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Description of key information

The substance [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide is considered as non-irritant for skin and eyes.  Analogous 25155-25-3 [1,3(or 1,4)-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide is not irritating to skin and is very slightly irritating to eyes.

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:
2012-03-06 to 2012-03-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-conducted and documented study performed under GLP according to current guideline.
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
human
Details on test animals and environmental conditions:
The EPISKIN(TM) model is a three-dimensional reconstructed human epidermis model consisting of adult human-derived epidermal keratinocytes seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. A highly differentiated and stratified epidermis model is obtained after a 13-day culture period comprising of the main basal, supra basal, spinous and granular layers and a functional stratum corneum.
Type of coverage:
other:
Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
Approximately 10 mg of the test item was applied to the epidermis surface. The epidermis surface had previously been moistened wtih 5 µl of sterile distilled water to improve contact between the solid test item and the epidermis.
Duration of treatment / exposure:
Triplicate tissues were treated wtih the test item for an exposure period of 15 minutes. Culbecco's Phosphate Buffered Saline (DPBS) with Ca++ and Mg++ was used as the negative control. Sodium Dodecyl Sulphate (SDS) 5% w/v was used as the positive control.
Observation period:
Rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.
Number of animals:
not applicable
Details on study design:
Pre-incubation: 2 ml maintenance medium, warmed to approximately 37 °C, was pipetted into the first column of 3 wells of a pre-labeled 12-well plate. Each epidermis unit was transferred into the maintenance medium felled wells (3 units per plate). A different 12-well plate wasused for the test item and each control item. The tissues were incubated at 37 °C, 5% CO2 in air overnight.
Application of test item and rinsing (Day 1): 2 ml maintenance medium, warmed to approximately 37 °C, was pipettted into the second column of 3 wells of the 12-well plate. Triplicate tissues were treated with the test item for an exposure period of 15 minutes. the test item was applied topically to the corresponsding tissues ensuring uniform covering. Approximately 10 mg of the test item ws applied to the epidermis surface. The epidermis surface had previously been moistened with 5 µl of sterile distilled water to improve contact between the solid test item and theepidermis. Triplicate tissues treated with 10 µl of DPBS served as the negative controls and triplicate tisues treated with 10 µl of SDS 5% w/v served as the positive controls. The plates were kept in the biological safety cabinet at romo temperature for 15 minutes.

At the end of the exposure period, each tissue was removed from teh well using forceps and rinsed using a wash bottle containing DPBS with Ca++ and Mg++. Rinsing was achieved uby filling and emptying each tissue insert for approximately 40 seconds using a constant soft stream of DPBS to gently remove any residual test item. The rinsed tissues were transferred to the second column of 3 wells containing 2 ml of maintenance medium in each well. The rinsed tissues were incubated at 37 °C, 5% CO2 in air for 42 hours.

Following the 42-hour post-exposure incubation period, each 12-well plate was placed onto a plate shaker for 15 minutes to homogenize the released mediators in the medium. 1.6 ml of the maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer at -14 to -30 °C for possible inflammatory mediator determination.

2 ml of a 0.3 mg/ml MTT solution, freshly prepared in assay medium, was pipetted into the third column of 3 wells of the 12 well plate(s). The tissues were transferred to the MTT filled wells, being careful to remove any excess maintenance medium from the bottom of the tissue insert by blotting on absorbent paper. The tissues were incubated for 3 hours at 37 °C, 5% CO2 in air. At the end of the 3-hour incubation period each tissue was placed onto absorbent paper to dry. A total biopsy of the epidermis was made using the EPISKIN(TM) biopsy punch. The epidermis was carefully separated from the collagen matrix using forceps and both parts (epidermis and collagen matrix) placed into labelled 1.5 ml micro tubes containing 500 µl acidified isopropanol, ensuring that both the epidermis and collagen matrix were fully immersed. Each tube was plugged to prevent evaporation and mixed throughly on a vortex mixer. The tubes we3re refrigerated at 1 to 10 °C until Day 6 of the experiment, allowing the extraction of the formazan crystals out of the MTT-loaded tissues.

At the end of the formazan extration period each tube was mixed throughly on a vortex mixer to produce a homogenous coloured solution.

For each tissue, duplicate 200 µl samples were transferred to the appropriate wells of a pre-labelled 96-well plate. 200 µl of acidified isopropanol alone was added to the two wells designated as "blanks". The optical density was measured (quantitative viability analysis) at 540 nm (without a reference filter) using the Anthos 2001 microplate reader.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean
Value:
99.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
The relative mean viability of the test item treated tissues was 99.5% after a 15-Minute exposure period.
Other effects:
The relative mean tissue viability for the positive control treated tissues was 7.8% relative to the negative control treated tissues and the standard deviation value of the percentage viability was 0.7%. The positive control acceptance criterion was tehrefore satisfied.

The mean OD540 for the negative control treated tissues was 0.847 and the standard deviation value of the percentage viability was 5.8%. The negative control acceptance criterion was therefore satisfied.

The standard deviation calculated from individual percentage tissue viabilities of three identically treated tissues was 3.3%. The test item acceptance criterion was therefore satisfied.

The MTT solution containing the test item did not turn blue which indicated that the test item did not directly reduce MTT.

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Tes test item is considered a Non-Irritant (NI).
Executive summary:

The purpose of this test was to evaluate the skin irritation potential of the test item using the EPISKIN(TM) reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-expousre incubation period of 42 hours. The principle of the assay was based on the measurement of cytotoxicity in reconstructed human epidermal cultures folowing topical exposure to thetest imte by means of the colourimetric MTT reduction assay. Cell viability is measured by enzymatic reduction of the yellow MTT tetrazolium salt (3 -[4,5 -dimethylthiazol-2 -yl]-2,5 -diphenyl-tetrazolium bromide) to a blue formazan salt (within the mitochondria of viable cells) in the test item treated tisssues relative to the negative controls. The concentration of the inflammatory mediator IL-1α in the culture medium retained following the 42 -hour post-exposure incubatino period is also determined for test items whicha re found to be borderline non-irritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result.

Triplicate tissues were treatd wtih the test item for an exposure period of 15 minutes. At the end of the exposure period each tissue was rinsed before incubating for 42 hours. At the end of the post-exposure incubation period each tissue was taken for MTT-loading. The maintenance medium from beneath each tissue was transferred to pre-labelled micro tubes and stored in a freezer for posssible inflammatory mediator determinatino. AFter MTTT loading a total biopsy of each epidermis wasmade and placed into micro tubes containing acidified isopropanol for extraction of formazan crystals out of the MTT-loaded tissues. At the end of the formazan extraction period each tube was mixed thoroughly and duplicate 200 µl samples were tranferred to the appropriate wells of a pre-labelled 96 -well plate. The optical density was measured at 540 nm. Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

The relative mean viability of the test item treated tissues was 99.5% after the 15 -minute exposure period. The quality criteria requried fora cceptance of results in the test were satisfied.

The test item was considered to be Non-Irritant (NI).

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:
20120-03-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-conducted and document study performed under GLP to current guideline.
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
yes
Remarks:
No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation.
GLP compliance:
yes (incl. certificate)
Species:
other: cow
Details on test animals or tissues and environmental conditions:
Not applicable
Vehicle:
other: mineral oil
Controls:
other: see above
Amount / concentration applied:
0.75 ml of a 20% w/v solution of test material in mineral oil
Duration of treatment / exposure:
240 minutes
Observation period (in vivo):
none
Number of animals or in vitro replicates:
Twelve corneas were used; three for the test material, three for the negative control, three for the positive control and three for the vehicle control.
Details on study design:
Source of Bovine Eyes
Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee and placed in Hanks' Balanced Salt Solution (HBSS), supplemented with Penicillin/Streptomycin, and transported to the laboratory on ice packs. The eyes were refrigerated on arrival and used within 24 hours of receipt. All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.

Preparation of Corneas
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed (epithelial side uppermost) in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders. The anterior and posterior chambers of each BCOP holder were filled with complete minimum essential medium (MEM) and plugged. The holders were incubated at 32 ±1°C for 60 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.

Selection of Corneas and Opacity Reading
The medium from both chambers of each holder was replaced with fresh complete MEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated. Three corneas were numerically allocated to the test item formulation. Three corneas were also numerically allocated to the negative control item, three corneas to the positive control item and three corneas to the mineral oil control.

Treatment of Corneas
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item at a concentration of 20% w/v in mineral oil or control items were applied to the cornea. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1°C for 240 minutes.

At the end of the exposure period the test item formulation and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. Due to the limited solubility in the MEM rinsing solutions, the corneas treated with the mineral oil and the corneas treated with the test item in mineral oil were initially swabbed with 1 % Tween 80 before rinsing with MEM.

The anterior chamber was refilled with fresh complete MEM. A post-treatment opacity reading was taken and each cornea was visually observed.

Application of Sodium Fluorescein and Permeability Determinations
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ±1°C for 90 minutes.
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 µL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492nm (00492) was measured. If values greater than 1.500 00492 were obtained a 1 in 5 dilution of the medium in complete MEM was performed and the measurement repeated. The modified value was multiplied by 5 to reflect the 1 in 5 dilution.

Histopathology
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
0.7
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
Treatment In Vitro Irritancy Score
Test Item 20% w/v in Mineral Oil 0.7
Negative Control 6.1
Positive Control 103.6
Mineral Oil 3.3

Corneal Epithelium Condition Post Treatment

Treatment  Cornea Number  Observation Post Treatment
 Negative Control  1

clear

 

 2

clear

 3

clear

 Positive Control

 4

cloudy

 

5

cloudy

 

6

cloudy

Mineral Oil

 7

 clear

 

 8

 clear

 

 9

 clear

 Test Item 20% w/v in Mineral Oil

 10

 clear

 

 11

 clear

 

 12

 clear

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The test item at a concentration of 20% w/v in mineral oil was considered not to be an ocular corrosive or severe irritant.
Executive summary:

A study was performed to assess the ocular irritancy potential of the test item to the isolated bovine cornea. The method was designed to be compatible with OECD Guidelines for the Testing of Chemicals No. 437 (2009) "Bovine Corneal Opacity and Permeability Assay" Method.

The test item was applied at a concentration of 20% w/v in mineral oil for 240 minutes. Negative, positive and mineral oil control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

The in vitro Irritancy scores are summarised as follows:

 Treatment

 In Vitro Irritancy Score

 Test Item 20% w/v in Mineral Oil

0.7 

 Negative Control

 6.1

 Positive Control

 103.6

 Mineral Oil Control

3.3 

The test item at a concentration of 20% w/v in mineral oil was considered not to be an ocular corrosive or severe irritant.

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation:

The purpose of this test was to evaluate the skin irritation potential of the 1,3 -bis(t-butylperoxy)benzene, CAS 2212 -81 -9 using the EPISKIN™ reconstructed human epidermis model after a treatment period of 15 minutes followed by a post-exposure incubation period of 42 hours. The concentration of the inflammatory mediator IL-1a in the culture medium retained following the 42-Hour post-exposure incubation period is also determined for test items which are found to be borderline nonirritant based upon the MTT reduction endpoint. This complimentary end-point will be used to either confirm a non-irritant result or will be used to override the non-irritant result. This method was designed to be compatible with the OECD Guidelines for the Testing of Chemicals No. 439 "In Vitro Skin Irritation"(adopted 22 July 2010) and Method 8.46 of Commission Regulation (EC) No. 440/200S/EC. The relative mean viability of the test item treated tissues was 99.5% after the 15-Minute exposure period. Therefore, the test item was considered to be Non-Irritant (NI).

The acute dermal irritation of structural analog [1,3(or 1,4)-phenylenebis(1 -methylethylidene)]bis[tert-butyl] peroxide

was evaluated in rabbits according to OECD 404 guideline (TNO, 1986). The substance was applied undiluted (purity 40%) and not pre-moistened to the skin of New-Zealand White albino rabbits and held in contact for 4 hours by means of an occlusive dressing. No irritation was observed. Under the experimental conditions, [1,3(or 1,4)-phenylenebis(1 -methylethylidene)]bis[tert-butyl] peroxide considered as non irritant when applied topically in rabbits.

Even though the purity was only 40% in the above noted acute dermal irritation study, data available from an acute dermal toxicity study (CIT, 1999) of structural analog [1,3(or 1,4)-phenylenebis(1 -methylethylidene)]bis[tert-butyl]peroxide where no dermal signs were observed at high doses (2000 mg/kg) indicate that the pure substance is not irritant.

Eye irritation

A study was performed to assess the ocular irritancy potential of 1,3 -bis(t-butylperoxy)benzene, CAS 2212 -81 -9 to the isolated bovine cornea according to OECD Guidelines for the Testing of Chemicals No. 437 (2009) "Bovine Corneal Opacity and Permeability Assay". The test item was applied at a concentration of 20% w/v in mineral oil for 240 minutes. Negative, positive and mineral oil control items were tested concurrently. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS). The IVIS for the test item was less than the negative control (physiological saline) and the vehicle control (mineral oil). Therefore, the test item at a concentration of 20% w/v in mineral oil was considered not to be an ocular corrosive or severe irritant.

The potential of structural analog [1,3(or 1,4)-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide

to induce ocular irritation was evaluated in rabbits (TNO, 1986) according to OECD 405 guideline (Klimisch 2 study). In this test, three male New Zealand White rabbits were used, 0.1 g of the substance (purity: 40%) was instilled in the conjunctival sac, and the eyes were not rinsed. After one hour, the eyes effects observed in all rabbits consisted of slight or moderate redness and slight or moderate swelling of the conjunctivae. All these clinical signs were fully reversible within 72 hours. All the mean scores calculated for each animal over 24, 48 and 72 hours were less than 1 (for chemosis, redness of the conjunctiva, iris lesions and corneal opacity). The purity of the substance was only 40 %; nevertheless, as only minimal signs of eye irritation were observed ( all scores were between 0 and 1, and as all clinical signs were fully reversible within 3 days), it is foreseeable that the substance with a higher purity is neither an eye irritant.

Conclusion

The substance [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide is considered as non-irritant for skin and eyes.

Justification for selection of skin irritation / corrosion endpoint:

In vitro irritation data from OECD 439 studies are now accepted as definitive; confirmation with in vivo data is no longer required.  These in vitro results are confirmed by in vivo data on a related substance (25155-25-3, a mixture of m- and p-isomers), which was found to be non-irritating.

Justification for selection of eye irritation endpoint:

In vitro irritation data from OECD 437 studies are now accepted as definitive; confirmation with in vivo data is no longer required.  These data are confirmed by in vivo studies on a related substance (25155-25-3, a mixture of m- and p-isomers), which was found to be slightly irritating.

Justification for classification or non-classification

According to the directive 67/548/EEC and EU Regulation (EC) N0. 1272/2008 (CLP), neither 1,3 -bis(t-butylperoxy)benzene, CAS 2212 -81 -9 nor its analog 25155 -25 -3 [1,3(or 1,4)-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide would be classified as a skin or eye irritant.