Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 DAYS

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Well documented study done according to OECD 406, with OECD GLP statement, QA statement, and test article C of A

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

conducted before LLNA was adopted

Test material

Test material information

Constituent 1

Reference substance name:

6731-36-8 (97.4%)

IUPAC Name:

6731-36-8 (97.4%)

Constituent 2

Reference substance name:

Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide

EC Number:

229-782-3

EC Name:

Di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide

Cas Number:

6731-36-8

Molecular formula:

C17H34O4

IUPAC Name:

1,1-bis(tert-butylperoxy)-3,3,5-trimethylcyclohexane

Details on test material:

The test substance LUPEROX 231 (1,1-DI-(tert-BUTYLPEROXY)-3,3,5 TRIMETHYLCYCLOHEXANE) used in the study was supplied by Elf Atochem
Deutschland. Documentation supplied by the Sponsor identified the test substance as follows:
-name:LUPEROX 231 (l,1-DI-(tert-BUTYLPEROXY)-3,3,5 TRIMETHYLCYCLOHEXANE)
- labelling: LUPEROX 231
-batch number: 530-9502-102
- protocol and labelling: 530-9502-102
- description: colourless to yellowish liquid,
-quantity and container: 2 plastic flasks each containing 0.1 kg
-date of receipt: 18.5.95
-storage conditions: at room temperature and protected from light
-purity: 97.4%.
Data relating to the characterization of the test substance are documented in a test article description and results of analysis provided by the Sponsor.

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

Species and strain: Dunkin-Hartley guinea-pigs.
Reason for this choice: species recommended by the international regulations for sensitization studies. The strain used has been shown to produce a satisfactory sensitization response using known positive sensitizers.
Breeder: Centre d'Elevage Lebeau, 78950 Gambais, France.
Number: 30 animals (15 males and 15 nulliparous and non-pregnant females). Allocation of the animals to the groups: on day -1, the animals were
weighed and randomly allocated to two groups: a control group 1 consisting of ten animals (five males and five females) and a treated group 2 consisting of 20 animals (ten males and ten females).
Weight: on day 1, the animals were approximately three months old and had a mean body weight ± standard deviation of 328 ± 17 g for the males and 323 ± 109 for the females.
Acclimatization: at least five days before the beginning of the study.
Identification of the animals: ear-tattoo.

Environmental conditions
During the acclimatization period and throughout the study, the conditions in the animal room were set as follows:
• temperature: 21 ± 2°C
• relative humidity: 30 to 70%
• light/dark cycle: 12 hl12 h
• ventilation: about 12 cycles/hour of filtered, non-recycled air.
The temperature and relative humidity were recorded continuously and records retained.
The housing conditions (temperature, relative humidity, light/dark cycle and ventilation) were checked regularly. During the acclimatization period
and throughout the study, the animals were housed individually in polycarbonate cages (48 cm x 27 cm x 20 cm) equipped with a polypropylene
bottle. Dust-free sawdust was provided as litter (SICSA, 92142 Alfortville, France). Bacteriological analysis of the sawdust and detection of possible
contaminants (pesticides, heavy metals) are performed periodically.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

paraffin oil

Induction 2

Concentration / amount:

Induction (treated group)
-intradermal injections: LUPEROX 231 (l,1-DI-(tert-BUTYLPEROXY)-3,3,5-TRIMETHYLCYCLOHEXANE)
at 10% (w/w) in paraffin oil.
-topical application: LUPEROX 231 (l,1-DI-(tert-BUTYLPEROXY)-3,3,5-TRIMETHYLCYCLOHEXANE)
at 50% (w/w) in paraffin oil.

Challenge (all groups)
-topical application: LUPEROX 231 (1,1-DI-(tert-BUTYLPEROXY)-3,3,5-TRIMETHYLCYCLOHEXANE)
at 50% (w/w) in paraffin oil.

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Challenge 2

Concentration / amount:

Induction (treated group)
-intradermal injections: LUPEROX 231 (l,1-DI-(tert-BUTYLPEROXY)-3,3,5-TRIMETHYLCYCLOHEXANE)
at 10% (w/w) in paraffin oil.
-topical application: LUPEROX 231 (l,1-DI-(tert-BUTYLPEROXY)-3,3,5-TRIMETHYLCYCLOHEXANE)
at 50% (w/w) in paraffin oil.

Challenge (all groups)
-topical application: LUPEROX 231 (1,1-DI-(tert-BUTYLPEROXY)-3,3,5-TRIMETHYLCYCLOHEXANE)
at 50% (w/w) in paraffin oil.

No. of animals per dose:

20 (ten male; ten female)

Details on study design:

Preliminary test
A preliminary test was conducted in order to determine the concentrations to be tested in themain study.

By intradermal route:
24 hours before treatment, the dorsal region of the animals was clipped, the test substance was prepared in an appropriate vehicle, intradermal administrations of the test substance (0.1 ml) at different concentrations were performed in the dorsal region between the shoulders, cutaneous reactions were evaluated approximately 24, 48 hours and six days after injection.

By cutaneous route:
24 hours before treatment, both flank regions of the animals were clipped, if necessary, the test substance was prepared in an appropriate vehicle, the test substance (0.5 ml for each concentration) was applied to a dry gauze pad of approximately 4 cm2 which was held in place by an occlusive dressing for 24 hours, cutaneous reactions were evaluated approximately 24 and 48 hours after removal of the dressings.

Criteria for selection of concentrations
The following criteria were used: the concentrations should be well-tolerated systemically and locally, intradermal injections should cause moderate irritant effect (no necrosis or ulceration of the skin), topical application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration, topical application for the challenge should be the highest concentration which does not cause irritant effect.

Main study
Preparation of the animals
For all animals and before each treatment, the application sites were: clipped on days -1 and 7 (scapular area 4 cm x 2 cm), clipped and shaved on day 21 (each flank 2 cm x 2 cm).

Induction phase by intradermal and cutaneous routes
Intradermal route: On day 1, six injections were made deep into the dermis of a clipped area (4 cm x 2 cm) in the dorsal region between the shoulders, using a needle (diameter: 0.50 x 16 mm, Terumo: C.M.L., 77140 Nemours, France) mounted on a 1 ml glass syringe (0.01 ml graduations, Record: Carrieri, 75005 Paris, France). Three injections of 0.1 ml were made into each side of this shoulder region, as follows:



Injection Site// Treated Group Control Group

Anterior// 1:FCA diluted at 50% (v/v) with 0.9% saline// 1:FCA diluted at 50% (v/v) with 0.9% saline


Middle// 2:test substance at 10% (w/w) in vehicle// vehicle


Posterior// mixture of 50/50 (w/v) of 1 and 2// mixture of 50/50 (w/v) of 1 and 2


Cutaneous route
Control group
On day 7, the scapular area was clipped. As the test substance was shown to be non-irritant during the preliminary tests, the animals were treated with 0.5 ml of sodium laurylsulphate (10% w/w) in vaseline in order to induce local irritation.

On day 8, a topical application to the region of the intradermal injections (4 cm x 2 cm) was performed.

Control group
-application of 0.5 ml of the vehicle.

Treated group
-application of 0.5 ml of the test substance at the chosen concentration.
The test substance and the vehicle were prepared on a dry gauze pad (Semes France, 54183 Heillecourt, France), which was then applied to the dorsal region between the shoulders and held in place for 48 hours by means of an adhesive hypoallergenic dressing (Laboratoires de Pansements et d'Hygiene, 21300 Chenove, France) and an adhesive anallergenic waterproof plaster (Laboratoire des Professions Medicales, 92240 Malakoff, France).
On removal of the dressing, if present, any residual test substance was removed by means of a dry or a moistened gauze pad. Cutaneous reactions were recorded one hour after removal of the occlusive dressing.

Challenge Phase
On day 22, the animals from both groups received an application of 0.5 ml of the test substance at the chosen concentration to the posterior right flank, and 0.5 ml of the vehicle to the posterior left flank. This application was performed using a 1 ml plastic syringe (0.01 ml graduations, Terumo: C.M.L., 77140 Nemours, France). The test substance and vehicle were prepared on a dry gauze pad (Semes France, 54183 Heillecourt, France), then applied to a 4 cm2 (2 cm x 2 cm) clipped area of the skin. The gauze pad was held in contact with the skin for 24 hours by means of an occlusive, hypoallergenic dressing (Laboratoires de Pansements et d'Hygiene, 21300 Chenove, France) and an adhesive anallergenic waterproof plaster (Laboratoire des Professions Medicales, 92240 Malakoff, France). On removal of the dressing, if present, any residual test substance was removed by means of a dry or a moistened gauze pad.

Positive control substance(s):

yes

Remarks:

The sensitivity of the guinea-pigs in experimental conditions were checked in a recent study with a positive sensitizer: 2,4-dinitro chlorobenzene. During induction period, the test substance was applied at 0.1 % (day 1) and 1 % (day 8) concentrations.

Results and discussion

Positive control results:

The sensitivity of the guinea-pigs in experimental conditions were checked in a recentstudy with a positive sensitizer: 2,4-dinitro chlorobenzene. During induction period, the testsubstance was applied at 0.1 % (day 1) and 1 % (day 8) concentrations. At cutaneous challenge application, 1 % (w/w) was tested on the right flank. The guinea-pigs which were used in a recent study, showed a satisfactory sensitization responsein 100% animals using a
positive sensitizer.

In vivo (non-LLNA)

Results

Reading:

other: all readings

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

3

Total no. in group:

20

Clinical observations:

maximum response was grade 1 erythema at 24 hrs: two males, one female

Remarks on result:

other: Reading: other: all readings. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 3.0. Total no. in groups: 20.0. Clinical observations: maximum response was grade 1 erythema at 24 hrs: two males, one female.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

Under experimental conditions and according to the maximization method of Magnusson and Kligman, no cutaneous reactions attributable to the sensitization potential of the test substance LUPEROX 231 (l,I-DI-(tert-BUTYLPEROXY)-3,3,5-TRIMETHYLCYCLOHEXANE at a concentration of 50% (w/w) were observed in guinea-pigs.

Executive summary:

No cutaneous reactions attributable to the sensitization potential of the test substance LUPEROX 231 (l,1-DI-(tert-BUTYLPEROXY)-3,3,5-TRIMETHYLCYCLOHEXANE at a concentration of 50% (w/w) were observed in guinea-pigs.