparation of Test Cell Cultures
The cells from flasks of actively growing cultures were detached and disaggregated as described above. The number of viable cells in the prepared cell suspension were determined by counting a trypan blue-stained cell preparation using an Improved Neubauer Haemocytometer. The cell suspension was diluted with maintenance medium without geneticin to give 1 x 105 viable cells/mL and 100 µL volumes pipetted into all wells except well H12 of sterile 96-well flat-bottomed microtitre plates. On each occasion four plates were prepared in parallel: three white plates for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay. Well H12 of each plate received 100 µL maintenance medium without geneticin with no cells. The plates were incubated for 24 ± 2 hours at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air, to allow the cells to attach.

Preparation of the Positive Control
Cinnamic aldehyde (Sigma, 239968, lot: STBG0250V, expiry: July 2020) was prepared by weighing between 20 – 40 mg into a tared glass container and diluted to a final concentration of 200 mM in DMSO using the following formula:

V=5×((p÷100)×w) / MW- (w/1000)
Where
V = volume of DMSO in mL to be added
p = purity of the chemical in %
MW = molecular weight of the chemical in g/mol
w = exact weight of the chemical added to the vial in mg
The 200 mM cinnamic aldehyde solution was further diluted to a final concentration of 6.4 mM by adding 32 µL of the 200 mM solution to 968 µL of DMSO.

Test Item Solubility
The test item was found to be soluble in DMSO at 200 mM.

Preparation of the Test Item
A stock solution of the test item was prepared by weighing between 20 – 40 mg into a tared glass container and diluting to 200 mM in DMSO using the formula above.

Treatment of Cultured Plates
Approximately 24 hours after the test cell culture plates were established, the medium was removed from the wells by careful inversion of the plates and blotting onto sterile paper towel. 150 µL of assay medium was added to every well of the 96 well plates. 50 µL from each well of the dilution plate was transferred to equivalent wells in the 96 well plates. Three white plates were dosed for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay.
The plates were then covered with a plate seal and placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air for 48 ± 2 hours.

Cell Viability Measurement
A kit (Molecular Probes Vybrant MTT kit V13154) was used to determine cell viability. 1 vial from the kit was reconstituted by adding 1 mL of sterile PBS (Gibco 10010) and vortexed mixed until dissolved to give 5 mg/mL MTT in DPBS. After incubation, the transparent plate was removed from the incubator and the plate seal discarded. The cell culture medium was removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 100 µL fresh assay medium was added to each well. 10 µL of MTT solution was added to each well of the 96-well plate. The plate was incubated at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air for 4 hours ± 10 minutes. The medium was then removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 50 µL of DMSO was added to each well. The plate was then placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air, protected from light, for at least 10 minutes. The absorbance value of each well was read using a plate reader with a 540 nm filter.


Luciferase Measurement
Luciferase was measured using the Steady Glo® Luciferase Assay system kit supplied by Promega (E2550). Steady-Glo® luciferase reagent was prepared by transferring the contents of one bottle of Steady-Glo® buffer to one bottle of Steady-Glo® substrate. The reagent was mixed by inversion until the substrate had dissolved. The reconstituted reagent was used on the same day it was prepared for the repeat of test 1 and for test 2. Frozen reconstituted reagent was used for test 1 and was thawed to room temperature before use.
After incubation the medium was removed from the wells of the triplicate white plates by careful inversion of the plates and blotting on sterile absorbent paper. 100 µL of fresh assay medium was added to each well before 100 µL of Steady-Glo® luciferase reagent was added to each well of the plate. The plates were shaken on a plate shaker for at least 5 minutes until the cells had lysed. Luminescence (emitted light) was measured using a SpectraMax L luminometer. Each plate was read for total photon count with an integration time of 1 second. The plates were dark adapted for 1 minute prior to measurement.

Number of Tests Required
Two independent tests each containing three replicates (i.e. n=6) were required to make a conclusion.

Positive control results:

The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was statistically significant above the threshold of 1.5 in at least one of the tested concentrations (4 to 64 μM) in both tests.

The EC1.5 values of the positive control, cinnamic aldehyde were 5.28 μM and 7.50 μM for test 1 and 2, respectively, which lay within the historical control range for this laboratory. The average induction in the three replicates for cinnamic aldehyde at 64 µM were 10.20 and 7.05 for test 1 and 2, respectively. The result of the first test (10.20) did not meet the acceptance criterion of between 2 and 8, however, there was a clear dose-response and therefore the test was accepted.

Key result

Run / experiment:

other: test 1

Parameter:

other: I max

Value:

2.4

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: The Imax tests was >1.5 fold and statistically significant as compared to the DMSO control.

Key result

Run / experiment:

other: test 2

Parameter:

other: I max

Value:

2.87

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: The Imax tests was >1.5 fold and statistically significant as compared to the DMSO control.

Key result

Run / experiment:

other: test 1

Parameter:

other: cellular viability in %

Value:

99.2

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: IC30 and IC50 could not be calculated.

Key result

Run / experiment:

other: test 2

Parameter:

other: cellular viability in %

Value:

96.12

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

other: IC30 and IC50 could not be calculated.

Key result

Run / experiment:

other: test 1

Parameter:

other: EC 1.5 in µM

Value:

87.42

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

5.28 μM

Key result

Run / experiment:

other: test 2

Parameter:

other: EC 1.5 in µM

Value:

89.76

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

7.50 μM

Other effects / acceptance of results:

Test item results:
The Imax for the test item was 2.40 in test 1 and 2.87 in test 2.
The Imax for both tests was >1.5 fold and statistically significant as compared to the DMSO control.
The cellular viability did not fall below 99.20% in test 1 and 96.12% in test 2 and therefore the IC30 and IC50 could not be calculated.
The EC1.5 for the test item was 87.42 µM and 89.76 µM for tests 1 and 2, respectively.

Negative solvent results.
The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 8.8% and 6.9% for test 1 and 2, respectively, which met the acceptance criterion of below 20%.

Results for the test item - Test 1

Test item conc. (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Mean fold induction	1.07	1.18	0.97	1.34	1.16	1.17	1.36	1.71	1.72	2.10	2.40	2.25
Statistically significant	No	No	No	No	No	No	No	Yes	Yes	Yes	Yes	Yes
Viability (%)	116.01	105.65	106.45	99.20	100.40	106.45	103.32	104.12	111.63	103.46	107.84	103.32
Imax	2.40
EC1.5(µM)	87.42
IC30(µM)	N/A
IC50(µM)	N/A

 

 

Determination criteria for the skin sensitisation potential of the test item	Result
Is the Imax>1.5 fold and statistically significant	Yes
Is the cellular viability >70% at the lowest concentration at the EC1.5determining concentration	Yes
Is the EC1.5value <1000µM	Yes
Is there an apparent overall dose-response for luciferase induction	Yes
KeratinoSens™ prediction	Positive

 

 

  Results for Cinnamic Aldehyde – Test 1

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.36	1.80	2.01	4.40	10.20
Statistically significant	No	Yes	Yes	Yes	Yes
Viability (%)	104.19	104.65	101.99	104.39	94.55
Imax	10.20
EC1.5(µM)	5.28
IC30(µM)	N/A
IC50(µM)	N/A

 

Test Acceptance Criteria	Result
Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations	Yes	Pass
Average induction of positive control at 64 µM between 2 – 8	No (10.20), however, there is a clear dose-response	Pass
EC1.5of positive control within two standard deviations of the historical mean (1.47 – 47.70)	Yes (5.28)	Pass
CV% of blank values < 20%	Yes (8.8%)	Pass

Results for the test item – Test 2

Test item conc. (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Mean fold induction	0.79	0.89	0.91	1.04	1.08	1.13	1.35	1.70	2.05	2.55	2.69	2.87
Statistically significant	No	No	No	No	No	No	No	Yes	Yes	Yes	Yes	Yes
Viability (%)	111.54	96.12	104.56	100.80	106.99	108.44	113.19	115.16	110.88	117.47	113.25	101.72
Imax	2.87
EC1.5(µM)	89.76
IC30(µM)	N/A
IC50(µM)	N/A

 

Determination criteria for the skin sensitisation potential of the test item	Result
Is the Imax>1.5 fold and statistically significant	Yes
Is the cellular viability >70% at the lowest concentration at the EC1.5determining concentration	Yes
Is the EC1.5value <1000µM	Yes
Is there an apparent overall dose-response for luciferase induction	Yes
KeratinoSens™ prediction	Positive

Results for Cinnamic Aldehyde – Test 2

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.29	1.53	1.85	3.06	7.05
Statistically significant	No	Yes	Yes	Yes	Yes
Viability (%)	110.35	107.79	112.46	113.58	105.15
Imax	7.05
EC1.5(µM)	7.50
IC30(µM)	N/A
IC50(µM)	N/A

 

Test Acceptance Criteria	Result
Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations	Yes	Pass
Average induction of positive control at 64 µM between 2 – 8	Yes (7.05)	Pass
EC1.5of positive control within two standard deviations of the historical mean (1.47 – 47.70)	Yes (7.50)	Pass
CV% of blank values < 20%	Yes (6.9%)	Pass

Historical Control Data for Cinnamic Aldehyde

n	Date	EC1.5(µM)
1	21-Jul-17	20.43
2	11-Aug-17	34.51
3	16-Nov-17	33.93
4	16-Nov-17	29.04
5	15-Dec-17	45.81
6	15-Dec-17	43.48
7	15-Feb-18	15.84
8	15-Feb-18	19.90
9	22-Feb-18	21.07
10	22-Feb-18	27.98
11	12-Jul-18	10.18
12	26-Jul-18	16.09
13	02-Aug-18	8.88
14	09-Aug-18	17.01
	Mean	24.58
	SD	11.56
Laboratory Historical Data Range (Mean +/- 2xSD)
1.47	to	47.70

Interpretation of results:

other: test item gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens™)

Conclusions:

It was concluded that the test item gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is a skin sensitizer.

Executive summary:

The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item, HER (Resorcinol bis-(2-Hydroxyethyl) ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol, is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens™).

The I_maxfor HER (Resorcinol bis-(2-Hydroxyethyl) ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol was 2.40 in test 1 and 2.87 in test 2. The I_maxfor both tests was >1.5 fold and statistically significant as compared to the DMSO control. The cellular viability did not fall below 99.20% in test 1 and 96.12% in test 2 and therefore the IC₃₀and IC₅₀could not be calculated. The EC_1.5for HER (Resorcinol bis-(2-Hydroxyethyl) ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol was 87.42 µM and 89.76 µM for tests 1 and 2, respectively. Graphs for HER (Resorcinol bis-(2-Hydroxyethyl) ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol showed an overall dose-response for luciferase induction.

 

The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was statistically significant above the threshold of 1.5 in at least one of the tested concentrations (4 to 64 μM) in both tests.

The EC_1.5values of the positive control, cinnamic aldehyde were 5.28 μM and 7.50 μM for test 1 and 2, respectively, which lay within the historical control range for this laboratory. The average induction in the three replicates for cinnamic aldehyde at 64 µM were 10.20 and 7.05 for test 1 and 2, respectively. The result of the first test (10.20) did not meet the acceptance criterion of between 2 and 8, however, there was a clear dose-response and therefore the test was accepted.

The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 8.8% and 6.9% for test 1 and 2, respectively, which met the acceptance criterion of below 20%.

It was concluded that HER (Resorcinol bis-(2-Hydroxyethyl) ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that HER (Resorcinol bis-(2-Hydroxyethyl) ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol is a skin sensitizer.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 25 September 2018 and Experimental completion date: 28 September 2018

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The OECD TG 442 C may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitizers and non-sensitizers for the purpose of hazard classification and labelling.

Details on the study design:

Test system:
- Synthetic peptide containing Cysteine: Ac-RFAACAA-OH (MW 752 gr/mol); supplier AnaSpec and lot 1658140.
- Synthetic peptide containing Lysine: Ac-RFAAKAA-OH (MW 777 gr/mol); supplier AnaSpec. and lot 1658141.
- Positive control: Cinnamic Aldehyde (Positive control), MW 132 gr/mol; Batch N°MKCB9907

Assessment of Test Item Solubility
The solubility of HER was assessed in acetonitrile at a nominal concentration of 100 mM.


Preparation of peptides stock solution:
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).

Preparation of Peptide Calibration Standards
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.

Preparation of Stability Controls and Precision Control
Stability controls (Reference Control B), precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile/buffer.

Preparation of Positive Control Solution and Test Item Stock Solution
The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution of HER was also prepared in acetonitrile.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Triplicate solutions each of the positive control and HER stock solutions were diluted with the Cysteine peptide stock solution so as to prepare solutions containing 0.5 mM Cysteine and 5 mM of Cinnamic Aldehyde or 5 mM HER. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co elution Controls
Triplicate solutions each of the positive control and HER stock solution were diluted with the Lysine peptide stock solution so as to prepare solutions containing 0.5 mM Lysine and 25 mM of Cinnamic Aldehyde or 25 HER. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

Incubation
The appearance of the HER and positive control samples in the HPLC vials was documented following preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis
The concentration of both the Cysteine and Lysine peptides in the presence of HER and the associated positive controls was quantified by HPLC using UV detection as detailed in the chromatographic section.

Calculations
The peak area response for the peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:

% Peptide depletion = 100 - (Peptide peak area in replicate depletion samples (x 100) / Mean Peptide peak area of reference control samples B)

Positive control results:

see table below

Key result

Run / experiment:

other: achieved results

Parameter:

other: Mean Cysteine depletion in the presence of the test item

Value:

-1.34

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

69.5% (positive comtrol depletion)

Key result

Run / experiment:

other: achieved results

Parameter:

other: Mean Lysine depletion in the presence of the test item

Value:

-2.68

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

61.5% (positive control depletion)

Solubility Assessment

Solubility of the test iem was achieved at a nominal concentration of 100 mM in acetonitrile.

Reactivity Assessment

	Peptide	Standard Linearity	Positive control depletion (%)	Reference controls	Test item
Acceptance criteria	Cysteine	r^2>0.99	60.8-100 (SD <14.9%)	0.45-0.55 mM (CV <15%)	SD <14.9%
	Lysine	r^2>0.99	40.2-69.0 (SD <11.6%)	0.45-0.55 mM (CV <15%)	SD <11.6%
Achieved results	Cysteine	r^2>0.999	69.5 (SD, 0.20%, n=3)	B: 0.505 mM (CV 0.31%, n=6)	SD 0.96% (n=3)
	Lysine	r^2>0.999	61.5 (SD, 0.53%, n=3)	B: 0.505 mM (CV 0.51%, n=6)	SD 1.08% (n=3)

CV: Coefficient of Variation

SD: Standard deviation

The depletion of peptide in the presence of the test iem was:

		Mean peak area of peptide with test item(µV.sec)	Mean peptide depletion by HER (%)
Cysteine	Control B: 803300 (n=6)	814050 (n=3)	-1.34
Lysine	Control B: 764810 (n=6)	785270^1(n=3)	-2.68

¹     Lysine peak integrated from co-eluting peak by dropping a perpendicular to the baseline

Applying the following Cysteine 1:10 reactivity prediction depletion model (below), reactivity is classed as “no to minimal” and the DPRA prediction is therefore negative and the test item is therefore predicted not to be a potential skin sensitizer. 

Mean of cysteine depletion (%)	Reactivity Class	DPRA Prediction
0%≤ Cys% depletion ≤13.89%	No or minimal reactivity	Negative
13.89%< Cys% depletion ≤23.09%	Low reactivity	Positive
23.09%< Cys% depletion ≤98.24%	Moderate reactivity
98.24%< Cys% depletion ≤100%	High reactivity

There were co-elution peaks in the Lysine assay and therefore the result from this assay is inconclusive, hence the prediction is made on the Cysteine assay only.   

Interpretation of results:

GHS criteria not met

Remarks:

Predicted by DPRA not to be a skin sensitizer. 

Conclusions:

Solutions of the test item were analyzed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. With no depletion of the cysteine peptide and co-elution of the test item in the Lysine peptide in the presence of the test item is therefore predicted by DPRA as negative and not to be a potential skin sensitizer based on this assay.

Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of HER (Resorcinol bis-(2-Hydroxyethyl) ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol (HER). 

Solutions of the test item were analyzed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. With co-elution of the test item with the Lysine peptide the result of only the Cysteine peptide is reported. There was no reactivity of the test item with the Cysteine peptide and therefore the test item is placed in the reactivity class of “no to minimal” and hence it is predicted by DPRA not to be a skin sensitizer. 

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 07 November 2018 and Experimental completion date: 21 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals: OECD 442E; In Vitro Skin Sensitisation: In Vitro Skin Sensitisation Assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation.

Version / remarks:

Annex I: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), June 2018.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

The OECD TG 442 E may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Details on the study design:

Controls for Cytotoxicity Test and h-CLAT.

Medium Control
- Name: Culture medium

Solvent Control for the Test Item
- Name: DMSO (final concentration 0.2%)

Positive Control (h-CLAT)
- Name: DNCB (2,4-dinitrochlorobenzene, CAS No.: 97-00-7, Sigma-Aldrich, Germany) final concentration: 2 and 3 µg/mL, Purity ≥ 99%)
- Solvent: DMSO (final concentration 0.2%), Fisher ScientificTM, Germany, diluted in culture medium.

Solvent Control for the Positive Control (h-CLAT)
- Name: DMSO (Dimethyl sulfoxide, CAS No. 67-68-5, Fisher ScientificTM, Germany) in culture medium, final concentration 0.2%, Purity ≥ 99%.

TEST ITEM PREPARATION
The test item was dissolved in DMSO and further diluted in culture medium to reach a concentration of 0.2% (v/v) DMSO in the medium.
The maximum concentration of test item was 1000 µg/mL (in 0.2% DMSO).
For the cytotoxicity test (dose finding assay) eight concentrations of the test item were analysed. For this, dilutions were prepared by 1:2 serial dilutions from 500 mg/mL (in DMSO).
Each solution was diluted with culture medium before application of the test solution to the cells to reach a final concentration of 0.2% (v/v) DMSO in the medium.

TEST SYSTEM AND SUPPORTING INFORMATION
Reasons for the Choice of THP-1 Cells
THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202 (provided by LGC Standards GmbH, Germany). THP-1 cells are used as a surrogate for human myeloid dendritic cells, because the THP-1 cells also show enhanced CD86 and/or CD54 expression when exposed to sensitisers.

THP-1 Cell Cultures
Stocks of the THP-1 cell line are stored in liquid nitrogen.
The cells are sub-cultured twice weekly.
The cell density should not exceed 1 E 106 cells/mL.
Cells can be used up to two months after thawing (passage number should not exceed 30).
The passage numbers of the used THP-1 cells were 15 and 17 in the cytotoxicity tests and 18 and 20 in the h CLAT for runs 1 and 2, respectively.

Culture Medium
RPMI 1640 Medium, GlutaMAXTM Supplement including 25 mM HEPES, supplemented with 10 % FBS (v/v), 0.05 mM 2 mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate and appropriate antibiotics (100 U/mL of penicillin and 100 µg/mL of streptomycin) is used to culture the cells during the assay.

Preparation and Seeding of THP-1 Cells
On the day of the cytotoxicity experiment directly before the application of the test item, solvent and medium control, a volume of 100 µL with a cell density of 0.9 - 1 x 10E6 THP-1 cells/mL was seeded in each well of a 96-well flat bottom plate.
For the main experiment (h-CLAT) 0.9 - 1 x 10E6 cells/well in a volume of 500 µL were seeded in a 24-well plate before the treatment.

Experimental Design and Procedures of the Cytotoxicity Test
Dose Finding Assay (Flow cytometer)
The test item concentrations investigated in the main experiment (h-CLAT) were determined with two cytotoxicity tests. The tests were performed with independent cell cultures on different days.


Treatment of the Cells
The test item dilutions were prepared freshly before each experiment.
Each DMSO solution was diluted with culture medium before application of the test solution to the cells to reach a final concentration of 0.2% (v/v) in the culture medium.
Each volume of the dilutions of the test item, culture medium and solvent control (e.g. 0.2% (v/v) DMSO in culture medium) was added to the cells.
Culture medium and the solvent control 0.2% (v/v) DMSO in the culture medium were tested additionally.
The treated THP-1 cells were incubated for 24 ± 0.5 hours.
All dose groups were tested in one replicate for each cytotoxicity test.
After incubation period, the cell cultures were microscopically evaluated for morphological alterations.


Staining of the Cells
Each test item-treated and not test item treated cells were collected, washed twice (2 - 8 °C) and re-suspended in a final volume of 1 mL/tube FACS buffer.
At least 10 minutes before the flow cytometry acquisition,7-AAD solution were added in each sample tube.

Flow Cytometry Acquisition (Cytotoxicity Test)
The following acquisition plots were prepared:
- 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
- Histogram plot of the FL-3 channel
The cell viability was measured by gating-out dead cells stained with 7-AAD. A total of 10,000 living cells were analysed.


Flow Cytometry Analysis (Cytotoxicity Test)
The cell viability is shown by the cytometry analysis program (% total) or is calculated according to the following equation:

Cell Viability [%]= (Number of living cells) / (Number of acquired cells) ×100

The CV75 value, a concentration showing 75% of THP-1 cell survival (25% cytotoxicity), is calculated by log-linear interpolation using the following equation:

Log CV75 = (((75-c)×Log (b)- (75-a) ×Log (d)) / (a-c ))

Where:
a is the minimum value of cell viability over 75%
c is the maximum value of cell viability below 75%
b and d are the concentrations showing the value of cell viability a and c respectively


Acceptability of the Cytotoxicity Assay
The cytotoxicity test is considered to be acceptable if it meets the following criteria:
- The cell viability of the medium and solvent control (if the test item is solved in DMSO) should be more than 90%.

Key result

Run / experiment:

other: 24 hours incubation (run 1 and run 2)

Parameter:

other: RFI of CD86 (Relative Fluorescence Intensity) in %

Remarks:

RFI expressed in %

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: prediction is considered positive for the test item in this h-CLAT

Other effects / acceptance of results:

Acceptance Criteria
The following acceptance criteria should be met when using the h-CLAT method:
• Cell viability of medium control and DMSO control should be more than 90%.
• In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
• For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of 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 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50% in at least one concentration of the two tested positive control concentrations.
• For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run.
Negative results are acceptable only for test items exhibiting a cell viability of < 90% at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90% the negative result should be discarded. In such case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability > 90% (OECD 442E guideline).

Prediction model
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs (OECD 442E guideline):
− The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%);
− The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%).
Otherwise, the h-CLAT prediction is considered NEGATIVE.
Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If, however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE. An h-CLAT prediction should be considered in the framework of an IATA (OECD 442E guideline).

 Resultsof the Dose Finding Assay (Cytotoxicity Test)

Results of the first Cytotoxicity Test for the Test Item

 

Test Group	Concentration [µg/mL]	Microscopic Evaluation / Cytotoxicity	Flow Cytometric Evaluation / Cell Viability [%]
Medium Control	-	no	98.16
Solvent Control	-	no	98.32
Test Item	7.8	no	98.22
	15.6	no	98.53
	31.3	no	98.37
	62.5	no	98.04
	125	no	98.40
	250	no	98.07
	500	no	97.89
	1000	no	98.00

Due to the lack of cytotoxicity in the Flow Cytometric Evaluation of the Cytotoxicity Test, a CV75 value could not be calculated.

Results of the second Cytotoxicity Test for the Test Item

Test Group	Concentration [µg/mL]	Microscopic Evaluation / Cytotoxicity	Flow Cytometric Evaluation / Cell Viability [%]
Medium Control	-	no	97.48
Solvent Control	-	no	97.08
Test Item	7.8	no	97.37
	15.6	no	97.15
	31.3	no	97.11
	62.5	no	97.61
	125	no	97.03
	250	no	97.44
	500	no	97.31
	1000	no	97.05

Due to the lack of cytotoxicity in the Flow Cytometric Evaluation of the Cytotoxicity Test, a CV75 value could not be calculated.

 Results of the h-CLAT Test

Results of the first h-CLAT run for the Test Item.

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	97.10
DMSO Control	-	100.0	100.0	97.13
Positive Control (DNCB)	2.0	159.3^#	178.2*	92.96
	3.0	326.0*	397.5*	89.70
Test Item	279	70.7	103.3	96.77
	335	95.1	193.7*	96.88
	402	86.2	122.2	96.03
	482	76.4	103.8	97.24
	579	82.1	127.6	96.86
	694	103.3	128.9	96.45
	833	103.3	186.6*	95.86
	1000	113.8	209.2*	96.61

*   RFI value of CD86 or CD54 fulfilled the positive criteria (CD86≥150% and CD54≥200%).

^#    CD54 RFI value of the positive control (2.0 µg/mL DNCB) did not fulfil the positive criterion (CD54 ≥ 200%).

Results of the second h-CLAT run for the Test Item

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability (%)
Medium Control	-	100.0	100.0	96.21
DMSO Control	-	100.0	100.0	95.76
Positive Control (DNCB)	2.0	139.8^#	187.7*	91.62
	3.0	310.7*	308.0*	85.44
Test Item	279	97.1	48.1	95.79
	335	18.4	17.5	95.84
	402	104.9	271.1*	96.50
	482	15.5	185.2*	96.48
	579	33.0	143.4	96.48
	694	139.8	98.6	96.19
	833	96.1	25.0	95.92
	1000	113.6	150.8*	95.49

*   RFI value of CD86 or CD54 exceeded the positive criteria (CD86≥150% and CD54≥200%).

^#    CD54 RFI value of the positive control (2.0 µg/mL DNCB) did not fulfil the positive criterion (CD54 ≥ 200%).

Interpretation of results:

other: The test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Conclusions:

The test item with an unknown log Pow 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:

Thisin 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 item dissolved in DMSO and further diluted 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 item was previously determined bytwo cytotoxicity tests.

Cytotoxic effects were not observed following incubation with the test item up to the highest tested concentration (1000 µg/mL). Due to the lack of cytotoxicity, a CV75 value could not be calculated.Therefore the OECD 442E guideline recommended maximal to be tested test item concentration (1000 µg/mL for DMSO) was used for the h-CLAT runs.

The following concentrations of the test item were tested in the main experiments (h-CLAT): 279, 335, 402, 482, 579, 694, 833 and 1000 µg/mL

The test item with an unknown log Pow was testedin 2 independentruns.The RFI of CD86 was greater than 150% in at least one concentration of both independent runs. A slight dose response could be observed in the two highest concentrations of the first h-CLAT run, but no clear dose response could be observed in the second h-CLAT run. However, since three test item concentrations of both runs showed an RFI of CD86 > 150%, 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%. Except the CD54 RFI value of the positive control (2.0 µg/mL DNCB) in the first and second h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the CD54 RFI value of the positive control (3.0 µg/mL DNCB) in the h-CLAT runs exceeded the positive criteria.

In conclusion, the test item with an unknown log Powactivated 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:

key study

Study period:

Experimental start date: 12 December 2018 and Experimental completion date: 15 January 2019.

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)

Version / remarks:

Adopted 22 July 2010

Deviations:

yes

Remarks:

Relative humidity in animal room was between approximately 14-65% instead of 45-65% for several hours. This deviation to the study plan does not affect the validity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Remarks:

Recognised as the recommended test system.

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc Postbus 6174 5960 AD Horst / The Netherlands

- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: (Pre-test and main study) 8-9 weeks
- Housing: Group, Makrolon Type II (pre-test) / III (main study) with wire mesh top.
- Bedding: Granulated soft wood bedding.
- Diet (e.g. ad libitum): 2018C Teklad Global 18% protein rodent diet (certified), ad libitum.
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: At least 5 days prior to the start of dosing under test conditions afetr health examination
- Indication of any skin lesions: Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 45-65% (except for deviation)
- Photoperiod (hrs dark / hrs light): Artificial light 6.00 a.m until 6.00 p.m.

Vehicle:

dimethylformamide

Remarks:

Purity 99.99%

Concentration:

LLNA test was performed using test item concentration of 10, 25 and 50 % (w/w)

No. of animals per dose:

- Number of animals for the pre-test: 2 females
- Number of animals for the main study: 16 females
- Number of animals per group: 4 females
- Number of test groups: 3
- Number of control (vehicle group): 1

Details on study design:

Vehicle and Dose Selection

Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 25 and 50% once daily each on three consecutive days. Prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6.
At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity.
Thus, the test item in the main study was assayed at 10, 25, and 50%. The highest concentration tested was the highest level that could technically be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.


Test Item Preparation
The test item was placed into an appropriate container on a tared balance and DMF was added (weight per weight).
The different test item concentrations were prepared individually.
The preparations were made freshly before each dosing occasion.


- CRITERIA USED TO CONSIDER A POSITIVE RESPONSE:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
• Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

Positive control substance(s):

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

Positive control results:

See below in "Any other information on results incl. tables."

Key result

Parameter:

EC3

Remarks on result:

not determinable

Remarks:

could not be calculated, since all S.I.'s are below the threshold value of 3.

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Test item concentration 0%

Key result

Parameter:

SI

Value:

0.76

Test group / Remarks:

Test item concentration 10%

Key result

Parameter:

SI

Value:

0.57

Test group / Remarks:

Test item concentration 25%

Key result

Parameter:

SI

Value:

0.44

Test group / Remarks:

Test item concentration 50%

Cellular proliferation data / Observations:

VIABILITY/MORTALITY:
No deaths occured during the study period.

CLINICAL SIGNS:
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.

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

DISCUSSION:
The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a -scintillation counter.
All treated animals survived the scheduled study period and no signs of systemic toxicity or local skin irritation were observed.
A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.
In this study Stimulation Indices of 0.76, 0.57, and 0.44 were determined with the test item at concentrations of 10, 25, and 50% in DMF. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.

Results of the GLP Positive Control:

Experiment performed in October 2018 (Envigo study number 1921100). Positive control substance: α-Hexylcinnamaldehyde

Vehicle: acetone:olive oil (4:1 v/v))

Test item concentration %	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	18	---	---	---	---
---	BG II	10	---	---	---	---
0	1	8090	8076	8	1009.5	1.00
5	2	11386	11372	8	1421.5	1.41
10	3	21836	21822	8	2727.8	2.70
25	4	83416	83402	8	10425.2	10.33

1 = Control Group

2-4 = Test Group

^a)= The mean value was taken from the figures BG I and BG II

^b)= Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

Calculation of the EC3 value:

	Test item concentration %	S.I.
Test Group 3	10 (a)	2.70 (b)
Test Group 4	25 (c)	10.33 (d)
EC3 = (a-c) [(3-d)/(b-d)] + c = 10.6% (w/v)

a,b,c,d = Co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot.

Historical Positive Control Data

These values represent historical control data of the last 10 periodic positive control experiments.

Positive Control Substance	Date	Concentration / Vehicle	S.I. values
alpha- hexyl-cinnamaldehyde	October 2018	25% in acetone:olive oil (4+1 v/v)	10.33
	April 2018		9.85
	October 2017		5.7
	April 2017		10.1
	October 2016		11.8
	April 2016		7.8
	October 2015		17.6
	April 2015		9.5
	October 2014		13.8
	April 2014		6.8

 

 Calculation and Results of Individual Data

Vehicle: DMF

Test item concentration %	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	11	---	---	---	---
---	BG II	20	---	---	---	---
0	1	7027	7011.5	8	876.4	1.00
10	2	5371	5355.5	8	669.4	0.76
25	3	3978	3962.5	8	495.3	0.57
50	4	3086	3070.5	8	383.8	0.44

1    = Control Group

2-4= Test Group

^a)   = The mean value was taken from the figures BG I and BG II

^b)       = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

RESULTS OF THE PRE-TEST:

Body Weights

Animal No.	Concentration %	Body Weight (g)
		prior 1^stApplication	prior to Sacrifice (Day 6)	Difference Day 1 to Day 6	Difference %
1	25	20.5	20.7	0.2	1.0
2	50	18.7	20.4	1.7	9.1

Ear Thickness

Animal No.	Conc. %	Ear Thickness
		prior to 1^stApplication (µm)			prior to 3^rdApplication (µm)			prior to Necropsy (µm)
		Right Ear	Left Ear	Mean	Right Ear	Left Ear	Mean	Right Ear	Left Ear	Mean
1	25	235	240	237.5	235	245	240.0	240	240	240.0
2	50	235	240	237.5	230	230	230.0	240	240	240.0

 

Animal No.	Difference Day 1 to Day 3 (µm)	Ear Swelling Day 3 (%)	Difference Day 1 to Day 6 (µm)	Ear Swelling Day 6 (%)
1	2.5	1.1	2.5	1.1
2	-7.5	-3.2	2.5	1.1

Ear Weights

Animal No.	Concentration %	Ear Weights after Necropsy (mg per animal)	% Increase Compared to Vehicle Values
1	25	24.83	-3.4
2	50	26.02	1.2

Mean of historical controls (DMF): 25.7 mg/ animal)

Ear Erythema

Animal No.	Score
	within 1 h after 1. appl.	24 h after 1. appl.	within 1 h after 2. appl.	24 h after 2. appl.	within 1 h after 3. appl.	24 h after 3. appl.	Day 5	Day 6
1	/	/	/*	/	/*	/	/	/
2	/*	/	/*	/*	/*	/*	/*	/

*substance residuals

Score:   / = No visible erythema
              1 = Very slight erythema
              2 = Well defined erythema             

              3 = Moderate to severe erythema

              4 = Severe erythema to formation of eschar which prevents grading of erythema

Interpretation of results:

GHS criteria not met

Conclusions:

The test item HER (Resorcinol bis-(2-Hydroxyethyl)ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol was not a skin sensitiser under the test conditions of this study.

Executive summary:

In the study the test item HER (Resorcinol bis-(2-Hydroxyethyl)ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol formulated in DMF (dimethylformamide) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 10, 25, and 50% (w/w).

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 0.76, 0.57, and 0.44 were determined with the test item at concentrations of 10, 25, and 50% in DMF, respectively.

The test item HER (Resorcinol bis-(2-Hydroxyethyl)ether, 2-[3-(2-hydroxyethoxy)phenoxy]ethanol was not a skin sensitiser under the test conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

According to the results obtained in all in vitro and in Chemico studies (OECD 442D, OECD 442E and OECD 442C), an OECD Guideline 429 study (LLNA) was performed and the result was negative. Therefore the test item does not need to be classified as skin sensitizer based on GHS criteria.