Registration Dossier

Administrative data

Description of key information

According to the maximization method of Magnusson and Kligman, the test item OO-TERT-AMYL-O-(2 -ETHYLHEXYL) MONOPEROXYCARBONATE (batch No. 25622 870403 -733) induced delayed contact hypersensitivity in 18/20 (90%) guinea pigs and should therefore be considered as a Category 1 sensitizer (extreme 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
Study period:
24 days
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: A GLP study done according to OECD guideline 406. The report has supporting documentation
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
on day 1, for the treated group, the third pair of intradermal injections was prepared in a mixture of FCA/0.9% NaCl (50/50,w/w). This minor deviation was not considered to have compromised the validity or integrity of the Study.
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
While LLNA was adopted in 2002, OECD 406 was still being commissioned in 2006 and was chosen as the method by the study sponsor
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Animals
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: . five males and five females for the preliminary test,
. 30 animals (15 males and 15 females) for the main test.
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 376 ± 14 g for the males and 363 ± 15 g for the females.
Acclimation: at least 5 days before the beginning of the study.
Identification: by individual ear-tattoo.

Environmental conditions
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. 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 autoclaved sawdust (SICSA, Alfortville, France).
Sawdust is analyzed by the supplier for composition and contaminant levels.

Food and water
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: corn oil for intradermal; acetone for epicutaneous
Concentration / amount:
5%
Route:
epicutaneous, semiocclusive
Vehicle:
other: corn oil for intradermal; acetone for epicutaneous
Concentration / amount:
5%
No. of animals per dose:
10 males; 10 females - test group
5 males; 5 females - control group
Details on study design:
2.1.3 Dosage form preparations
All dosage form preparations were made freshly on the morning of administration and any
unused material was discarded that same day.

2.1.4 Other materials
The other materials used were sterile isotonic saline solution (0.9% NaCl), batch No. EVD09B
(Laboratoire Fresenius, Sèvres, France) and Freund's complete adjuvant, batch No. 93K8932
(Sigma, Saint-Quentin-Fallavier, France).
2.3.1 Preparation of the animals
For both the preliminary test and the main test, the application sites of all animals were:
• clipped before intradermal injections (interscapular region 4 cm x 3 cm),
• clipped before topical applications of the induction phase (same region),
• clipped and shaved before topical applications of the challenge phase (each flank
3 cm x 3 cm),
• shaved before the 48-hour reading of the challenge phase and before the 24-hour reading of
the induction phase (preliminary test),
• clipped before skin sampling.

2.3.2 Preliminary test
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: 2.5%, 5%, 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.

2.3.3 Main test
2.3.3.1 Induction phase by intradermal and cutaneous routes
2.3.3.1.1 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 plastic syringe (0.01 mL
graduations).
Three injections of 0.1 mL were made into each side of this interscapular region (i.e. three pairs
of sites), as follows:
Injection Site Treated group Control group
1 Anterior FCA at 50% (v/v) in 0.9% NaCl FCA at 50% (v/v) in 0.9% NaCl
2 Middle test item at 2.5% (w/w)
in corn oil
corn oil
3 Posterior* test item at 2.5% (w/w) in the mixture
FCA/0.9% NaCl (50/50)
vehicle at 50% (w/v) in a 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.
The anterior and middle pairs of injections were performed close to each other and nearest the
head, while the posterior pair was performed towards the caudal part of the test area.

2.3.3.1.2 Cutaneous route
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
undiluted test item 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.

2.3.3.2 Challenge phase
On day 22, the animals of treated and control groups received an application of the test item and
vehicle. The filter paper of a chamber (Finn Chamber®) was fully-loaded with the test item at
the concentration of 5% (w/w) and was then applied to a shaved area of the skin of the posterior
right flank of all animals.
The vehicle was applied under the same experimental conditions to the skin of the posterior left
flank.
The chambers were held in contact with the skin for 24 hours by means of an adhesive
anallergenic waterproof plaster.

Positive control substance(s):
yes
Remarks:
mercaptobenothiazole; historical control
Positive control results:
The sensitivity of the experimental technique is regularly assessed using a known
moderate sensitizer, MERCAPTOBENZOTHIAZOLE. In a recent study performed under
CIT experimental conditions, the strain of guinea pigs used showed a satisfactory sensitization
response in 80% animals
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
5%
No. with + reactions:
18
Total no. in group:
20
Clinical observations:
moderate erythema (1-2) and dryness of skin
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5%. No with. + reactions: 18.0. Total no. in groups: 20.0. Clinical observations: moderate erythema (1-2) and dryness of skin.
Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the experimental conditions and according to the maximization method of Magnusson and Kligman, the test item OO-TERT-AMYL-O-(2-ETHYLHEXYL) MONOPEROXYCARBONATE (batch No. 25622 870403-733) induced delayed contact hypersensitivity in 18/20 (90%) guinea pigs and should therefore be considered as an extreme sensitizer (Category 1).
Executive summary:

Under the experimental conditions and according to the maximization method of Magnusson and Kligman, the test item OO-TERT-AMYL-O-(2-ETHYLHEXYL) MONOPEROXYCARBONATE (batch No. 25622 870403-733) induced delayed contact hypersensitivity in 18/20 (90%) guinea pigs and should therefore be considered as a Category 1, extreme sensitizer (R43).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Migrated from Short description of key information:

Under the experimental conditions and according to the maximization method of Magnusson and Kligman, the test item OO-TERT-AMYL-O-(2-ETHYLHEXYL) MONOPEROXYCARBONATE (batch No. 25622 870403-733) induced delayed contact hypersensitivity in 18/20 (90%) guinea pigs and should therefore be considered as a Category 1 sensitizer (extreme sensitizer).

Justification for selection of skin sensitisation endpoint:

A well-conducted GLP study.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The data from the skin sensitization test are conclusive, and the test substance is classified as Category 1 skin sensitizer.