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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
Study period:
to 1998-10-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted 17 July 1992
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study report from 1998 readily available

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Name: LUPEROX 188 M 70 (or PEROXYNEODECANOATE DE CUMYLE)
- Source and lot/batch No. of test material: XX200001 (supplied by Elf Atochem S.A.)
- Expiration date of the lot/batch: September 1998
- Appearance: liquid
- Purity: 75.6% in isododecane

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at -20 °C and protected from light

Appearance: colourless liquid
Container: one plastic flask

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, 76410 Saint-Aubin-lés-Elbeuf, France.
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
- Age at study initiation: approx. 3 months
- Weight at study initiation: 345 ± 12 g (males), 336 ± 8 g (females)
- Housing: 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).
- Diet (e.g. ad libitum): "106 pelleted diet" (UAR, 91360 Villemoisson-sur-Orge, France); ad libitum
- Water (e.g. ad libitum): Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 °C
- Humidity (%): 30 to 70%
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 h/12 h

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
corn oil
Concentration / amount:
0.1 mL of the test substance 10% (w/w) in corn oil/ see details on study design
Day(s)/duration:
day 1/single application
Adequacy of induction:
other: highest concentration evaluated in the pre-test
Route:
epicutaneous, open
Vehicle:
other: vaseline
Concentration / amount:
10% (w/w)
Day(s)/duration:
day 7
Adequacy of induction:
other: topical application of sodium lauryl sulfate in vaseline to induce local irritation
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.5 ml of the undiluted test substance/see details on study design
Day(s)/duration:
day 8/single application, 48 hours
Adequacy of induction:
highest technically applicable concentration used
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.5 ml of the undiluted test substance
Day(s)/duration:
day 22/24 hours
Adequacy of challenge:
other: highest technical concentration used (undiluted)
No. of animals per dose:
in total 30 (15 males/15 females)
- control group: five males and 5 females
- treatment group: ten males and 10 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:
- 24 hours before treatment, the dorsal region of the animals was clipped,
- intradermal administrations of the test substance formulation (0.1 mL) at different concentrations were performed in the interscapular region,
- cutaneous reactions were evaluated approximately 24, 48 hours and 6 days after the injections
By cutaneous route:
- 24 hours before treatment, both flank regions of the animals were clipped,
- 0.5 mL of the undiluted test substance or test substance formulation at the chosen concentration were placed on a dry gauze pad (approximately 4 s) which was then applied to the skin and held in place by an occlusive dressing for 24 hours,
- cutaneous reactions were evaluated approximately 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.

MAIN STUDY
- Application sites were clipped on days -1 and 7 (interscapular region 4 x 2 cm) and clipped and shaved on day 21 (each flank 2 x 2 cm)
A. INDUCTION EXPOSURE
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 16mm) mounted on a 1 mL glass syringe (0.01 mL graduations).
Three injections of 0.1 mL were made into each side of this interscapular region (i.e. three pairs ofsites), as follows:
- Anterior injection site: Treated group: Freund’s complete adjuvant (FCA) diluted at 50% (v/v) with 0.9% NaCl; Control group: FCA diluted at 50% (v/v) with 0.9% NaCl
- Middle injection site: Treated group: Test substance at 10% (w/w) in corn oil; Control group: vehicle undiluted
- Posterior injection site: Treated group: Test substance at 10% (w/w) in a 1:1 mixture FCA/ 0.9% NaCl; Control group: Vehicle at 50% (w/w) in a 1:1 mixture FCA/ 0.9% NaCl. The test substance was suspended in FCA prior to combining with the aqueous phase.The final concentration of the test substance was equal to that used in the middle injection site.

Cutaneous route:
On day 7, the interscapular area was clipped. As the test substance was shown to be non-irritant during the preliminary test, the animals were treated with 0.5 mL of sodium lauryl sulfate (10%,w/w) in vaseline in order to induce local irritation.
On day 8, a cutaneous application to the region of the intradermal injections (4 cm x 2 cm) was performed as follows:
Control group
- application of 0.5 mL of the vehicle.
Treated group
-application of 0.5 mL of the undiluted test substance.
The test substance or the vehicle was placed on a dry gauze pad, which was then applied to the interscapular region. 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, no residual test substance was observed. Cutaneous reactions were recorded 1 hour after removal of the occlusive dressing.

B. CHALLENGE EXPOSURE
On day 22, the animals of both groups received an application of 0.5 mL of the undiluted test substance to the posterior right flank and 0.5 mL of the vehicle to the posterior left flank. This application was performed using a 1 mL plastic syringe (0.01 mL graduations). The test substance or the vehicle was placed on a dry gauze pad, which was then applied to a 4 cm² (2 cm x 2 cm) clipped area of the skin. The pads were held in contact with the skin for 24 hours by means of an occlusive, hypoallergenic dressing and an adhesive anallergenic waterproof plaster. On removal of the dressing, no residual test substance was observed.

- Evaluation (hr after challenge): Twenty-four and 48 hours after removal of the dressing of the challenge application, both flanks of the treated and control animals were observed to evaluate cutaneous reactions.

OTHER: Determination of the Allergenicity level:
The treated animals show a positive reaction if macroscopic cutaneous reactions are clearly visible (erythema and/or oedema greater or equal 2) and if the treated animals have a greater intensity or duration of response than the maximum reaction seen in control animals, or if macroscopic reactions are confirmed at microscopic examination as being due to the sensitisation process.
Challenge controls:
10 animals (5 male/5 female) previosly treated with the vehicle were challenged together with the test group using the same test article concentration as for the test group.
Positive control substance(s):
yes
Remarks:
DCNB and Mercaptobenzothiazole

Results and discussion

Positive control results:
According to this study used the Magnusson and Kligman method, the test substance DNCB at the concentration of 1% (w/w) induced positive skin sensitization reactions in 90% of the guinea-pigs. 30% of the animals treated with mercaptobenzothiazole showed a sensitization response.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no clinical signs and no mortality were observed
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no clinical signs and no mortality were observed
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no clinical signs and no mortality were observed
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no clinical signs and no mortality were observed
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
1% DNCB
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
20% Mercaptobenzothiazole
No. with + reactions:
3
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Any other information on results incl. tables

Table 2: Individual results of the control group for skin reaction after challenge

Sex Animal no. 24 hours 48 hours
Male 1 1 0
2 1 0
3 1 0
4 0 0
5 0 0
Female 6 0 0
7 0 0
8 0 0
9 0 0
10 0 0

Table 3: Individual results of the test group for skin reaction after challenge

Sex Animal no. 24 hours 48 hours
Male 11 1 0
12 0 0
13 0 0
14 1 0
15 0 0
16 0 0
17 0 0
18 1 0
19 1 0
20 1 0
Female 21 0 0
22 1 0
23 0 0
24 1 0/Dryness of the skin
25 0 0
26 0 0
27 0 0
28 1 0
29 0 0
30 0 0

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, in an in vivo skin sensitisation test conducted in accordance to OECD 406 the test material did not induce delayed contact hypersensitivity in guinea pigs.
Executive summary:

In an in vivo skin sensitization study conducted according to OECD 406 with the test item in corn oil, young adult Dunkin-Hartley guinea pigs (10/sex) were tested using the Guinea Pig Maximisation Test of Magnusson and Kligman. DCNB and mercaptobenzothiazole served as positive controls. On day 1, intradermal injections were performed using a 10% suspension of the test material. On day 8, an occlusive epicutaneous induction was performed with the undiluted test item. On day 22 all animals were challenged by a cutaneous application of the undiluted test item for 24 hours under occlusive conditions. Skin reactions were examined 24 and 48 h following removal of the dressing.

No clinical signs and no deaths were noted during the study. No well-defined cutaneous reactions were observed after the challenge application. Based on the results from this study, the test item did not induce delayed contact hypersensitivity in guinea pigs.