Cyclohexylidenebis[tert-amyl] peroxide

Endpoint summary

Description of key information

The potential of 1,1-DI(t-AMYLPEROXY)CYCLOHEXANE 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 (96/54/EEC, B.6, 30 July 1996) guidelines (Ollivier, 2006). The study was conducted in compliance with the principles of Good Laboratory Practice Regulations. 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 2.5% in corn oil (treated group) or vehicle alone (control group),

·      test item at the concentration of 2.5% 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 undiluted test item 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 (acetone) 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 10% (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 5% (w/w) in acetone to the left flank and at the concentration of 1% (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 2/10 and 3/10 animals of the control group at the 24 and 48-hour readings, respectively. In the treated group, a discrete erythema was noted in 2/20 and 7/20 animals at the 24 and 48-hour readings, respectively. Dryness of the skin was also observed in 1/20 animals at the 48-hour reading. After the second challenge application, no relevant cutaneous reactions were recorded.

Under these experimental conditions and according to the maximization method of Magnusson and Kligman, 1,1-DI(t-AMYLPEROXY)CYCLOHEXANE should not be considered as a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Study performed before the implementation of the REACH regulation.

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L’Arbresle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: Caesarian obtained, Barrier sustained - Virus Antibody Free (COBS - VAF®).
- Age at study initiation: 1-2 months old
- Weight at study initiation: 354 ± 13 g for the males and 349 ± 22 g for the females
- Housing: individually in polycarbonate cages with stainless steel lid
- Diet: free access to 106 pelleted diet (SAFE, Villemoisson, Epinay-sur-Orge, France)
- Water: Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 30 to 70
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12

Route:

intradermal

Vehicle:

corn oil

Concentration / amount:

2.5%

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:

unchanged (no vehicle)

Concentration / amount:

100%

Day(s)/duration:

day 8

Adequacy of induction:

highest technically applicable concentration used

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Concentration / amount:

10%

Day(s)/duration:

Day 22

Adequacy of challenge:

highest non-irritant concentration

No.:

#2

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Concentration / amount:

5%

Day(s)/duration:

day 36

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

test: 10 males & 10 females
control: 5 males & 5 females

Details on study design:

RANGE FINDING TESTS:
A preliminary test was conducted in order to determine the concentrations to be tested in the main study.
By intradermal route (tested concentrations: 5%, 10% and 25% (w/w)):
• intradermal injections of the dosage form preparations (0.1 mL) were performed in the interscapular region,
• local reactions were evaluated approximately 24, 48 hours and 6 days after the injections.
By cutaneous route
Under the conditions of the induction phase (tested concentrations: 50% (w/w) and 100%):
• a filter paper (approximately 8 cm2) was fully-loaded with a dosage form preparation and was then applied to the clipped area of the skin. The filter paper was held in place by means of an occlusive dressing for 48 hours,
• cutaneous reactions were evaluated 24 and 48 hours after removal of the dressing.
Under the conditions of the challenge phase (tested concentrations: 10%, 25%, 50% (w/w) and 100%):
• the filter paper of a chamber (Finn Chamber®) was fully-loaded with a dosage form preparation. The chamber was then applied to the clipped area of the skin (one concentration per flank). The chamber was held in place by means of an occlusive dressing for 24 hours,
• cutaneous reactions were evaluated 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 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.

Challenge controls:

corn oil

Positive control substance(s):

yes

Positive control results:

Mercaptobenzothiazole (induction phase 1% (w/w) on day 1 (intradermal route) and 20% (w/w) on day 8 (cutaneous route); challenge phase 20% (w/w) on day 22 (cutaneous route)) induced positive skin sensitization reactions in 80% (8/10) guinea pigs.

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

10%

No. with + reactions:

2

Total no. in group:

10

Clinical observations:

All graded 1

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

10%

No. with + reactions:

3

Total no. in group:

10

Clinical observations:

All graded 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10%

No. with + reactions:

2

Total no. in group:

20

Clinical observations:

All graded 1

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

7

Total no. in group:

20

Clinical observations:

All graded 1

Reading:

rechallenge

Hours after challenge:

24

Group:

negative control

Dose level:

5 and 1%

No. with + reactions:

0

Total no. in group:

10

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

Dose level:

5 and 1%

No. with + reactions:

0

Total no. in group:

10

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

5%

No. with + reactions:

1

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

Dose level:

1%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

5 and 1%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

no indication of skin sensitisation

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

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

1%

No. with + reactions:

8

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Interpretation of results:

GHS criteria not met

Conclusions:

Under these experimental conditions and according to the maximization method of Magnusson and Kligman, 1,1-DI(t-AMYLPEROXY)CYCLOHEXANE should not be considered as a skin sensitizer.

Executive summary:

The potential of 1,1-DI(t-AMYLPEROXY)CYCLOHEXANE 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 (96/54/EEC, B.6, 30 July 1996) guidelines. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations. 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 2.5% in corn oil (treated group) or vehicle alone (control group),

·      test item at the concentration of 2.5% 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 undiluted test item 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 (acetone) 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 10% (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 5% (w/w) in acetone to the left flank and at the concentration of 1% (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 2/10 and 3/10 animals of the control group at the 24 and 48-hour readings, respectively. In the treated group, a discrete erythema was noted in 2/20 and 7/20 animals at the 24 and 48-hour readings, respectively. Dryness of the skin was also observed in 1/20 animals at the 48-hour reading. After the second challenge application, no relevant cutaneous reactions were recorded.

Under these experimental conditions and according to the maximization method of Magnusson and Kligman, 1,1-DI(t-AMYLPEROXY)CYCLOHEXANE should not be considered as a skin sensitizer.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Not classified as skin sensitizer according to CLP and GHS criteria.