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Administrative data

Description of key information

Skin irritation

The skin irritation potential of4,4-bis(tert-butyldioxy)valerate (Luperox 230) was evaluated using the EpiskinTM reconstructed human epidermis model (Valin, 2016). 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 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,4,4-bis(tert-butyldioxy)valerate(Luperox 230) is considered to be non-irritant to skin.

 

Eye irritation

The potential irritant and corrosive properties of4,4-bis(tert-butyldioxy)valerate(Luperox 230) the eye was evaluated in the Bovine Corneal Opacity and Permeability (BCOP) test (Gerbeix, 2016. This method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage. The design of this study was based on the guideline OECD Guideline 437 and the study was performed in compliance with the OECD Principles of Good Laboratory Practice. Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C.A single validated experiment was performed using three corneas for each treated series (test item, positive control and negative control).Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test item, tested undiluted, and the negative and positive controls were applied in a single validated experiment using a treatment time of 10 minutes and using the closed-chamber. At completion of the treatment period, all formulations were removed from the front opening of the anterior chamber and the epithelia were rinsed. The corneas were then incubated for 2 hours (± 10 minutes) at +32°C before a second opacity measurement was performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at +32°C.At the end of the incubation period, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities. No notable opaque spots or irregularities were observed on all test item-treated corneas. All acceptance criteria were fulfilled. The study was therefore considered as valid. The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 0.As the mean IVIS was = 3, the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).Under the experimental conditions of this study,4,4-bis(tert-butyldioxy)valerate(Luperox 230) was identified as a test chemical not requiring classification for eye irritation.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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
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 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.
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
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 December 2015 -- 04 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Species:
cattle
Strain:
other: not applicable
Details on test animals or tissues and environmental conditions:
Species: bovine cattle.
Origin: bovine eyes were obtained from freshly slaughtered cattle at the abattoir EVA, Saint Pierre sur Dives, France.
Age: bovine cattle were up to 12 months old.
Reason for choice: bovine corneas are recommended by regulatory authorities for this type of study. They are adapted for the evaluation of potential ocular irritants since they are part of the target organ.
Transport from supplier to CiToxLAB France: the eyes were transported to CiToxLAB France at ambient temperature, immerged in buffered Hanks medium containing an antibiotic [Hank’s Balanced Salts Solution (HBSS) plus penicillin/streptomycin (100 units/100 µg/mL final)]. A container with smooth internal surfaces was used for the transport to avoid damage to the corneas.

Upon arrival at CiToxLAB France, the selection and preparation of corneas was performed as soon as possible. At each step of the preparation procedure, care was taken to avoid touching the corneas in order not to damage them.

Selection: a careful macroscopic examination was performed on all eyes to detect the presence of any defects (opacity, scratches, pigmentation, etc). Any eyes with defects were discarded. The examination was performed under a lamp, using HBSS in order to keep the eyes moistened and shiny. Particular attention was paid to the corneas and the eyes were swivelled in order to observe the fringe areas and any scratches directly under the light.
Preparation of the selected corneas: the tissues surrounding the eyeball were carefully pulled away and the cornea, surrounded by approximately 2 to 3 mm of sclera, was dissected out. The isolated corneas were stored in HBSS until all corneas had been prepared.
Washing of the corneas: the corneas were washed for 15 minutes, three times, in HBSS plus penicillin/streptomycin (100 units/100 µg/mL final) at room temperature. The corneas were used within a maximum of 24 hours.

(Pre)Incubation T°C: 32°C
Dates of experimental phase: from 08 January 2016 to 04 February 2016
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
Amount applied (volume or weight with unit): 750 µL
Duration of treatment / exposure:
Exposure period of 10 minutes (± 30 seconds) followed by rinsing
Observation period (in vivo):
Opacity measurement:
- before treatment
- after 2-hour incubation in water bath at +32°C (± 1°C).

Permeability measurement:
- after 90-min incubation in water (and other procedures), following the 2nd opacity measurement
Number of animals or in vitro replicates:
Not applicable
Triplicate corneas for each timepoint and tested substance (test item, negative control, positive control)
Details on study design:
REMOVAL OF TEST SUBSTANCE
- Rinsing: - the anterior chamber was emptied using a metal gavage tube attached to a vacuum pump,
- the corneas were rinsed six times with pre-warmed cMEM containing phenol red (i.e. until the dose formulation had been completely removed from the chamber or until the phenol red was not discoloured). Then, the corneas were finally rinsed with pre-warmed cMEM without phenol red.

NEGATIVE CONTROL:
As the test item was tested undiluted (i.e. in its original form), 0.9% Sodium Chloride (0.9% NaCl) was used as negative control.

Since several test items were assayed concurrently, the negative control was shared.

POSITIVE CONTROL:
As the test item was tested using a 10-minute treatment, the positive control was absolute ethanol. It was used neat and sampled on the day of use.

Since several test items were assayed concurrently, the positive control was shared.

SCORING SYSTEM/TOOL
- Opacity:
Using an opacitometer
The average change in opacity during exposure is determined. It is corrected by subtracting the average negative control value from values of positive control and test item.
- Permeability:
Using a spectrophotometer: optical density (OD) at 490 nm wavelength
The optical density is corrected by subtracting the average negative control value from values of positive control and test item.
- Scoring:
In vitro irritancy score (IVIS) = Corrected Opacity + (15 x Corrected OD)

Interpretation: see below
Irritation parameter:
in vitro irritation score
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
39
Irritation parameter:
cornea opacity score
Value:
0.3
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
10
Irritation parameter:
fluorescein leakage
Value:
0.006
Vehicle controls validity:
valid
Remarks:
0.005
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks:
1.911
Interpretation of results:
GHS criteria not met
Conclusions:
Under the experimental conditions of this study, the test item was identified as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category).
Executive summary:

The potential irritant and corrosive properties of Luperox 230 the eye was evaluated in the Bovine Corneal Opacity and Permeability (BCOP) test. This method can identify chemicals inducing serious eye damage and chemicals not requiring classification for eye irritation or serious eye damage. The design of this study was based on the guideline OECD Guideline 437 and the study was performedin compliance with CiToxLAB France standard operating proceduresand with the OECD Principles of Good Laboratory Practice. Corneas obtained from freshly slaughtered calves were mounted in corneal holders. Both chambers of the corneal holder were filled with complemented MEM culture media (cMEM) and pre-incubated for 1 hour and 5 minutes (± 5 minutes) at +32°C. A single validated experiment was performed using three corneas for each treated series (test item, positive control and negative control). Before the treatment, a first opacity measurement was performed on each cornea using an opacitometer. The test item, tested undiluted, and the negative and positive controls were applied in a single validated experiment using a treatment time of 10 minutes and using the closed-chamber. At completion of the treatment period, all formulations were removed from the front opening of the anterior chamber and the epithelia were rinsed. The corneas were then incubated for 2 hours (± 10 minutes) at +32°C before a second opacity measurement was performed. After the second opacity measurement, the medium of the anterior chamber was removed and filled with a fluorescein solution. The holders were then incubated vertically for 90 minutes (± 5 minutes) at +32°C. At the end of the incubation period, the optical density of the solution from the posterior chamber of each holder was measured in order to determine the permeability of the cornea. Each cornea was then observed for opaque spots and other irregularities. No notable opaque spots or irregularities were observed on all test item-treated corneas. All acceptance criteria were fulfilled. The study was therefore considered as valid. The mean In Vitro Irritancy Score (IVIS) of the test item-treated corneas was: 0. As the mean IVIS was = 3, the test item was considered as a test chemical not requiring classification for eye irritation or serious eye damage (UN GHS No Category). Under the experimental conditions of this study, Luperox 230 was identified as a test chemical not requiring classification for eye irritation.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

No classification is warranted for skin and eye irritation according to CLP/GHS criteria.