4-bromo-3,3,4,4-tetrafluorobut-1-ene

Endpoint summary

Description of key information

In chemico covalent binding to proteins: No reactivity (OECD TG 442C Direct Peptide Reactivity Assay (DPRA)

In Vitro Skin Keratinocyte Activation: No activity (OECD TG 442D, Induction of Antioxidant‐Response‐Element Dependent Gene Activity and Cytotoxicity in the Keratinocyte ARE‐Reporter Cell Line Keratinosens™

In Vitro Skin dendritic cell sensitization: non-sensitizer (OECD TG 442E, Human Cell Line Activation Test (H-CLAT))

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:

10 March 2020 to 13 March 2020

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:

25 June 2018

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.

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 highthroughput 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 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, two of which were valid and used, to determine the maximal induction (Imax), the concentration for maximal gene induction (CImax), the EC1.5 value (concen tration 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 article. For each definitive assay, the KeratinoSens™ cells were cultured in quadruplicate plates for approximately 24 hours, treated with the test article for 48±1 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 BTFB Induction of Antioxidant Response Element-Dependent Gene Activity and Cytotoxicity (Using M TT) in the Keratinocyte ARE-Reporter Cell Line KeratinoSens™ Assay was performed to determine the skin sensitization potential of the test article, supplied by The Chemours Company. The laboratory phase of this study was conducted from 10 March 2020 to 13 March 2020 at the Institute for In Vitro Sciences, Inc. (IIVS).

Evaluation of Test Results

A test article was predicted to have sensitization potential if:1) The EC1.5 value fell below 1000 μM in at least 2 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 a mean EC 1.5 of 9.08 μM and mean IC50 of >64 μM.

Key result

Run / experiment:

other: Mean of 2 definitive runs

Parameter:

other: Gene fold induction above the solvent control. There was no induction above 1.5 and viability was greater than 70%.

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

other: N/A see vehicle controls

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

The value of "0" is incorrect. The EC1.5 for test article BTFB was not determinable. There was no gene induction above 1.5-fold.

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 results of the three definitive trials for each plate were assessed using similar criteria outlined in the validation ring trial . Those acceptance criteria included: 1) variability in DMSO solvent control wells for each definitive assay was <20%; and 2) the positive control produced a statistically significant induction above 1.5 fold below 64 μM in each definitive assay. The second definitive trial was not valid due to the variability in the DMSO solvent control wells in excess of 20%.

The test article, BTFB, was tested in 3 definitive assays, 2 of which were valid and used; the second definitive assay did not meet the acceptance criteria and was not considered valid due to excessive solvent control variability. Each definitive assay included a set of 4 plates (3 for gene induction, 1 for cytotoxicity assessment). The test article, BTFB, was tested at 12 concentrations ranging from 0.977 to 2000 µM. The positive control, Cinnamic Aldehyde, was tested at 5 concentrations ranging from 4 to 64 µM. A summary of the EC_1.5 values (concentration inducing luciferase activity 1.5-fold (i.e., 50% above) that of the solvent controls) and the IC₃₀ and IC₅₀ values (concentrations leading to a 30% and 50% reduction in viability relative to solvent controls, respectively) of the definitive assays are presented in Table 1. Additional luciferase induction information (which was not used for the current prediction model) that includes the I_max (the maximal fold induction) and the CI_max (the concentration at which the maximal fold induction occurs), is also presented in Table 1.

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 was predicted to be a skin non-sensitizer.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The laboratory phase of this study was conducted from 21 February 2020 to 14 May 2020 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: EURL ECVAM DB ALM Protocol No. 158

Version / remarks:

2019

Deviations:

yes

Remarks:

See "Any other information on materials and methods incl. tables"

Qualifier:

according to guideline

Guideline:

other: OECD Test Guideline 442E

Version / remarks:

2018

Deviations:

yes

Remarks:

See "Any other information on materials and methods incl. tables"

GLP compliance:

yes

Type of study:

activation of dendritic cells

Justification for non-LLNA method:

Non-animal test method OECD approved. Activation of dendritic 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 or risk assessment.

Details on the study design:

The h-CLAT is an in vitro assay which measures the changes in the expression of cell surface markers CD54 (ICAM 1) and CD86 associated with the process of dendritic cell activation in the human acute monocytic leukemia cell line, THP 1 (American Type Culture Collection, ATCC, Manassas, VA, TIB 202™). 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 2.0×106 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 are measured by flow cytometry following cell staining with fluorescein isothiocyanate (FITC) labelled antibodies. Cytotoxicity measurement, using propidium iodide (PI) staining, is conducted concurrently to assess whether upregulation of surface marker expression occurs at sub-cytotoxic concentrations.

Positive control results:

Valid for all trials

Key result

Run / experiment:

other: 3 definitive trials Mean of EC200 trials - 816 μg/mL Mean of EC150 trials - >1000 μg/mL

Parameter:

other: EC200 cut-off at 200% for CD54 and EC150 cut-off at 150% for CD86

Value:

816 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

The results of the dose range assay and valid definitive trials are shown below. The results of the positive control (DNCB) are shown below. The criteria for a valid assay was achieved for each definitive trial.

Interpretation of results:

study cannot be used for classification

Conclusions:

All acceptance criteria for a valid assay were met for the definitive trials presented in this report. The test substance, BTFB, was considered a non-sensitizer according to the h-CLAT.

Executive summary:

The Human Cell Line Activation Test (h-CLAT) was used to assess the skin sensitization potential of test substance, BTFB (Lot# B236). 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 dendritic cell activation in the human leukemia cell line, THP-1, following exposure to a test substance. 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 Vitro Skin Sensitization: Human Cell Line Activation Test (h-CLAT)" [1]. Based upon the results of this study, the test substance, BTFB, was a potential non-sensitizer.

[1] OECD Test Guideline 442E "In Vitro Skin Sensitization: Human Cell Line Activation Test (h-CLAT)," Adopted 27 June 2018.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The laboratory phase of the study was conducted from 24 February 2020 to 27 February 2020 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))

Version / remarks:

June 2019

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 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.

Details on the study design:

The Direct Peptide Reactivity Assay was used to assess the skin sensitization potential of the test article. Synthetic peptides containing cysteine or lysine were reacted with the test article for 24 ± 2 hours. After the incubation period, the extent of peptide depletion was analyzed using High Performance Liquid Chromatography (HPLC) coupled with ultra-violet (UV) spectrometric detection.

Key result

Run / experiment:

other: single run

Parameter:

other: % Mean peptide depletion of Cysteine and Lysine

Value:

0.36 %

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Interpretation of results:

study cannot be used for classification

Conclusions:

Based on the results of the Direct Peptide Reactivity Assay, the test substance, BTFB (Lot/Batch#: B236), was predicted to be a non-sensitizer.

Executive summary:

The Direct Peptide Reactivity Assay was used to assess the skin sensitization potential of the test article, BTFB (Lot/Batch#: B236). The skin sensitization potential of the test article was evaluated by measuring the depletion of synthetic peptides containing either cysteine or lysine amino acids following incubation with the test article, using the protocol that is consistent with the OECD Test Guideline 442C "Key-Event-Based Test Guideline For In Chemico Skin Sensitisation Assays Addressing The Adverse Outcome Pathway Key Event On Covalent Binding To Proteins" [1]. Based upon the results of this study, the test article, BTFB, was classified as a non-sensitizer.

[1] OECD Test Guideline 442C "Key-Event-Based Test Guideline For In Chemico Skin Sensitisation Assays Addressing The Adverse Outcome Pathway Key Event On Covalent Binding To Proteins," 18 June 2019

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Three studies were performed addressing three steps in the Adverse Outcome Pathway for skin sensitisation.  The potential to initiate was addressed by measuring covalent binding to proteins, keratinocyte activation by measuring binding to the antioxidant/electrophile response element (ARE) and dendritic cell activation by monitoring the upregulation of the cell surface markers CD54 and CD86.

• In the DPRA assay, the test substance was predicted to be a non-sensitizer based in both the Cysteine only prediction model and the Cysteine and Lysine prediction model. The positive control confirmed that the assay functioned as expected.


• In the Ketatinosens™ assay, the test substance was predicted to be a non-sensitizer in both trials during the assay. An MTT direct reduction test showed that the test substance was not cytotoxic, and the positive control confirmed that the assay functioned as expected.


• In the h-CLAT assay, the test substance was predicted to be a non-sensitizer in all three trials during the assay. The positive control confirmed that the assay functioned as expected.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

No signs of irritation were noted in acute or repeated dose whole-body inhalation exposure studies.

Justification for classification or non-classification

Based on in chemico and in vitro tests performed in accordance with OECD guidelines 439, 431, and 437, the test substance not a skin sensitizer. Therefore, the substance does not need to be classified for skin sensitisation according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.  No assessment can be made for respiratory sensitisation.