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Description of key information

According to EU REACH regulations, the use of non-animal alternative test methods to address skin sensitization are prefered to in vivo methods. Additionally, EURL ECVAM has published a guidance document recommending the use of non-animal approaches for skin sensitization assessment (March 2017) using a weight-of-evidence approach. Therefore, to evaluate the sensitization potential of FRET 11-0539, three validated alternative models were used. These models incuded the DPRA, KeratinoSens, and hCLAT. The results of each assay were positive, indicating FRET 11-0539 is a potential skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
The laboratory phase of the study was conducted from 18 December 2014 to 25 June 2015 at the Institute for In Vitro Sciences, Inc.
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
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
Non animal test method - OECD approved. The Direct Peptide Reactivity assay provides an in vitro procedure used for supporting the discrimination between skin sensitizers and non sensitizers in accordance with the UN GHS. According to REACH, In vivo methods can only be used if the in chemico or in vitro test methods are not adequate for the substance or cannot be used for classification and risk assessment.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
-Source and lot/batch No of test material: Sponsor Lot No. RB272-88
-Expiration date of the lot/batch: 31 December 2015
-Purity: >99%
-Appearance: clear colorless liquid
-Storage condition of test material: Ambient temperature, dark
Details on the study design:
The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 hours incubation with the test chemical at 25+/- 2.5ºC. The synthetic peptides contain phenylalanine to aid in the detection. Relative peptide concentration is measured by high performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm. Cysteine and lysine peptide percent depletion values are then calculated and used in a prediction model which allows assigning the test chemical to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.
Positive control results:
Cinnamic aldehyde % peptide depletion for cysteine and lysine were 81.8% and 63.0%, respectively.
Key result
Run / experiment:
other: Single run
Parameter:
other: % mean peptide depletion of cysteine and lysine
Value:
10.5
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Prediction Model:

Mean Peptide Depletion of Cysteine and Lysine (%)

DPRA Prediction

Peptide Depletion of Cysteine (%)

0 - 6.38

Minimal Reactivity

Negative

0 - 13.89

Minimal Reactivity

6.39 - 22.62

Low Reactivity

Positive

13.90 - 23.09

Low Reactivity

22.63- 42.47

Moderate Reactivity

23.10- 98.24

Moderate Reactivity

42.48 - 100

High Reactivity

98.25 - 100

High Reactivity

DPRA Results:

IIVS Test Article Number

Sponsor’s Designation

% Mean Peptide Depletion

% Mean Peptide Depletion of Cysteine and Lysine

Reactivity (Cysteine and Lysine)

Potential Sensitizer?

Cysteine

Lysine

14AQ07

FRET 11-0539

17.0

4.1

10.5

Low

Yes

Positive Control

Cinnamic Aldehyde

81.8

63.0

 

 

Interpretation of results:
study cannot be used for classification
Conclusions:
Based on the results of the Direct Peptide Reactivity Assay for the test article, FRET 11-0539 (Lot# RB272-88), was predicted to be a potential skin sensitizer.
Executive summary:

The Direct Peptide Reactivity Assay was used to assess the skin sensitization potenetial of the test substance, FRET 11-0539 (Lot# RB272 -88). The skin sensitization potential of the test substance was evaluated by measuring the depletion of synthetic peptides containing either cysteine or lysine amino acids following incubation with the test substance, using the protocol that is consistent with the OECD Test Guideline 442C “In ChemicoSkin Sensitization: Direct Peptide Reactivity Assay (DPRA)”[1]. Based upon the results of this study, the test substance, FRET 11-0539, was classified as a skin sensitizer.


[1]           OECD Test Guideline 442C “In ChemicoSkin Sensitization: Direct Peptide Reactivity Assay (DPRA)”, Adopted 4 February 2015.

 

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
The laboratory phase of this study was conducted from 23 August 2017 to 28 February 2018 at the Institute for In Vitro Sciences, Inc.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Test Guideline 442E
Version / remarks:
July 2016
Deviations:
yes
Remarks:
Cell pellet resuspension was wrongly worded in the protocol. The procedure prescribed in the protocol was an error which was amended. The deviation did not affect the study because the correct procedure was used in the assay.
GLP compliance:
yes
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
Non animal test method - OECD approved. Activation of dentritic cells by the human Cell Line Activation Test (h-CLAT) provides an in vitro procedure used for supporting the discrimination between skin sensitizers and non sensitizers in accordance with the UN GHS. According to REACH, In vivo methods can only be used if the in chemico or in vitro test methods are not adequate for the substance or cannot be used for classification and risk assessment.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
-Source and lot/batch No of test material: Sponsor Lot No. RB272-88
-Expiration date of the lot/batch: January 2019
-Purity: 99.1%
-Appearance: clear colorless liquid
-Storage condition of test material: Ambient temperature, dark
Details on the study design:
The Human Cell Line Activation Test (h-CLAT) was used to identify potential skin sensitizers and non-sensitizers in accordance with the United Nations Globally Harmonized System (UN GHS). The skin sensitization potential of a test article was evaluated by measuring the changes in the expression of cell surface markers CD54 and CD86 associated with the process of dendritic cell activation in the human leukemia cell line, THP-1 (American Type Culture Collection, ATCC, Manassas, VA, TIB-202™), following exposure to a test article. Dendritic cell activation is considered one of the key biological events in the adverse outcome pathway for skin sensitization, where CD54 and CD86 are subsequently involved in dendritic cell migration to the lymph nodes and T-cell priming. THP-1 cells, seeded at a density of 2.0×10^6 cells/mL in culture medium in 24-well plate format define the Test System. After treatment of the test or control articles to the Test System, the expression of cell surface markers were measured by flow cytometry following cell staining with fluorescein isothiocyanate (FITC) labelled antibodies. Cytotoxicity measurement, using propidium iodide (PI) staining, was conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations.
Positive control results:
Pass
Key result
Run / experiment:
other: 5 definitive assays
Parameter:
other: EC200 cut-off of 200% for CD54 and 150% for CD86
Value:
27.9
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid

Positive Control:

Positive Control

Trial

CD54 RFI

CD86 RFI

Cell Viability (%)

Pass/Fail

2,4-Dinitrochlorobenzene

Preliminary Assay

Not Tested

Not Tested

65.74

Pass

B1

443.9

212.4

59.94

Pass

B2

665.2

264.0

62.18

Pass

    B5   1025.1   198.1   67.35   Pass

Test Article:

IIVS Test Article Number

Sponsor’s Designation

CV75 (µg/mL)

Definitive Trial

EC200

(µg/mL)

EC150

(µg/mL)

Sensitization Potential

17AA07

FRET 11-0539

83.4

B1

<28.0

NA

Yes

B2

<27.9

NA

Yes

        B5   42.0   NA   Yes
Interpretation of results:
study cannot be used for classification
Conclusions:
Based on the results of the human Cell Line Activation test for the test article, FRET 11-0539 (Lot# RB272-88), was predicted to be a potential skin sensitizer.
Executive summary:

The human Cell Line Activation Test was used to assess the skin sensitization potenetial of the test substance, FRET 11-0539 (Lot# RB272-88). The skin sensitization potential of the test substance was evaluated by measuring the changes in the expression of cell surface markers CD54 and CD86 associated with the process of dendritic cell activation in the human leukemia cell line, THP-1, following exposure to a test article.  Dendritic cell activation is considered one of the key biological events in the adverse outcome pathway for skin sensitization, where CD54 and CD86 are subsequently involved in dendritic cell migration to the lymph nodes and T-cell priming. The testing was done using the protocol that is consistent with the OECD Test Guideline 442E “In VitroSkin Sensitization: human Cell Line ActivationTest (h-CLAT)”[1]. Based upon the results of this study, the test substance, FRET 11 -0539, was classified as a skin sensitizer.


[1]           OECD Test Guideline 442E “In VitroSkin Sensitization: human Cell Line activation Test (h-CLAT)”, Adopted 26 July 2016.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
The laboratory phase of this study was conducted from 16 December 2014 to 23 January 2015 at the Institute for In Vitro Sciences, Inc. (IIVS).
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
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
Non animal test method - OECD approved. Activation of keratinocytes by the Induction of Antioxidant-Response-Element Dependent Gene Activity and Cytotoxicity (Using MTT) in the Keratinocyte ARE Reporter Cell Line provides an in vitro procedure used for supporting the discrimination between skin sensitizers and non sensitizers in accordance with the UN GHS. According to REACH, In vivo methods can only be used if the in chemico or in vitro test methods are not adequate for the substance or cannot be used for classification and risk assessment.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
-Source and lot/batch No of test material: Sponsor Lot No. RB272-88
-Expiration date of the lot/batch: 31 December 2015
-Purity: >99%
-Appearance: clear colorless liquid
-Storage condition of test material: Ambient temperature, dark
Details on the study design:
The Induction of Antioxidant-Response-Element Dependent Gene Activity and Cytotoxicity (Using MTT) in the Keratinocyte ARE-Reporter Cell Line KeratinoSens skin sensitization assay is a high-throughput cell-based in vitro test to screen for the skin sensitization potential of chemicals. The KeratinoSens cells (Givaudan, Switzerland) were propagated as a reporter cell line. The KeratinoSens cells are transfected HaCaT keratinocytes that include a 56-base-pair insertion containing the ARE sequence from the AKR1C2 gene, a SV40 promoter, the luciferase gene in the vector pGL4 from Promega, and a stable insertion allowing for cells to be selected in the presence of Geneticin (G418). The signalling pathway with the repressor protein Keap1 and the transcription factor Nrf2, which binds to the antioxidant / electrophile response element (ARE / EpRE), was shown to be a valuable cellular endpoint to detect skin sensitizers in vitro. The induction of luciferase directly indicates the activation of ARE dependent genes.
Cytotoxicity of a test article was assessed using cell MTT (3-[4,5 - dimethylthiazol-2-yl] - 2,5 - diphenyl tetrazolium bromide). Cytotoxicity was determined by measuring the relative conversion of MTT in test article-treated cultures compared to the solvent control at 570nm absorbance.

Experimental Design

The experimental design of this study consisted of three definitive assays to determine the maximal induction (Imax), the concentration for maximal gene induction (CImax), the EC1.5 value (concentration for a statistically significant induction of 50% above solvent controls), and a mean IC50 (concentration leading to 50% viability as compared to solvent controls) for the test articles. For each definitive assay, the KeratinoSens cells were cultured in quadruplicate plates for 24 hours, treated with the test articles for 48 hours, and assessed for luciferase induction (3 plates) and cytotoxicity (1 plate). The procedures that were performed in this assay were a modification of the procedures previously described by Natsch, et al. (2008) and were performed similar to those procedures performed by the Institute for In Vitro Sciences, Inc. in the KeratinoSens ring-study. The Induction of Antioxidant-Response-Element Dependent Gene Activity and Cytotoxicity (Using MTT) in the Keratinocyte ARE- Reporter Cell Line KeratinoSens Assay was performed to determine the skin sensitization potential of the test articles, supplied by International Flavors & Fragrances Inc.

Evaluation of Test Results

A test article was predicted to have sensitization potential if:1) The EC1.5 value fell below 1000 μM or 200 μg/ml 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 apparent overall dose response which was similar between repetitions.
Positive control results:
The positive control cinnamic aldehyde had an EC 1.5 of 8.43 μM and mean IC50 of > 64μM.
Key result
Run / experiment:
other: mean of 3 definitive runs
Parameter:
other: EC 1.5 value (uM)
Value:
26.06
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
Criteria for Determination of a Valid Definitive Assay: The KeratinoSens assay was accepted when the positive control (cinnamic aldehyde) caused an EC1.5 value that fell within two standard deviations of the historical mean. Additionally, the test article was predicted to have sensitization potential if: 1) The EC1.5 value fell below 1000 μM 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.

The test article, FRET 11-0539 was tested in three definitive assays. Each definitive assay included a set of 4 plates (3 for gene induction, 1 for cytotoxicity assessment). The test article, was tested at 12 concentrations ranging from 0.978 to 2000 μM. The positive control, Cinnamic Aldehyde, was tested at 5 concentrations ranging from 4 to 64 μM. A summary graph representing the luciferase fold induction and the cell viability for each tested concentration of the test article is included. The test article was predicted to have sensitization potential if: 1) The EC1.5 value fell below 1000 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, FRET 11-0539 was predicted to be a sensitizer.

Interpretation of results:
study cannot be used for classification
Conclusions:
Based on the data from The Induction of Antioxidant-Response-Element Dependent Gene Activity and Cytotoxicity (Using MTT) in the Keratinocyte ARE- Reporter Cell Line KeratinoSens assay, the test article FRET 11-0539 was predicted to be a skin sensitizer.
Executive summary:

The Induction of Antioxidant-Response-Element Dependent Gene Activity and Cytotoxicity (Using MTT) in the Keratinocyte ARE- Reporter Cell Line KeratinoSens was used to assess the skin sensitization potential of the test article, FRET 11-0539 (Lot# RB272-88). The skin sensitization potential of the test article was evaluated using the protocol that is consistent with the OECD Test Guideline 442D “In VitroSkin Sensitisation: ARE-Nrf2 Luciferase Test Method”[1]. Based upon the results of this study, the test article, was classified as a skin sensitizer.

[1] OECD Test Guideline 442D “In VitroSkin Sensitisation: ARE-Nrf2 Luciferase Test Method)”,

Adopted 4 February 2015.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Justification for classification or non-classification

Three different validated, non-animal alternative models were used to evaluate the sensitiation potential of FRET 11-0539 (DPRA, KeratinoSens, and hCLAT). The results of these assays consistently demonstrated FRET 11-0539 to be a contact sensitizer. Therefore, based on the weight of evidence, it is appropriate to classify FRET 11-0539 as a skin sensitizer.