Registration Dossier

Administrative data

Description of key information

The potential of the test substance to induce delayed contact hypersensitivity was evaluated in guinea pigs according to the maximization method of Magnusson and Kligman (OECD406). No deaths and no clinical signs were noted during the main test. After the challenge application of the test item, a dryness of the skin was noted on both flanks of 1/10 control animals at the 48-hour reading, only. In the treated group, on the right flank (test item application), a discrete or moderate erythema was noted in 4120 and 5/20 animals at the 24 and 48-hour readings, respectively. A dryness of the skin was observed in 1/20 and 4/20 animals at the 24 and 48-hour readings, respectively. On the left flank (vehicle application), a dryness of the skin was noted in 1/10 animals at the 48-hour reading, only. The cutaneous reactions observed in 5/20 animals of the treated group, which were of higher incidence and severity than those recorded in the animals of the control group, were attributed to delayed contact hypersensitivity. However the test article does not meet the criteria for classification as a sensitizer. Under CLP a substance is classified as a sensitizer if > 30 % of the test animals have a positive response in the GPMT.

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:
other: Apparently well conducted GLP study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
Method of Magnusson and Kligman

Magnusson B. and Kligman A.M. The identification of contact allergens by animal assay.
The guinea pig maximization test. J. Invest. Derm., 52: 268-276 (1969).
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
This was the standard at this time
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.
Strain and sanitary status: Hartley Crl: (HA) BR, Caesarian obtained, Barrier sustained - Virus
Antibody Free (COBS - VAF@).
Reason for this choice: species generally accepted by regulatory authorities for this type of
study. The strain used has been shown to produce a satisfactory sensitization response using
known sensitizers.
Breeder: Charles River Laboratories France, L' Arbresle, France.
Number: 7 males and 7 females for the preliminary test,
30 animals (15 males and 15 females) for the main test.
Allocation to the groups: on day -1 of the main test, 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; their body weight at the
beginning of the study is presented in appendix 3.
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 (7:00-19:00)
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 filed. In addition to these daily checks, the housing conditions
and corresponding instrumentation and equipment are verified and calibrated at regular
intervals.
During the acclimation period and throughout the study, the animals were housed individually in
polycarbonate cages with stainless steel lid (48 cm x 27 cm x 20 cm) equipped with a
polypropylene bottle.
Each cage contained autoc1aved sawdust (SICSA, Alfortville, France).
Sawdust is analyzed by the supplier for composition and contaminant levels.

During the study, the animals had free access to 106 pelleted diet (SAFE, Villemoisson,
Epinay-sur-Orge, France).
Food is analyzed regularly by the supplier for composition and contaminant levels.
The diet formula is presented in appendix 2.
Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum.
Bacteriological and chemical analyses of water are performed regularly by external laboratories.
These analyses include the detection of possible contaminants (pesticides, heavy metals and
nitrosamines ).
No contaminants were known to have been present in the diet, drinking water or bedding
material at levels which may be expected to have interfered with or prejudiced the outcome of
the study.
Route:
intradermal and epicutaneous
Vehicle:
other: Acetone for challenge application
Concentration / amount:
See below.
Route:
epicutaneous, occlusive
Vehicle:
other: Acetone for challenge application
Concentration / amount:
See below.
No. of animals per dose:
Control: 10 animals/dose
Treated: 20 animals/dose
Details on study design:
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.

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% NaCI (both groups),
test item at the concentration of 0.5% in corn oil (treated group) or vehicle alone (control
group),
test item at the concentration of 0.5% in a mixture FCA/0.9% NaCI (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 7, a topical application of sodium lauryl sulfate at 10% (w/w) in vaseline was performed
to the same area of the animals of both groups, in order to induce a local irritation.
On day 8, the animals of the treated group received a topical application of the test item at the
concentration of 25% (w/w) in ethanol/purified water (80/20, w/w) 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 1 % (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.
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.

The choice of the vehicle was based on tests to check the homogeneity (visual check) of the
preparation (for cutaneous application and intradermal injections) and its free passage through a
needle (for intradermal injections). The highest concentrations which satisfied these criteria
were called the maximal practicable concentrations.
The vehicle used for intradermal injections was com oil, batch No. 015KOl15 (Sigma,
Saint-Quentin-Fallavier, France).
For topical applications, the vehicles used were:
*for the induction phase: a 80/20 (w/w) mixture of ethanol/purified water: ethanol,
batch No. V7B948027B (Carlo Erba, Val de Reuil, France) and purified water (prepared at
CIT by reverse osmosis),
*for the challenge application: acetone, batch No. 1317112638 (Merck, Chelles, France).

Challenge controls:
See above.
Positive control substance(s):
yes
Remarks:
MERCAPTOBENZOTHIAZOLE
Positive control results:
In a recent study performed under CIT experimental conditions, the strain of guinea pigs used showed a satisfactory sensitization response in 78% animals.
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
test chemical
No. with + reactions:
1
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
No. with + reactions:
4
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation

No deaths and no clinical signs were noted during the main test.

A dryness of the skin was noted on both flanks of 1/10 control animals at the 48-hour reading, only. In the treated group, on the right flank (test item application), a discrete or moderate erythema (grade 1 or 2) was noted in 4/20 and 5/20 animals at the 24 and 48-hour readings, respectively. A dryness of the skin was observed in 1/20 and 4/20 animals at the 24 and 48-hour readings, respectively. On the left flank (vehicle application), a dryness of the skin was noted in 1/1 0 animals at the 48-hour reading, only.

The cutaneous reactions observed in 5/20 animals of the treated group, which were of higher incidence and severity than those recorded in the animals of the control group.

Under the experimental conditions of this study and according to the maximization method of Magnusson and Kligman, the test item CYCLOHEXANONE PEROXIDE (batch No. 317100114) induced delayed contact hypersensitivity in 5/20 (25%) guinea pigs.

However the test article does not meet the criteria for classification as a sensitizer. Under CLP a substance is classified as a sensitizer if > 30 % of the test animals have a positive response in the GPMT (Guidance to Regulation (EC) No 1272/2008 on classification, labelling and packaging (CLP) of substances and mixtures Version 4.0 November 2013).

Interpretation of results:
GHS criteria not met
Remarks:
While 25% of the treated animals had more intense reactions than controls, this percentage does not meet the criteria for classification
Conclusions:

The cutaneous reactions observed in 5/20 animals of the treated group, which were of higher incidence and severity than those recorded in the animals of the control group. However the test article does not meet the criteria for classification as a sensitizer. Under CLP a substance is classified as a sensitizer if > 30 % of the test animals have a positive response in the GPMT.
Executive summary:

The potential of the test item CYCLOHEXANONE PEROXIDE (batch No. 317100114) 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, 30th July 1996) guidelines. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

No deaths and no clinical signs were noted during the main test. After the challenge application of the test item, a dryness of the skin was noted on both flanks of 1/10 control animals at the 48-hour reading, only. In the treated group, on the right flank (test item application), a discrete or moderate erythema was noted in 4120 and 5/20 animals at the 24 and 48-hour readings, respectively. A dryness of the skin was observed in 1/20 and 4/20 animals at the 24 and 48-hour readings, respectively. On the left flank (vehicle application), a dryness of the skin was noted in 1/10 animals at the 48-hour reading, only.

The cutaneous reactions observed in 5/20 animals of the treated group, which were of higher incidence and severity than those recorded in the animals of the control group, were attributed to delayed contact hypersensitivity.

However the test article does not meet the criteria for classification as a sensitizer. Under CLP a substance is classified as a sensitizer if > 30 % of the test animals have a positive response in the GPMT.

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

In the GPMT cutaneous reactions were observed in 5/20 animals of the treated group, which were of higher incidence and severity than those recorded in the animals of the control group, these findings were attributed to delayed contact hypersensitivity. However the test article does not meet the criteria for classification as a sensitizer. Under CLP a substance is classified as a sensitizer if > 30 % of the test animals have a positive response in the GPMT.