Reaction mass of bisisopropyl peroxydicarbonate and bis-sec-butyl peroxydicarbonate and isopropyl-secbutylperoxydicarbonate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

April 15-19, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

other: Preliminary unaudited data

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

February 2015

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:

Bis-sec-butyl peroxydicarbonate

EC Number:

243-424-3

EC Name:

Bis-sec-butyl peroxydicarbonate

Cas Number:

19910-65-7

Molecular formula:

C10H18O6

IUPAC Name:

2,2'-[dioxybis(carbonyloxy)]dibutane

Test material form:

liquid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
-Source and lot/batch No of test material: Sponsor Lot No. 17121B3105
-Expiration date of the lot/batch:27 November 2018
Purity test date:10 February 2018
Purity:99.3%
Appearance:Clear colorless non-viscous liquid
Storage condition of test material:-15to -25 degrees celcius

In vitro test system

Details on the study design:

Test system
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 (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
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 perfo
rmed 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 M
TT) in the Keratinocyte ARE- Reporter Cell Line KeratinoSens Assay was performed to determine the
skin sensitization potential of the test articles, supplied by AkzoNobel.
The laboratory phase of this study was conducted from 15 to 19 April 2018 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 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.

Results and discussion

Positive control results:

The positive control cinnamic aldehyde had an EC 1.5 of <4 μM and IC50 of >64μM.

In vitro / in chemico

Results

Key result

Run / experiment:

other: Mean of 3 definitive runs

Parameter:

other: EC 1.5 Value (uM)

Value:

2 000

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

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

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 are 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.

Any other information on results incl. tables

The test article, Di-sec-butyl peroxydicarbonate 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 0.977 to 2000 μM. The positive control, Cinnamic Aldehyde,

was tested at 5 concentrations ranging from 4 to 64 μM. A summary of the EC1.5 (concentration

for a statistically significant induction of 50% above solvent controls) and IC(concentration leading to

50% viability as compared to solvent controls) results 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 Imax(the maximal fold induction) and the CImax(the concentration at which the maximal

fold induction occurs), is also presented . A summary graph representing the luciferase fold induction

and the cell viability for each tested concentration of the test article is included .

A 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, Di-sec-butyl peroxydicarbonate was predicted

to be a non sensitizer.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

DPRA and H-CLAT still in progress

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 Di-sec-butyl peroxydicarbonate was predicted to be a skin non-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, Di-sec-butyl peroxydicarbonate (Lot# 17121B3105). 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,Di-sec-butly peroxydicarbonate (Lot# 17121B3105) was classified

as a skin non sensitizer.

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

Adopted 4 February 2015.