[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 March 2020 to 3 April 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

other: EPA TSCA (40 CFR 792)

Deviations:

yes

Remarks:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Version / remarks:

25 June 2018

Deviations:

yes

Remarks:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Study was conducted in compliance with EPA TSCA (40 CFR 792). Study designed based off of OECD Guideline 442D.

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

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:

Reaction mass of 2(R/S)‐1‐{[(1R,2S,5R,8S)‐4,4,8‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐1‐yl]oxy}pentan‐2‐o and 2(R/S)‐1‐{[(1S,2S,5R,8R)‐1,4,4‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐8‐yl]oxy}pentan‐2‐ol

Molecular formula:

C20H36O2

IUPAC Name:

Reaction mass of 2(R/S)‐1‐{[(1R,2S,5R,8S)‐4,4,8‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐1‐yl]oxy}pentan‐2‐o and 2(R/S)‐1‐{[(1S,2S,5R,8R)‐1,4,4‐trimethyltricyclo[6.3.1.0~2,5~]dodecan‐8‐yl]oxy}pentan‐2‐ol

Test material form:

liquid: viscous

Details on test material:

Chemical name: 1‐[(4,4,8‐trimethyltricyclo[6.3.1.02,5]dodecan‐1‐yl)oxy]pentan‐2‐ol and 1‐[(1,4,4‐trimethyltricyclo[6.3.1.02,5]dodecan‐8‐yl)oxy]pentan‐2‐ol and isomers

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 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 signaling pathway with the repressor protein Keap1 and the transcription fact or 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 considered valid and used 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 AND IC30 (concentrations leading to 50% and 30% viability, respectively, as compared to solvent controls) for the test substance. 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 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 article, FRET 18-0091, supplied by International Flavours & Fragrances Inc.. The laboratory phase of this study was conducted from 31 March 2020 to 3 April 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.

Vehicle / solvent control:

DMSO

Positive control:

cinnamic aldehyde [442D]

Results and discussion

Positive control results:

The positive control, cinnamic aldehyde, had a mean EC 1.5 of 5.81 μM and mean IC50 of >64 μM.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

IC30 [442D]

Value:

14.93 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC 1.5 [442D]

Value:

3.71 µM

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Results 3

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

IC50 [442D]

Value:

13.23 µM

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 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 first definitive trial was not valid due to excess variability in the DMSO solvent control wells.

Any other information on results incl. tables

The test article, FRET 18-0091, was tested in 3 definitive assays. The first definitive assay was not considered valid due to excessive variability in the solvent control wells. Each definitive assay included a set of 4 plates (3 for gene induction, 1 for cytotoxicity assessment). The test article, FRET 18-0091, 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. A summary graph representing the luciferase fold induction and the cell viability for each tested concentration of the test article is included following Table 1.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Based upon 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 18-0091, was predicted to be a skin sensitizer.