Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1998-06-04 to 1998-07-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): POLYRAM SL
- Physical state: pale yellow translucent liquid
- Analytical purity: 100 % (expressed in complex substance)
- Purity test date: not stated
- Lot/batch No.: 6108
- Expiration date of the lot/batch: 1999 /03
- storage conditions : at room temperature; protected from light

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 les Elbeuf, FRANCE
- Age at study initiation: approximately three months old
- Weight at study initiation: 374 +/- 22g for the males and 354 +/- 16g for the females
- Housing: individually in polycarbonate cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature : 21 +/- 2°C
- Humidity : 30 to 70%
- Air changes : approximately 12 cycles/hour of filtered, non recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
physiological saline
Concentration / amount:
- Induction (treated group):
* intradermal injections : test item at the concentration of 0.1% (w/w) in sterile isotonic saline solution (0.9% NaCl)
* topical application : test item at the concentration of 10% (w/w) in sterile isotonic saline solution (0.9% NaCl)

- Challenge (all groups):
topical application : test item at the concentration of 1% (w/w) in sterile isotonic saline solution (0.9% NaCl)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
- Induction (treated group):
* intradermal injections : test item at the concentration of 0.1% (w/w) in sterile isotonic saline solution (0.9% NaCl)
* topical application : test item at the concentration of 10% (w/w) in sterile isotonic saline solution (0.9% NaCl)

- Challenge (all groups):
topical application : test item at the concentration of 1% (w/w) in sterile isotonic saline solution (0.9% NaCl)
No. of animals per dose:
_ a control group 1 : 10 animals (5 males and 5 females)
_ a treated group 2 : 20 animals ( 10 males and 10 females)
Details on study design:
RANGE FINDING TESTS:
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 2 cm),
• clipped before topical applications of the induction phase (same region),
• clipped and shaved before topical applications of the challenge phase
(each flank 2 cm x 2cm),
• shaved before the challenge phase.

Concentrations tested in the range finding test:
By intradermal route (tested concentrations: 10 %, 5 %, 1% and 0.1% (w/w)):
• intradermal injections of the dosage form preparations (0.1 mL) were performed in the
interscapular region,
• cutaneous 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: 100%, 50%, 10%, 5%
and 1 % (w/w)):
• a filter paper (approximately 4 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 24 hours,
• cutaneous reactions were evaluated 24 and 48 hours after removal of the dressing.
Under the conditions of the challenge phase (tested concentrations: 100%, 50%, 10%, 5%
and 1 % (w/w)):
•a filter paper (approximately 4 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 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.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal exposure and 1 epicutaneous (occlusive) exposure
- Exposure period: intradermal on day 1, epicutaneous on day 8
- Test groups: 10 males and 10 females
- Control group: 5 males and 5 females
- Site: the interscapular region, same site for both induction exposures
- Frequency of applications: single treatment for both intradermal and epicutaneous exposures
- Duration: intradermal exposure: single injection, epicutaneous exposure: 48h under occlusive dressing
- Concentrations:
intradermally for induction: Freund's complete adjuvant (FCA) diluted at 50% (v/v) with 0.9% NaCl, 0.1% (w/w) test item in 0.9% NaCl and 0.1% test item in a mixture FCA/0.9% NaCl (50/50, v/v),
epicutaneous for induction: 10% (w/w) in 0.9% NaCl

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day 22
- Exposure duration: 24 h under occlusive dressing
- Test groups: 10 males and 10 females
- Control group: 5 males and 5 females
- Site: treatment with test item on the right flank, treatment with vehicle on the left flank
- Concentrations: 1% (w/w) in 0.9% NaCl (0.9% NaCl as control)
- Evaluation (hr after challenge): 24 , 48 hours after removal of the dressing according to the method of Draize (See table 1 in results and discussion free-text for details)

GENERAL
- The animals were observed at least once a day during the study in order to check for clinical signs and mortality.
- The animals were weighed individually on the day of allocation into the groups, on the first day of the study (day 1) and on the last day of the study (day 25).


Challenge controls:
Yes-see above.
Positive control substance(s):
yes
Remarks:
2,4-Dinitrochlorobenzene (DNCB) and Mercaptobenzothiazole

Results and discussion

Positive control results:
The species and strain which were used showed a satisfactory sensitization response in 90 % animals treated with DNCB and in 30% animals treated with Mercaptobenzothiazole.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
1.0% w/w test item
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 1.0% w/w test item. No with. + reactions: 0.0. Total no. in groups: 20.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
1.0% w/w test item
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No clinical signs and no mortality were observed during the study. The body weight gain of the treated animals was normal when compared to that of the control animals.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1.0% w/w test item. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No clinical signs and no mortality were observed during the study. The body weight gain of the treated animals was normal when compared to that of the control animals..
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the experimental conditions and according to the maximization method of Magnusson and Kligman, the test substance should not be considered as a skin sensitizer.
Executive summary:

The potential of the substance to induce delayed contact hypersensitivity was assessed in guinea pigs accordingto the OECD (n°406, 17th july 1992) and Commission Regulation (EC) (n°96/54/E.E.C., B.6, 30 july 1996) guidelines. The study was performed in compliance with the principle of Good Laboratory Practices 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. The induction phase was realized both by intradermal route on day 1 (Test material 0.1 % w/w in 0.9% NaCl) and by cutaneous route on day 8 (Test material 10% w/w in 0.9% NaCl). The challenge phase was realized on day 22 by cutaneous application of the test material at 1% w/w in 0.9% NaCl. The cutaneous reactions were scored 24 and 48 after the challenge phase.

No clinical signs and no deaths related to treatment were noted during the study. After the challenge application, no cutaneous reactions were observed in the treated animals nor in the control group.

The animals treated with positive control showed a satisfactory sensitisation response of 90% for 2,4-Dinitrochlorobenzene and 30% for mercaptobenzothiazole.

According to the Magnusson & Kligman maximization method, the test substance does not induce delayed contact hypersensitivity in guinea pigs.