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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:
1999
Report date:
1999

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:
test was conducted in 1999, LLNA was not adopted until 2002

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
additive
Test material form:
other: colorless liquid
Details on test material:
* name:
- protocol: t-AMYL PEROXYPN ALATE (LUPEROX 554M75) ·
-labelling: t-AMYL PEROXYPN ALATE LUPEROX 554M75
*batch number:
- protocol and labelling: 646·9803-702 (13/03/98)
* Elf Atochem filing number: CAL 4100/98
*description: colorless liquid
*container: one plastic flask
*date of receipt: 16 December 1998
* storage conditions: at ·20°C and protected from light
*purity: approximately 75%
* expiry date: June 1999.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
Animals
Species and strain: Dunkin-Hartley guinea-pigs.
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
positive sensitizers.
Breeder: Charles River France, 76410 Saint-Aubin-les-Elbeuf, France.
Number: two males and two females for the preliminary test,
30 animals (IS males and 15 females) for the main test.
Females were nulliparous and non-pregnant.
Allocation of the animals to the groups: on day -I, 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 I, the animals of the main test were approximately 3 months old and had a mean
body weight ± standard deviation of 339 ± 11 g for the males and 349 + 8 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 hl12 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 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 and chemical analyses of the sawdust, including the detection of possible
contaminants (pesticides, heavy metals), are performed regularly by external laboratories.
The results of these analyses are archived at CIT.

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.
The diet formula is presented in appendix 2.
Drinking water filtered by a FO Millipore membrane (0.22 micron) was provided ad libitum.
Bacteriological and chemical analyses of the water and diet, including the detection of possible
contaminants (pesticides, heavy metals and nitrosamines), are performed regularly by external
laboratories.
The results of these analyses are archived at CIT.
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.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
unchanged (no vehicle)
Concentration / amount:
Induction (treated group)
· intradermal injections: t-AMYL PEROXYPN ALATE (LUPEROX 554M75) at the
concentration of 5% (w/w) in com oil,
· topical application: t-AMYL PEROXYPN ALATE (LUPEROX 554M75) undiluted.
Challenge (all groups)
· topical application: t-AMYL PEROXYPN ALATE (LUPEROX 554M75) undiluted.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
Induction (treated group)
· intradermal injections: t-AMYL PEROXYPN ALATE (LUPEROX 554M75) at the
concentration of 5% (w/w) in com oil,
· topical application: t-AMYL PEROXYPN ALATE (LUPEROX 554M75) undiluted.
Challenge (all groups)
· topical application: t-AMYL PEROXYPN ALATE (LUPEROX 554M75) undiluted.
No. of animals per dose:
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).
Details on study design:
On day 1, intradermal injections of Freund's complete adjuvant mixed with the test substance
(treated group) or the vehicle (control group) were performed 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.
Challenge controls:
Control groups were challenged by a cutaneous application of the test substance to the right flank.

The sensitivity of the guinea-pigs in CIT experimental conditions was checked with a positive
sensitizer, 2,4-Dinitro Chlorobenzene (DNCB). During the induction period, the reference
substance DNCB was applied at the concentrations of 0.1 % (w/w) (day I) and 1 % (w/w) (day 8)
in corn oil. For the challenge application, the reference substance DNCB was applied at the
concentration of 1% (w/w) in corn oil.
Positive control substance(s):
yes
Remarks:
2,4-Dinitro Chlorobenzene (DNCB)

Results and discussion

Positive control results:
Sensitization response in 90% animals treated with DNCB.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100%
No. with + reactions:
20
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100%
No. with + reactions:
12
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
1 % (w/w) in corn oil
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
90% sensitization
Remarks on result:
positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under our experimental conditions and according to the maximization method of Magnusson and Kligman, the test substance t-AMYL PEROXYPIV ALATE (LUPEROX 554M75) (batch No. 646-9803-702 (13/03/98» induces delayed contact hypersensitivity in 12/20 (60%)
In this test 60% response at 5% intradermal induction dose and this reults in a classification as skin sensitizer 1b (guinea-pigs. ≥ 30 % responding at > 1 % intradermal induction dose.).
Executive summary:

The study was conducted to evaluate the potential of the test substance tert-amyl peroxypivalate to induce delayed contact hypersensitivity 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.

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, intradermal injections of Freund's complete adjuvant mixed with the test substance (treated group) or the vehicle (control group) were performed 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.

Under our experimental conditions and according to the maximization method of Magnusson and Kligman, the test substance t-AMYL PEROXYPIV ALATE (LUPEROX 554M75) (batch No. 646-9803-702 (induced delayed contact hypersensitivity in 12/20 (60%) guinea-pigs.