Di-tert-butyl peroxide

Endpoint summary

Description of key information

Di-tert-butyl peroxide has been tested for skin irritation in vivo and eye irritation in vitro and in vivo.  It is not irritating to skin or eyes in any of these studies.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well-conducted GLP study performed according to established protocol. Test article is well characterized and a certificate of analysis is included in the report.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

Three animals were used as recommended by international guidelines. Animals were identified individually with a metal tag in the ear. On the day of treatment, the animals had a mean body weight of 2.4 +/- 0.1 kg. Animals were acclimated at least 5 days before the beginning of the study. Conditions in the animal room were as follows: temperature, 18 +/- 3 °C; relative humidity, 30 to 70%; light/dark cycle, 12 hr/12 hr; ventilations, approximately 12 cycles/hour of filtered, non-recycled air. Animals were housed individually in polystyrene cages. Each cage was equipped with a food container and a water bottle. During the study, the animals had free access to 112C pelleted diet. Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum.

Type of coverage:

semiocclusive

Preparation of test site:

shaved

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

Doses of 0.5 ml were placed on a 6 cm2 dry gauze pad, which was then applied to the right flank (application for 4 hours) or the left flank (application for 3 minutes) of the animals.

Duration of treatment / exposure:

Three minutes (one animal) or four hours (three animals).

Observation period:

One hour, 24, 48 and 72 hours after removal of the dressing or until reversibility of cutaneous reactions.

Number of animals:

Three

Details on study design:

The test substance was evaluated in one animal in a first assay. The duration of exposure was 3 minutes on one flank and 4 hours on the other flank. Since the test substance was not irritant in this first assay, it was then applied for 4 hours to two other animals in a second assay. The test substance was used undiluted. Doses of 0.5 ml were placed on a 6 cm2 dry gauze pad, which was then applied to the right flank (application for 4 hours) or the left flank (application for 3 minutes) of the animals. The test substance and the gauze pad were held in contact with the skin by means of an adhesive hypoallergenic aerated semi-occlusive dression and a restraining bandage. The untreated skin sered as control No residual test substance was observed on removal of the dressing.

Irritation parameter:

erythema score

Basis:

mean

Time point:

24/48 h

Score:

0

Irritation parameter:

erythema score

Basis:

mean

Time point:

72 h

Score:

1.7

Irritation parameter:

edema score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Irritant / corrosive response data:

After a 3-minute exposure (one animal), no cutaneous reactions were observed. After a 4-hour exposure (three animals), a very slight erythema (grade 1) was observed in one animal on day 1. A well-defined erythema (grade 2) was observed in the second animal between days 1 and 3, then a very slight erythema (grade 1) was noted on day 4. It was replaced by dryness ofthe skin from day 5 up to day 8. No cutaneous reactions were observed in the third animal.

Other effects:

None recorded.

Mean scores over 24, 48 and 72 hours for each animal were 0.0, 0.0 and 1.7 for erythema and 0.0, 0.0 and 0.0 for oedema.

Interpretation of results:

not irritating

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under our experimental conditions, the test substance PEROXYDE DE DI-t-BUTYLE (batch No. 990-9801-124) is non-irritant when applied topically to rabbits.

Executive summary:

The potential of the test substance PEROXYDE DE DI-t-BUTYLE (batch No. 990-9801-124) to induce skin irritation was evaluated in rabbits according to OECD (No. 404, 17th July 1992) and EC (92/69/EEC, B.4, 31st July 1992) guidelines. After a 3 -minute exposure (one animal), no cutaneous reactions were observed. After a 4-hour exposure (three animals), a very slight erythema (grade 1) was observed in one animal on day 1. A well-defined erythema (grade 2) was observed in the second animal between days 1 and 3, then a very slight erythema (grade 1) was noted on day 4. It was replaced by dryness of the skin from day 5 up to day 8. No cutaneous reactions were observed in the third animal. Under our experimental conditions, the test substance PEROXYDE DE DI-t-BUTYLE (batch No. 990-9801-124) is non-irritant when applied topically to rabbits.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2010-03-31 to 2010-09-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well-documented study performed under GLP according to established guidelines with no deviations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Test Animals:
Animals: Young Adult New Zealand White Rabbit, SPF
Rationale: Recognized by international guidelines as a recommended test system.
Breeder: Charles River Laboratories France, BP 0109, 69592 L’Arbresle / France
Number of Animals per Test: 3 (Animals of both sexes were used)
Age (when treated): 14 weeks (male), 18 weeks (females)
Body Weight Range (when treated): 2826 g (male), 2706 - 2707 g (females)
Identification: By unique cage number and corresponding ear number.
Acclimatization: Under laboratory conditions after health examination. Only animals without any visual signs of illness were used for the study.
Randomization: Selected by hand at time of delivery. No computer generated randomization program.

Environmental Conditions:
Conditions: Standard Laboratory Conditions. Air-conditioned with 10-15 air changes per hour, and continuously monitored environment with a room temperature of 17-23 °C and a relative humidity between 30-70%, automatically controlled light cycle of 12 hours light and 12 hours dark, music during the daytime light period.
Accommodation: Individually in stainless steel cages equipped with feed hoppers and drinking water bowls.
Diet: Pelleted standard Kliba Nafag 3418 rodent maintenance diet (batch no. 05/10) provided by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) available ad libitum. Results of analyses for contaminants are archived at Harlan Laboratories Ltd. A piece of wood (imported by Indulab AG, Gams / Switzerland from ABEDD® - LAB & VET GmbH, 1160 Vienna / Austria) and a haystick 4642 (batch no. 54/09, Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) were also provided for environmental enrichment.
Water: Community tap water from Füllinsdorf ad libitum. Results of bacteriological, chemical and contaminant analyses are archived at Harlan Laboratories Ltd.

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.1 mL of di-tert-butyl peroxide (CAS# 110-05-4) was withdrawn with a syringe and applied undiluted. The test item was applied at 0.1 mL/animal, the dose specified in the test guidelines for a liquid test item.

Duration of treatment / exposure:

Single exposure.

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3 (1 male and 2 females)

Details on study design:

Di-tert-butyl peroxide (CAS# 110-05-4) was used as delivered by the Sponsor.


The pH of the test item was measured from an aliquot of the test item, before the study initiation date. By using pH strips , the pH was found to be 4.

According to Commission Regulation (EC) No 440/2008 B.5. and OECD Guidelines 405, a test item is not required to be tested if the pH-value is less than 2 or greater than 11.5, owing to its predictable corrosive properties.

0.1 mL of di-tert-butyl peroxide (CAS# 110-05-4) was withdrawn with a syringe and applied undiluted. The test item was applied at 0.1 mL/animal, the dose specified in the test guidelines for a liquid test item.

The eyes of the animals were examined one day prior to test item administration. The test item was placed in the conjunctival sac of the left eye of each animal after gently pulling the lower lid away from the eyeball. The lids were then gently held together for about one second to prevent loss of test item. The right eye remained untreated and served as the reference control.

A single animal (one female) was treated first. As neither a corrosive effect nor a severe irritant effect was observed after the 1- and 24-hour examinations, the test was completed using the two remaining animals.

Rationale: The application form and dose were used to detect an irritating potential of the test item applied.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Irritation parameter:

iris score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0.33

Reversibility:

fully reversible

Irritation parameter:

conjunctivae score

Basis:

animal #3

Time point:

24/48/72 h

Score:

0.33

Reversibility:

fully reversible

Irritation parameter:

chemosis score

Basis:

mean

Time point:

24/48/72 h

Score:

0

Irritant / corrosive response data:

The instillation of di-tert-butyl peroxide (CAS# 110-05-4) into the eye resulted in mild, early onset and transient ocular changes, such as slight to moderate reddening of the conjunctivae and slight to moderate reddening of the sclerae. Also, slight swelling of the conjunctivae was noted, although exclusively 1 hour after instillation. All effects were reversible and were no longer evident 72 hours after treatment, the end of the observation period for all animals. No abnormal findings were observed in the cornea or for the iris light reflex of any animal at any of the examinations. No corrosion was observed at any of the measuring intervals. No staining of the treated eyes by the test item was observed and no clinical signs were observed.

Other effects:

Coloration: No staining produced by the test item was observed in the treated eyes.
Corrosion: No corrosion of the cornea was observed at any of the reading times.

Viability / Mortality: No intercurrent deaths occurred during the course of the study.

Clinical Signs: No clinical signs were recorded throughout the entire observation period.

Body Weights: The body weight of the animals was within the range commonly recorded for this strain and age.

Interpretation of results:

GHS criteria not met

Conclusions:

Based upon the referred classification criteria (Regulation (EC) No 1272/2008), di-tert-butyl peroxide (CAS# 110-05-4) is not classified with respect to eye irritation in rabbits.

Executive summary:

The primary eye irritation potential of di-tert-butyl peroxide (CAS# 110-05-4) was investigated according to OECD test guideline no. 405. The test item was applied by instillation of 0.1 mL into the left eye of each of three young adult New Zealand White rabbits. Scoring of irritation effects was performed approximately 1, 24, 48 and 72 hours after test item instillation.

The mean score was calculated across 3 scoring times (24, 48 and 72 hours after instillation) for each animal for corneal opacity, iris light reflex and redness and chemosis of the conjunctivae. The individual mean scores for corneal opacity and iris light reflex were 0.00 for all three animals. The individual mean scores for the conjunctivae were 1.00, 0.33 and 0.33 for reddening and 0.00, 0.00 and 0.00 for chemosis, respectively.

The instillation of di-tert-butyl peroxide (CAS# 110-05-4) into the eye resulted in mild, early onset and transient ocular changes, such as reddening and swelling of the conjunctivae and reddening of the sclerae. These effects were reversible and were no longer evident 72 hours after treatment, the end of the observation period for all animals. No abnormal findings were observed in the cornea or for the iris light reflex of any animal at any of the examinations. No corrosion was observed at any of the measuring intervals. No staining of the treated eyes by the test item was observed and no clinical signs were observed.

Thus, the test item did not induce significant or irreversible damage to the rabbit eye.

Based upon the referred classification criteria (Regulation (EC) No 1272/2008), di-tert-butyl peroxide (CAS# 110-05-4) is not classified with respect to eye irritation in rabbits.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well-documented study performed under GLP according to established guidelines with no deviations.

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:

other: cow (Bos bovis)

Strain:

not specified

Vehicle:

unchanged (no vehicle)

Controls:

other: three vehicle controls and three positive controls were tested simultaneously

Amount / concentration applied:

1.0 ml

Duration of treatment / exposure:

Ten minutes

Observation period (in vivo):

One hundred twenty minutes

Number of animals or in vitro replicates:

Three corneas per group

Details on study design:

Collection of Bovine Eyes
Freshly isolated bovine eyes were collected from the abattoir. Excess tissue was removed from the excised eyes. The isolated eyes were transported to the laboratory in Hank’s buffered salt solution (HBSS) supplemented with streptomycin / penicillin at ambient temperature. The corneae were isolated on the same day after delivery of the eyes, inserted in pre-cooled preservation medium composed of Medium 199 supplemented with Lglutamine,
Na-bicarbonate and Taurine, and stored in the refrigerator at 2 – 8 °C until the following day. Shortly before use, Dextran was added to the medium.

Preparation of Corneae
All eyes were carefully examined macroscopically for defects. Those presenting defects such as vascularization, pigmentation, opacity and scratches were discarded. The cornea was carefully removed from the eye using scalpel and rounded scissors. A rim of about 2 mm of tissue (sclera) was left for stability and handling of the isolated cornea. All corneae used in the experiment were collected in Hank’s BSS supplemented with streptomycin / penicillin and checked finally with a view box for defects listed above. Each cornea was mounted in a cornea holder with the endothelial side against the sealing ring (O-ring) of the posterior part of the holder. The cornea was gently flattened over the Oring but stretching was avoided. After the anterior part of the holder was positioned on top of the cornea and fixed in place with screws, both compartments of the holder were filled with complete medium. The posterior compartment was filled first to return the cornea to its natural convex position. Care was taken to assure no air bubbles were present within the compartments. For equilibration, the corneae in the holder were incubated in a vertical position for one hour at 32 ± 2 °C in a water-bath.
At the end of the incubation period, the complete medium was removed from both compartments and replaced with fresh cMEM, and the basal opacity was determined (t0). For measurement, the posterior compartment was plugged and the anterior compartment was unplugged.

Exposure of the test item to the corneae:
The test item was taken out of the freezer directly prior to application. The anterior
compartments were washed several times with 0.9% (w/v) NaCl in deionized water (saline).
The remaining saline on the anterior compartment was removed by aspiration. Saline was
placed in the posterior compartment, while the anterior compartment received the test item at
a volume of 1.0 mL on the surface of the corneae using plastic pipettes, and was incubated
at 32 ± 2 °C in the water-bath, while the corneae were in a horizontal position.

Exposure of the controls to the corneae:
Fresh cMEM was placed in the posterior compartment, while the anterior compartment received the negative or positive control at a volume of 1.0 mL on the surface of the corneae and was incubated at 32 ± 2 °C in the water-bath, while the corneae were in a horizontal position. The test item was tested undiluted. The positive control 2-Ethoxyethanol was tested neat. 0.9% (w/v) saline was used as negative control item. The incubation time lasted ten minutes (± 30 seconds).

Post-exposure handling of the corneae:
After incubation, the test item was rinsed off from the application side using saline. The saline in the posterior compartment was removed by aspiration, the posterior compartment was washed at least three times with cMEM and then filled with cMEM, and opacity was measured (t10). The corneae exposed to the negative and positive controls were treated like the test item corneae, but the application side was rinsed with cMEM. The corneae were then futher incubated at 32 ± 2 °C for further two hours in a vertical position, followed by a third opacity reading (t130). In the second step of the assay, permeability of the cornea was determined. Fresh complete medium was added to the posterior compartment and 1 mL of a Na-fluorescein solution, 0.5 % dissolved in HBSS, was placed in the anterior compartment. Corneae were incubated again in a horizontal position for an additional 90 minutes at 32 ± 2 °C in the water-bath. The optical density of an aliquot of the mixed complete medium from the posterior chamber was measured spectrophotometrically at 490 nm (OD490).

Irritation parameter:

cornea opacity score

Run / experiment:

10 mins

Value:

0.81

Remarks on result:

no indication of irritation

Irritation parameter:

other: corneal permeability

Run / experiment:

130 min

Value:

0.009

Remarks on result:

no indication of irritation

Interpretation of results:

GHS criteria not met

Conclusions:

The test item di-tert-butyl peroxide (CAS No. 110-05-4) did not cause any opacity or permeability of the corneae compared with the results of the negative control. The calculated in vitro score was 0.81 and therefore, the test item was classified as non eye irritant.

Executive summary:

This in vitro study was performed to assess the corneal irritation and damage potential of ditert- butyl peroxide (CAS No. 110-05-4) by means of the BCOP assay using fresh bovine corneae. After a first opacity measurement of the fresh bovine corneae (t0), the neat test item di-tertbutyl peroxide (CAS No. 110-05-4), the positive, and the negative controls were applied to corneae and incubated for 10 minutes at 32 ± 2 °C. After the incubation phase the test item, the positive, and the negative controls were each rinsed from the corneae and opacity was measured again (t10). Further, the corneae were incubated for another 120 minutes at 32 ± 2 °C in complete medium, and opacity was measured a third time (t130). After the opacity measurements permeability of the corneae was determined while application of 1 mL of a fluorescein solution for 90 minutes at 32 ± 2 °C in a horizontal position. The liquid coming out was measured spectrophotometrically. With the negative control (0.9% NaCl solution) neither an increase of opacity nor permeability of the corneae could be observed. The positive control (2-Ethoxyethanol) showed clear opacity and distinctive permeability of the corneae and therefore, is classified as very severe eye irritant. The test item di-tert-butyl peroxide (CAS No. 110-05-4) did not cause any opacity or permeability of the corneae compared with the results of the negative control. The calculated in vitro score was 0.81 and therefore, the test item was classified as non eye irritant. In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item di-tert-butyl peroxide (CAS No. 110-05-4) is not considered to be an eye irritant.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Justification for selection of skin irritation / corrosion endpoint:
Apparently well conducted GLP study.

Justification for selection of eye irritation endpoint:
Apparently well conducted GLP study.

Justification for classification or non-classification

Di- tert- butyl peroxide does not cause irritation when tested using in vitro or in vivo methods; therefore, it is not classified for either skin or eye irritation.