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

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

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.

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

242-440-8

EC Name:

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

Cas Number:

18599-22-9

Molecular formula:

C4H3BrF4

IUPAC Name:

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

Test material form:

liquid

In vitro test system

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.

Results and discussion

Positive control results:

The positive control cinnamic aldehyde had a mean EC 1.5 of 9.08 μM and mean IC50 of >64 μM.

In vitro / in chemico

Results

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%.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.