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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report date:
2006

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:
The study was performed before the implementation of the REACH regulation.

Test material

Constituent 1
Chemical structure
Reference substance name:
Diisopropylbenzene hydroperoxide
EC Number:
247-988-1
EC Name:
Diisopropylbenzene hydroperoxide
Cas Number:
26762-93-6
Molecular formula:
C12H18O2
IUPAC Name:
reaction mass of 1-(3-isopropylphenyl)-1-methylethyl hydroperoxide and 1-(4-isopropylphenyl)-1-methylethyl hydroperoxide
Test material form:
liquid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Species and sex: male and nulliparous and non-pregnant female guinea pigs.
Breeder: Charles River Laboratories France, L’Arbresle, France.
Allocation to the groups: on day -1, the animals were weighed and allocated to two groups: a
control group of ten animals (five males and five females) and a treated group of 20 animals
(ten males and ten females).
Age/weight: on day 1, the animals of the main test were 1-2 months old and had a mean
body weight ± standard deviation of 369 ± 16 g for the males and 360 ± 13 g for the females.
Acclimation: at least 5 days before the beginning of the study.
Identification: by individual ear-tattoo

The conditions in the animal room were set as follows:
• temperature: 22 ± 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.
• Food and water ad libitum

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
corn oil
Concentration / amount:
1%
Day(s)/duration:
Day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
other: ethanol/water (80/20)
Concentration / amount:
10%
Day(s)/duration:
Day 8
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: acetone
Concentration / amount:
2.5%
Day(s)/duration:
Day 22
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
other: acetone
Concentration / amount:
1% (left flank) and 0.5% (right flank)
Day(s)/duration:
Day 36
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
5 males and 5 females for the preliminary test,
30 animals (15 males and 15 females) for the main test.
Details on study design:
Three injections of 0.1 mL were made into each side of this interscapular region (i.e. three pairs
of sites), as follows:
1 Anterior FCA at 50% (v/v) in 0.9% NaCl
2 Middle test item at 1% (w/w) in corn oil
3 Posterior test item at 1% (w/w) in the mixture FCA/0.9% NaCl (50/50)
FCA: Freund's complete adjuvant
* : The test item was suspended in FCA prior to be combined with the aqueous phase. The final concentration of the test item was equal to that used in injection 2.

As the test item was shown to be irritant during the preliminary test, a topical application of sodium lauryl sulfate was not necessary on day 7.

On day 8, a pad of filter paper (approximately 8 cm2) was fully-loaded with the test item at the concentration of 10% (w/w) and was then applied to the interscapular region of the animals of the treated group. The animals of the control group received an application of the vehicle alone under the same
experimental conditions. 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 (day 10), no residual test item was observed.
A local irritation was recorded in the animals of both groups.
Positive control substance(s):
yes
Remarks:
Mercaptobenzothiazole

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
2.5 %
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
score:1
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
2.5 %
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
Score: 2 or 3
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
2.5 %
No. with + reactions:
15
Total no. in group:
20
Clinical observations:
Score: 1 or 2
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
2.5 %
No. with + reactions:
11
Total no. in group:
20
Clinical observations:
Score: 1 or 2
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
0.5 %
No. with + reactions:
0
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
0.5 %
No. with + reactions:
0
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
0.5 %
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
Score 1
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
0.5 %
No. with + reactions:
0
Total no. in group:
20
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
1 %
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Score 1
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
1 %
No. with + reactions:
0
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
1 %
No. with + reactions:
4
Total no. in group:
20
Clinical observations:
Score 1 or 2
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
1 %
No. with + reactions:
10
Total no. in group:
20
Clinical observations:
Score 1 or 2
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
1%
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

 

A discrete erythema (grade 1) was observed in 4/10 animals of the control group at the 24-hour reading. At the 48-hour reading, a moderate or intense erythema (grade 2 or 3) was noted in 2/10 and 1/10 animals, respectively. Dryness of the skin, together with crusts in one animal, was also recorded at the 48-hour reading in 5/10 animals.

In the treated group, at the 24-hour reading, a discrete or moderate erythema (grade 1 or 2), together with dryness of the skin in one animal, was noted in 13/20 and 2/20 animals, respectively. At the 48-hour reading, a discrete or moderate erythema (grade 1 or 2) was recorded in 3/20 and 9/20 animals, respectively. Dryness of the skin was also observed in 13/20 animals at the 48-hour reading.

First challenge application

Scoring of skin reactions was as follows:

 

 

Control group

 

Sex

Animal number

 

LF

24 hours

 

RF

 

 

LF

48 hours

 

RF

Male

286

0

 

0

 

0

 

0/S

 

287

0

 

0

 

0

 

0

 

288

0

 

1

 

0

 

2/S

 

289

0

 

0

 

0

 

0

 

290

0

 

0

 

0

 

0

Female

301

0

 

0

 

0

 

0

 

302

0

 

1

 

0

 

0

 

303

0

 

1

 

0

 

3/S/A

 

304

0

 

1

 

0

 

2/S

 

305

0

 

0

 

0

 

0/S

LF: left flank (vehicle)

RF: right flank (test item at the concentration of 2.5% (w/w))

A : crusts

S : dryness of the skin

 

 

 

Treated group

 

Sex

Animal number

 

LF

24 hours

 

RF

 

 

LF

48 hours

 

RF

Male

291

0

 

1/S

 

0

 

2/S

 

292

0

 

1

 

0

 

1/S

 

293

0

 

0

 

0

 

0

 

294

0

 

0

 

0

 

2/S

 

295

0

 

1

 

0

 

0

 

296

0

 

1

 

0

 

1/S

 

297

0

 

1

 

0

 

0/S

 

298

0

 

2

 

0

 

2/S

 

299

0

 

1

 

0

 

2

 

300

0

 

2

 

0

 

2/S

Female

306

0

 

0

 

0

 

0

 

307

0

 

1

 

0

 

2/S

 

308

0

 

1

 

0

 

2/S

 

309

0

 

1

 

0

 

2/S

 

310

0

 

1

 

0

 

1/S

 

311

0

 

0

 

0

 

0

 

312

0

 

1

 

0

 

0/S

 

313

0

 

1

 

0

 

2/S

 

314

0

 

0

 

0

 

0

 

315

0

 

1

 

0

 

0

LF: left flank (vehicle)

RF: right flank (test item at the concentration of 2.5% (w/w))

S : dryness of the skin

In order to determine whether the observed cutaneous reactions are attributable to delayed contact hypersensitivity or to an irritant effect of the test item, a second challenge application was performed.

For this second challenge application, the lower concentrations of 1% and 0.5% (w/w) were chosen.

The observed cutaneous reactions were as follows:

Left flank: test item at the concentration of 1%

No cutaneous reactions were noted in the animals of the control group.

In the treated group, a discrete or moderate erythema (grade 1 or 2) was noted in 3/20 and 1/20 animals, respectively. At the 48-hour reading, a discrete or moderate erythema (grade 1 or 2) was observed in 9/20 and 1/20 animals, respectively. Dryness of the skin was also recorded in 10/20 animals at the 48-hour reading.

Right flank: test item at the concentration of 0.5%

A discrete erythema (grade 1) was noted in 1/10 animals of the control group at the 24-hour reading only.

In the treated group, a discrete erythema (grade 1) was observed in 1/20 animals at the 24-hour reading; at the 48-hour reading, the evaluation of the erythema was masked by dryness of the skin in this animal. Dryness of the skin was also recorded in another animal at the 48-hour reading.

The cutaneous reactions observed at the 48-hour reading of the second challenge application on the left flank of 10/20 animals, which were of higher incidence and severity than those recorded in the control group, were attributed to delayed contact hypersensitivity.


Second challenge application

Scoring of skin reactions was as follows:

 

 

 

 

Control group

 

 

 

 

 

Sex

Animal number

before treatment LF                      RF

 

LF

24 hours

 

RF

 

LF

48 hours

RF

Male

286

0

0

0

0

0

0

 

287

0

0

0

1

0

0

 

288

0

0

0

0

0

0

 

289

0

0

0

0

0

0

 

290

0

0

0

0

0

0

Female

301

0

0

0

0

0

0

 

302

0

0

0

0

0

0

 

303

0

0

0

0

0

0

 

304

0

0

0

0

0

0

 

305

0

0

0

0

0

0

 

 

 

 

Treated group

 

 

 

 

 

Sex

Animal number

before treatment LF                      RF

 

LF

24 hours

 

RF

 

LF

48 hours

RF

Male

291

0

0

1

1

1/S

LS

 

292

0

0

2

0

2/S

0

 

293

0

0

0

0

0/S

0

 

294

0

0

0

0

0

0

 

295

0

0

0

0

0

0

 

296

0

0

0

0

0

0

 

297

0

0

1

0

1/S

0

 

298

0

0

1

0

1/S

0

 

299

0

0

0

0

0/S

0

 

300

0

0

0

0

1

0

Female

306

0

0

0

0

0/S

0

 

307

0

0

0

0

0

0

 

308

0

0

0

0

0

0

 

309

0

0

0

0

1/S

0

 

310

0

0

0

0

1

0

 

311

0

0

0

0

0

0

 

312

0

0

0

0

1/S

0/S

 

313

0

0

0

0

0

0

 

314

0

0

0

0

1/S

0

 

315

0

0

0

0

1

0

LF : left flank (test item at the concentration of 1% (w/w))

RF: right flank (test item at the concentration of 0.5% (w/w))

LS : reading masked by an important dryness of the skin

S : dryness of the skin

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
According to the maximization method of Magnusson and Kligman, DIISOPROPYLBENZENE HYDROPEROXIDE in diisopropylbenzen (purity :55.9%) induces delayed contact hypersensitivity in 10/20 (50%) guinea pigs and should therefore be considered as a mild sensitizer.
Executive summary:

The potential of diisopropylbenzene hydroperoxide to induce delayed contact hypersensitivity was evaluated in guinea pigs according to the maximization method of Magnusson and Kligman and to OECD406 guideline. Thirty guinea pigs were allocated to two groups: a control group of five males and five females and a treated group of ten males and ten females. On day 1, three pairs of intradermal injections were performed in the interscapular region of all animals: Freund's complete adjuvant (FCA) diluted to 50% (v/v) with 0.9% NaCl (both groups), test item at the concentration of 1% in corn oil (treated group) or vehicle alone (control group), test item at the concentration of 1% in a mixture FCA/0.9% NaCl (50/50, w/w) (treated group) or vehicle at the concentration of 50% (w/v) in a mixture FCA/0.9% NaCl (50/50, v/v) (control group).

On day 8, the animals of the treated group received a topical application of the test item at the concentration of 10% (w/w) in ethanol/water (80/20) to the same test site, which was then covered by an occlusive dressing for 48 hours. The animals of the control group received an application of the vehicle under the same experimental conditions. On day 22, all animals of both groups were challenged by a cutaneous application of the test item at the concentration of 2.5% (w/w) in acetone to the right flank. The test item was maintained under an occlusive dressing for 24 hours. The vehicle was applied to the left flank under the same experimental conditions. Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing.

As equivocal cutaneous reactions were noted after the first challenge, a second challenge application was performed on day 36. The test item was applied at the concentration of 1% (w/w) in acetone to the left flank and at the concentration of 0.5% (w/w) in acetone to the right flank of the animals of both groups, under the same experimental conditions as for the first challenge application.

At the end of the study, the animals were killed without examination of internal organs. No skin samples were taken from the challenge application sites. No systemic clinical signs and no deaths were noted during the study.

After the first challenge application, a discrete erythema was observed in 4/10 animals of the control group at the 24-hour reading. At the 48-hour reading, a moderate or intense erythema was noted in 2/10 and 1/10 animals, respectively. Dryness of the skin, together with crusts in one animal, was also recorded at the 48-hour reading in 5/10 animals. In the treated group, at the 24-hour reading, a discrete or moderate erythema, together with dryness of the skin in one animal, was noted in 13/20 and 2/20 animals, respectively. At the 48-hour reading, a discrete or moderate erythema was recorded in 3/20 and 9/20 animals, respectively. Dryness of the skin was also observed in 13/20 animals at the 48-hour reading.

After the second challenge application with 1%, no cutaneous reactions were noted in the animals of the control group. In the treated group, a discrete or moderate erythema was noted in 3/20 and 1/20 animals, respectively. At the 48-hour reading, a discrete or moderate erythema was observed in 9/20 and 1/20 animals, respectively. Dryness of the skin was also recorded in 10/20 animals at the 48-hour reading. At the concentration of 0.5%, a discrete erythema was noted in 1/10 animals of the control group at the 24-hour reading only. In the treated group, a discrete erythema was observed in 1/20 animals at the 24-hour reading; at the 48-hour reading, the evaluation of erythema was masked by dryness of the skin in this animal. Dryness of the skin was also recorded in another animal at the 48-hour reading.

The cutaneous reactions observed at the 48-hour reading of the second challenge application on the left flank of 10/20 animals, which were of higher incidence and severity than those recorded in the control group, were attributed to delayed contact hypersensitivity.

Diisopropylbenzene monohydroperoxide in diisopropylbenzene (purity 55.9 %) induces delayed contact hypersensitivity in 10/20 (50%) guinea pigs and should therefore be considered as a mild sensitizer.