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Diss Factsheets

Toxicological information

Skin irritation / corrosion

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted from 1 May 2018 to 4 May 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 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
29 July 2016
Deviations:
yes
Remarks:
see Principles of method if other than guideline
Principles of method if other than guideline:
Deviations:
1. On 2 May 2018, there was a power failure at the Testing Facility. The power was restored around 7:30 am. The temperatures of the Kenmore and Thermo Scientific Freezer were between +2ºC to -4ºC at 7:42 am. This is a deviation from SOP0513.R7 that specifies acceptable freezer temperatures as -20±5ºC. This deviation affected storage conditions of any reagents stored at -20±5ºC. The temperature was in range (-16oC) at 10:14 am. The temperatures of the Fridgidaire, VWR, and Thermo Scientific refrigerators were between 9 to 10ºC at 7:41 to 7:43 am. This power failure affected storage conditions of any tissues and reagents stored at 2-8ºC. The responses of the assay positive and negative control were in line with the historical ranges at IIVS. Therefore, the deviations are not considered to have adversely affected the accuracy of the study.

2. On 2 May 2018, there was a power failure. The power was restored around 7:30 am. The temperatures of the Thermo Scientific Freezer were between +2ºC to -4ºC at 7:42 am. This is a deviation from SOP0513.R7 that specifies acceptable freezer temperatures as -20±5ºC. This deviation affected storage conditions of the test article included in this study and stored at -20±5ºC. The temperature was in range (-16oC) at 10:14 am. The Sponsor was informed of the power outage and was asked whether the test article can be used for testing. The Sponsor shared that storage at 2oC for 8 hours would have lost only 0.03% of the substance based on kinetic equation for thermal decomposition. Therefore, the Sponsor advised that the test material can be used for testing. The data analysis showed comparable responses of the tissues to the test article treatment at both exposure times. The deviation is not considered to have an adverse effect on the study quality and integrity.

3. The protocol states that to test for residual test article-mediated MTT reduction, two killed tissues will be treated with the positive control or test article concurrently with the viable tissues. During the study conduct, a significant difference in the MTT reduction on the second killed control tissue treated with the assay positive control for 3 minutes (raw OD550 value of 0.115 vs. 0.341 for the duplicate tissue). Therefore, another killed control experiment was conducted in the same day using duplicate tissues from the same lot. The raw OD550 values for the additional killed control tissues included in the study were 0.339 and 0.411, respectively, and comparable with the historical results. Under the circumstances, only the 3 OD550 corrected values for the 3 minutes killed control positive control that were comparable with historical values were averaged. The deviation is not considered to have an adverse impact on the study accuracy.
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
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-[({[(butan-2-yloxy)carbonyl]peroxy}carbonyl)oxy]butane
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:-15 to -25 degrees celcius

In vitro test system

Test system:
human skin model
Remarks:
human reconstructed skin
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
foreskin from a single donor
Source strain:
other: Contact manufacturer-MatTek
Details on animal used as source of test system:
N/A
Justification for test system used:
See guideline
Details on test system:
The EpiDerm™ Model (EPI-200) (MatTek Corporation, Ashland, USA) that is used in this study
consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi
layered, highly differentiated model of the human epidermis. It consists of organized basal, spinou
s and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid l
ayers arranged in patterns analogous to those found in vivo. The EpiDerm™ Model incorporates
several features which make it advantageous in the study of potential dermal corrosivity. First, the
test system uses a serum-free medium which eliminates the possibility of serum protein and test
article interaction (Shopsis and Eng, 1988). Secondly, the target cells are epithelial, derived from
human skin (Cannon et al., 1994). Third, since the tissue has a functional stratum corneum, the test
materials are applied directly to the tissue surface, at air interface, so that undiluted and/or end use
dilutions can be tested directly (Harbell et al., 1994).
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
Neat
Duration of treatment / exposure:
3 mins and 60 mins
Duration of post-treatment incubation (if applicable):
N/A
Number of replicates:
2 per exposure time

Test system

Type of coverage:
other: Topical

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
1 experiment
Value:
36.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: 3 min tissue vialbilty=83.0% and 60 minutes tissue viability = 36.9%
Other effects / acceptance of results:
CRITERIA FOR DETERMINATION OF A VALID TEST
The assay will be accepted if the following criteria are met: 1) the positive control results in a corrosive
classification (i.e., <50% cell viability compared to negative controls, after a 3-minute exposure, and/
or <15% after a 60-minute exposure). 2) the mean OD550 value of the negative control tissues is ≥
0.8 and < 2.8.

Any other information on results incl. tables

The test article Di-sec-butyl peroxydicarbonate was tested in the EpiDerm™ Corrosivity Assay. Two

tissues were used to assess viability after a 3-minute exposure, and two tissues were used to assess

viability after a 60-minute exposure. Negative and positive controls were tested in parallel. Table 1

summarizes the mean % viability results for the test article and the positive control. The raw and analyzed

data are presented in attached report. The classification of the positive control, 8N KOH, was determined

to be corrosive thereby meeting the acceptance criterion.

The positive control, 8N potassium hydroxide (8N KOH), is known to directly reduce MTT in the absence

of viable cells. Therefore, a killed-control experiment was performed. The results of the killed control

experiment showed that there was significant direct MTT reduction in the positive control-treated killed

controls. Additional calculations were performed to correct for the amount of MTT reduced directly by

the positive control residues as described in the Presentation of Data section.

The test article as not determined to be a colorant (was not considered to have potential interference

with the MTT measurement) and was not observed to directly reduce MTT in the absence of viable cells.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based upon the results of this assay, the test substance, Di-sec-butyl peroxydicarbonate, was predi
cted to be non corrosive according to the prediction model of the OECD TG 431. Accordingly, the te
st substance, Di-sec-butyl peroxydicarbonate, would be labeled as a non corrosive material within the
Globally Harmonized System.
Executive summary:

The MatTek Corporation’s EpiDerm reconstituted human epidermis model was used to assess the

potential skin corrosivity of the test substance, Di-sec-butyl peroxydicarbonate.The skin corrosivity

potential of the testsubstancewas evaluated by measuring the relative cell viability in treated tissues after

a 3-minute and 60-minute exposure to the test substance according to theOECD Test Guideline 431

“In VitroSkin Corrosion: Human Skin Model Test” (TG 431)[1].The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-

diphenyltetrazolium bromide) conversion assay, which measures the NAD(P)H-dependent microsomal

enzyme reduction of MTT (and to a lesser extent, the succinate dehydrogenase reduction of MTT) to

a blue formazan precipitate, was used to assess cellular metabolism after test substance exposure[2].

Under the conditions specified inthis study, the testsubstance,Di-sec-butyl peroxydicarbonate, resulted

in a cell viability of 83% and 16.9% after the 3 minute and the 60 minute exposures, respectively;

thus, the test substance was predicted to be corrosive. Accordingly, the test substance,Di-sec-butyl

peroxydicarbonate, would not require classification and labelling for skin corrosivity[KJ(1] .Results of

the positive control and negative control met the criteria of a valid assay.

[1] OECD Test Guideline 431 “In Vitro Skin Corrosion: reconstructed human epidermis (RHE)

test method”, Adopted 29 July 2016.

[2] Berridge, M.V., Tan, A.S., McCoy, K.D., Wang, R. (1996) The Biochemical and Cellular Basis

of Cell Proliferation Assays That Use Tetrazolium Salts.Biochemica4:14-19.