Registration Dossier

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 December 2015 -- 21 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Butyl 4,4-bis(tert-butyldioxy)valerate
EC Number:
213-626-6
EC Name:
Butyl 4,4-bis(tert-butyldioxy)valerate
Cas Number:
995-33-5
Molecular formula:
C17H34O6
IUPAC Name:
butyl 4,4-bis(tert-butylperoxy)pentanoate
Test material form:
other: Colorless to yellow liquid

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: EpiskinTM Model Kit (0.38 cm2 tissues)
Cell source:
other: SkinEthic Laboratories, Lyon, France
Vehicle:
unchanged (no vehicle)
Details on test system:
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37°C
- Temperature of post-treatment incubation (if applicable): 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Number of washing steps: several
- Observable damage in the tissue due to washing: no
- Modifications to validated SOP: no

DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Spectrophotometer: no data
- Wavelength: 570 nm
- Filter: no data
- Filter bandwidth: no data

NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION: 1

SCORING SYSTEM:
- Optical density (OD) was measured at 570 nm:
Relative mean viability (%) = 100 x mean cOD(test item) / mean cOD(negative control)
where:
- mean cOD Negative Control = mean ODNC – mean ODblank
- mean cOD Test Item = mean ODTI – mean ODblank
- mean cOD Positive Control = mean ODPC - mean ODblankPREDICTION MODEL / DECISION CRITERIA
Mean relative viability is = 50%: category 2 (H315)
Mean relative viability is > 50% and Mean IL-1a released concentration > 60 pg/mL: category 2 (H315)
Mean relative viability is > 50% and Mean IL-1a released concentration = 60 pg/mL: no category
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 µL

VEHICLE
- Amount(s) applied (volume or weight with unit): 10µL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 10µL
- Concentration (if solution): 5% (w/v) aqueous solution
Duration of treatment / exposure:
15 min
Duration of post-treatment incubation (if applicable):
MTT-loading after a 42h-incubation period following rinsing. Observation of MTT-> formazan transformation by viable cells.
Number of replicates:
3.

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Time point: 15 min exposure + 42h expression.
Value:
103
Vehicle controls validity:
valid
Remarks:
100%
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
12%
Remarks on result:
no indication of irritation
Remarks:
IL-1a concentrations < 0.5 pg/mL
Other effects / acceptance of results:
- OTHER EFFECTS:
- Visible damage on test system: no
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline: no

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item is considered to be non-irritant to skin.
Executive summary:

The skin irritation potential of Luperox 230 was evaluated using the EpiskinTM reconstructed human epidermis model. The study design was based upon international guidelines (OECD Guideline No. 439 and Commission Regulation (EC) No. 761/2009, B.46). The study was conducted in compliance with CiToxLAB France standard operating procedures and the principles of Good Laboratory Practice. Preliminary tests were performed to detect the ability of the test item to directly reduce MTT as well as its colouring potential. Following the preliminary tests, the skin irritation potential of the test item was tested in the main test. The test item and both the negative and positive controls were applied topically on triplicate tissues and incubated at room temperature for 15 minutes. At the end of the treatment period, each tissue was rinsed with D-PBS and incubated for 42 hours at +37°C, 5% CO2in a humidified incubator. The cell viability was then assessed by means of the colourimetric MTT reduction assay. Relative viability values were calculated for each tissue and expressed as a percentage of the mean viability of the negative control tissues which was set at 100% (reference viability). In addition, the concentration of the inflammatory mediator IL-1a was evaluated in the culture medium retained following the 42-hour recovery period. This quantification, based on an ELISA assay, was performed since the mean relative viability of the test item-treated tissues was > 50% following the MTT reduction assay.

In the preliminary tests, the test item was found not to have direct MTT reducing properties or colouring potential. All acceptance criteria for the negative and positive controls were fulfilled. The study was therefore considered to be valid. Following a 15 minutes exposure and 42 hours of recovery period, the relative mean viability of the tissues treated with the test item was 103% with a standard deviation of 3%. As the mean viability was > 50% after the MTT reduction, the IL-1a concentrations in culture media samples retained from the three negative controls and the three test item-treated tissues were analyzed by ELISA. The mean IL-1a concentration for treated tissues was found below the limit of quantification (i.e. < 5 pg/mL). Due to this value being below 60 pg/mL, the results met the criteria for an in vitro classification as non-irritant to skin.  Under the experimental conditions of this study, Luperox 230 is considered to be non-irritant to skin.