Di-tert-butyl sec-butylidene diperoxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The study procedure was conducted between 18 June 1998 and 20 July 1998

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study conducted in 1998

Test material

Test material information

Constituent 1

Reference substance name:

Di-tert-butyl sec-butylidene diperoxide

EC Number:

218-507-2

EC Name:

Di-tert-butyl sec-butylidene diperoxide

Cas Number:

2167-23-9

Molecular formula:

C12H26O4

IUPAC Name:

2,2-bis(tert-butylperoxy)butane

additive 1

Reference substance name:

Hydrocarbons, C4, 1,3-butadiene-free, polymd., triisobutylene fraction, hydrogenated

EC Number:

297-629-8

EC Name:

Hydrocarbons, C4, 1,3-butadiene-free, polymd., triisobutylene fraction, hydrogenated

Cas Number:

93685-81-5

Molecular formula:

not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

isododecane

Test material form:

liquid

Specific details on test material used for the study:

The test substance is referred to as LUPEROX 220 M 50.
Batch number: 512-9800-001
Description: colourless liquid
date of receipt: 7 April 1998
Storage conditions: at room temperature and protected from light
Purity: 50.1 %
Expiry date: September 1998.

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

Breeder: Charles River France, 76410 Saint-Aubin-les-Elbeuf, France.
Number: . two males and two females for the preliminary test
. 30 animals ( 1 S males and 15 females) for the main test.
Females were nulliparous and non-pregnant.
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 of the main test were approximately 3 months old and had a mean body weight ± standard deviation of 377 ± 17 g for the males and 366 ± 21 g for the females.
Acclimatization: at least 5 days before the beginning of the study.
Identification of the animals: ear-tattoo.

Environmental conditions
The conditions in the animal room were set as follows:
temperature: 21 ± 2°C
relative humidity: 30 to 70%
light/dark cycle: 12 h/12 h
ventilation: approximately 12 cycles/hour of filtered, non-recycled air
The temperature and relative humidity were under continuous control and recording. The records were checked daily and retained. In addition to these daily checks, the housing conditions and corresponding instrumentation and equipment are verified and calibrated at regular intervals.

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, including the detection of possible contaminants (pesticides, heavy metals), are perfonned regularly by external laboratories.

Food and water
During the study, the animals had free access to "106 pelleted diet" (UAR, 91360 Villemoissonsur-Orge, France).
Food is analysed regularly by the supplier for composition and contaminant levels.

Drinking water filtered by a FG MilJipore membrane (0.22 micron) was provided ad libitum. Bacteriological and chemical analysis of the water and diet, including the detection of possible contaminants (pesticides, heavy metals and nitrosamines), are performed regularly by external laboratories.

No contaminants are known to be present in the diet, drinking water or bedding material at levels which may be expected to interfere with or prejudice the outcome of the study.

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

corn oil

Concentration / amount:

25 % (w/w) for intradermal route
0.5 ml undiluted test substance for the cutaneous route

Day(s)/duration:

Six intradermal injections on Day 1, follwed by 48 hour cutaneous exposure starting on Day 7.

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Concentration / amount:

50 % (w/w)

Day(s)/duration:

24 hrs

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Preliminary study:
2 males and 2 females had 3 doses tested via the intra dermal route on each animal. One male and one female of the same animals were than treated at 2 doses via the cutaneous route

Main study:
5 males and 5 females were in the control group
10 males and 10 females were in the treated group

Details on study design:

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

By intradennal route:
- 24 hours before treatment, the dorsal region of the animals was clipped,
- intradermal administrations of the test substance formulation (0.1 ml) at different concentrations were performed in the interscapular region,
- cutaneous reactions were evaluated approximately 24, 48 hours and 6 days after the injections.

By cutaneous route:
- 24 hours before treatment, both flank regions of the animals were clipped,
- 0.5 ml of the undiluted test substance or test substance formulation at the chosen concentration were placed on a dry gauze pad (approximately 4 cm2) which was then applied to the skin and 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,
- intradennal injections should cause moderate irritant effects (no necrosis or ulceration of the skin),
- cutaneous application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration,
- cutaneous application for the challenge phase should be the highest concentration which does not cause irritant effects.

Main study

Preparation of the animals
For all animals and before each treatment, the application sites were:
- clipped on days 1 and 7 (interscapular region 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 4 cm x 2 cm clipped interscapular area, using a needle (diameter: 0.50 x 16 mm) mounted on a 1 ml glass syringe (0.01 ml graduations).
Details on these injections can be found in the table in section 'any othe information on materials and methods'
The anterior and middle pairs of injections were performed close to each other and nearest the head, while the posterior pair was perfo nned towards the caudal part of the test area.

Cutaneous route
On day 7, the interscapular area was clipped.
As the test substance was shown to be non-irritant during the preliminary test, the animals were treated with 0.5 ml of sodium lauzyl sulfate (10%, w/w) in vaseline in order to induce local irritation.
On day 8, a cutaneous application to the region of the intradermal injections (4 cm x 2 cm) was performed as follows:
Control group: application of 0.5 ml of the vehicle.
Treated group: application of 0.5 ml of the undiluted test substance.

The test substance or the vehicle was placed on a dry gauze pad, which was then applied to the interscapular region.
The pad was held in place for 48 hours by means of an adhesive hypoallergenic dressing and an adhesive anallergenic waterproof plaster.
On removal of the dressing, no residual test substance was observed.
Cutaneous reactions were recorded 1 hour after removal of the occlusive dressing.

Challenge phase
On day 22, the animals of both groups received an application of 0.5 ml of the test substance at the concentration of 50% (w/w) 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). The test substance or the vehicle was placed on a dry gauze pad, which was then applied to a 4 cm2 (2.cm x 2 cm) clipped area of the skin.
The pads were held in contact with the skin for 24 hours by means of an occlusive, hypoallergenic dressing and an adhesive anallergenic waterproof plaster.
On removal of the dressing, no residual test substance was observed.

Challenge controls:

The control group was treated with the same induction phase but with vehicle instead of test substance

Positive control substance(s):

no

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50% (w/w) challenge application

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No clinical signs and no mortality were observed during the study.

Remarks on result:

no indication of skin sensitisation

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50% (w/w) challenge application

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

No clinical signs and no mortality were observed during the study.

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

PRELIMINARY STUDY

Administration by intradermal route

In order to determine the concentration to be used in the main study, the following concentrations were tested:

Animal number	Concentration of the test substance % (w/w)	24 hours	Scoring after treatment 48 hours	6 days
Male 01	10 + FCA 10	I I	I LI	I LI
Female 01	10 + FCA 10	I I	I LI	I LI
Male 01	5 + FCA 5	I LI	I LI	I LI
Female 01	5 + FCA 5	I LI	I LI	I LI
Male 01	1 + FCA 1	I LI	I LI	I LI
Female 01	1 + FCA 1	I LI	I LI	I LI
Male 02	75 + FCA 75	I I	N N	N N
Female 02	75 + FCA 75	I I	N N	N N
Male 02	50 + FCA 50	I I	N N	A A
Female 02	50 + FCA 50	I I	N N	A A
Male 02	25 + FCA 25	I I	I I	I I
Female 02	25 + FCA 25	I I	I I	I I

FCA: Freund's Complete Adjuvant

N : necrosis

I : irritant

LI : slightly irritant

A : crust

In order to respect the criteria for the selection of concentrations (the concentration should be well-tolerated systemically and locally, intradermal injections should cause moderate initant effect but no necrosis or ulceration of the skin), concentration chosen for the main study was 25% (w/w).

Application by cutaneous route

Several concentrations were tested in order to determine the concentrations to be used in the main study.

 

Animal number	Concentration of the test substance %		Scoring after removal of the dressing
			24 hours		48 hours
			E	O	E	O
Male 01	100	RF	1	0	0	0
	50 (w/w)	LF	0	0	0	0
Female 01	100	RF	0	0	0	0
	50 (w/w)	LF	0	0	0	0

E : erythema

0 : oedema

RF : right flank

LF : left flank

After removal of the dressing, no residual test substance was observed.

In order to respect the criteria for the selection of concentrations (the concentrations should be well-tolerated systemically and locally, cutaneous application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration, cutaneous application for the challenge phase should be the highest concentration which does not cause irritant effect), concentration chosen for the topical application of the induction phase (day 8) was 100%. For the challenge application (day 22), it was 50% (w/w).

MAIN STUDY

Clinical examinations

No clinical signs and no mortality were observed during the study.

The body weight gain of the treated animals was normal when compared to that of the control animals.

Scoring of cutaneous reactions

End of the induction period

On day 10, after the cutaneous application of the induction period, signs of initation were observed at the interscapular test site in the control and treated groups.

Challenge application

The score for skin reactions was 0 in all animals at all treatment sites

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental conditions and according to the maximization method of Magnusson and Kligman, the test substance LUPEROX 220 M 50 (batch No. 512-9800-001 (MXL)) does not induce delayed contact hypersensitivity in guinea-pigs.

Executive summary:

The potential of the test substance LUPEROX 220 M 50 (batch No. 512-9800-001 (MXL)) to induce delayed contact hypersensitivity was evaluated in guinea-pigs according to the maximization method of Magnusson and Kligman and to OECD (No. 406, 17th July 1992) and EC (92/69/EEC, B.6, 31st July 1992) guidelines.

The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

Methods

Thirty guinea-pigs were allocated to two groups: a control group 1 (five males and five females) and a treated group 2 (ten males and ten females).

On day 1, intradennal injections of Freund's complete adjuvant mixed with the test substance (treated group) or the vehicle (control group) were perfonned in the interscapular region.

On day 7, the same region received a topical application of sodium lauryl sulfate in vaseline (10%, w/w) in order to induce local irritation.

On day 8, the test substance (treated group) or the vehicle (control group) was applied to the same test site which was then covered by an occlusive dressing for 48 hours.

On day 22, after a rest period of 12 days, all animals of the treated and control groups were challenged by a cutaneous application of the test substance to the right flank. The left flank served as control and received the vehicle only. Test substance and vehicle were maintained under an occlusive dressing for 24 hours.

Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing.

Test substance concentrations were as follows:

Induction (treated group)

- intradennal injections: LUPEROX 220 M 50 at the concentration of 25% (w/w) in corn oil,

- topical application: LUPEROX 220 M 50 undiluted.

Challenge (all groups)

- topical application: LUPEROX 220 M 50 at the concentration of 50% (w/w) in com oil.

At the end of the study, animals were killed without examination of internal organs.

No skin samples were taken from the challenge application sites.

The sensitivity of the guinea-pigs in CIT experimental conditions was checked with positive sensitizers 2,4-Dinitro Chlorobenzene (DNCB) and Mercaptobenzothiazole.

During the induction period, the reference substance DNCB was applied at the concentrations of 0.1 % (w/w) (day 1) and 1 % (w/w) (day 8). The reference substance Mercaptobenzothiazole was

applied at the concentrations of 1 % (w/w) (day 1) and 20% (w/w) (day 8).

For the challenge application, the reference substance DNCB was applied at the concentration of 1 % (w/w). The reference substance Mercaptobenzothiazole was applied at the concentration of 20% (w/w).

Results

No clinical signs and no deaths were noted during the study.

No cutaneous reactions were observed after the challenge application.

The species and strain which were used showed a satisfactory sensitization response in 90% animals treated with DNCB and in 30% animals treated with Mercaptobenzothiazole.

Conclusion

Under our experimental conditions and according to the maximization method of Magnusson and Kligman, the test substance LUPEROX 220 M 50 (batch No. 512-9800-001 (MXL)) does not induce delayed contact hypersensitivity in guinea-pigs.