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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:
From 20 Aug 1991 to 20 Sep 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD 406), GLP compliant

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
reduced number of animals (sufficient, if unambiguous result)
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The test was performed before the adoption of OECD 429 LLNA in 2002.

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[2,4-dihydro-5-methyl-2-(p-tolyl)-3H-pyrazol-3-one]
EC Number:
239-898-6
EC Name:
4,4'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[2,4-dihydro-5-methyl-2-(p-tolyl)-3H-pyrazol-3-one]
Cas Number:
15793-73-4
Molecular formula:
C34H28Cl2N8O2
IUPAC Name:
4,4'-[(3,3'-dichlorobiphenyl-4,4'-diyl)didiazene-2,1-diyl]bis[5-methyl-2-(4-methylphenyl)-2,4-dihydro-3H-pyrazol-3-one]
Test material form:
solid: nanoform

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Pirbright-White
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hoechst AG, Kastengrund, SPF Zucht
- Weight at study initiation: 295-352 g
- Housing: groups of 5 animals
- Diet (ad libitum): Altromin §112 Haltungsdiät für Meerschweinchen, Altromin GmbH, Lage/Lippe, Germany
- Water (ad libitum): tap water
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 20
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
petrolatum
Remarks:
for epicutaneous treatment
Concentration / amount:
1% (intradermal induction), 25% (epicutaneous induction), 1% (challenge)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Remarks:
for epicutaneous treatment
Concentration / amount:
1% (intradermal induction), 25% (epicutaneous induction), 1% (challenge)
No. of animals per dose:
6 for determination of the primary non-irritating concentration
3 for determination of the tolerance of the intradermal injections
10 for treated group
5 for control group
5 for accompanying group

Details on study design:
RANGE FINDING TESTS:
The primary non-irritating concentration was determined in a pre-test in concentrations of 1, 5 and 25% test item in petrolatum, applied to the shaved left flanks of 2 animals each (0,5 g, 2 x 2 cm, occlusive, 24 h). 24 h after removal of the patch the treated areas were examined for erythema and edema.
The tolerance of the intradermal injections was tested with two injections of 0,1 ml of 0.2, 1.0 and 5.0% test item in paraffin. The injection sites (sites 1, 2 and 3) were all within a dorsal area measuring 2 x 4 cm in the vicinity of the shoulders.


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: two intradermal injections per animal, one epicutaneous treatment
- Exposure period: 48 h occlusive for dermal exposure
- Test groups: 10 females
Intradermal Treatment: The injection sites (site 1, 2 and 3) were all within a shaved dorsal area of 2 x 4 cm. The injection sites were left uncovered. Two intradermal injections of 0.1 ml per animal of the following preparations were applied (day 1 of study): site 1: 50% FCA, site 2: 1% test item in paraffin, site 3: test item in 50% FCA
Epicutaneous treatment: An amount of 0.5 g of the test item preparation (25% test substance in petrolatum) was administered to a 2 x 4 cm cellulose patch. This patch covered the area where the intradermal injection had been placed. The administration area was then kept under an occlusive bandage covered with an impermeable film and an elastic bandage for 48 hours.
- Control group and accompanying group: 5 females each
Controls received the same treatment with: intradermal site 1: 50% FCA ( Freund's Complete Adjuvants); site 2: paraffin; site 3: 50% FCA;
epicutaneous: petrolatum
- Site: dorsal, in vicinity of the shoulders
- Frequency of applications: day 1: intradermal treatment, day 8: epicutaneous treament

B. CHALLENGE EXPOSURE
- No. of exposures: one
- Day(s) of challenge: day 22 of study
- Exposure period: 24 h, occlusive
- Test groups: An amount of 0.5 g of the test item preparation was administered to a 2 x 2 cm cellulose patch.
- Site: left flank
- Concentrations: 1% in petrolatum (test and control group)
- Evaluation (hr after challenge): 24 and 48 h after removal of the patch

The accompanying group was challenged in the same way as the test group, but on days 15-18 of the study

OTHER:
Evaluation of effects: The decisive criterion for evaluation of the sensitizing properties of a test substance is the number of sensitized test animals, not the intensity of the dermal reaction. The substance is considered to be sensitizing if 30 % or more of the animals in the treatment group definitely showed a positive skin reaction and at the same time no irritant effects have emerged in the control group.
Scoring system for dermal reactions: according to guideline EU B.6 and OECD 406

Positive control substance(s):
not specified
Remarks:
According to guideline, a periodic test with a positive control substance is necessary. These data are not stated in study report, but also not mentioned under "deviations from guidelines"

Results and discussion

Positive control results:
not stated

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
1%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no effects
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no effects.
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
1%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no effects
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no effects.
Reading:
1st reading
Hours after challenge:
48
Group:
other: accompanying group
Dose level:
1%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no effects
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: other: accompanying group. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no effects.
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
1%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no effects
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no effects.
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
1%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no effects
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no effects.
Reading:
2nd reading
Hours after challenge:
72
Group:
other: accompanying group
Dose level:
1%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no effects
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: other: accompanying group. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no effects.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this guinea pig maximisation test, the test substance was not sensitising.
Executive summary:

Skin sensitization of the test item was performed in 15 female guinea pigs (10 test group, 5 control group) according to the method of Magnusson & Kligman according to guidelines EU B.6 and OECD 406. Intradermal induction was performed with 1% test item in paraffin, dermal induction with 25% test item in petrolatum. The challenge treatment was conducted with 1% test item in petrolatum. Under the conditions of this guinea pig maximisation test, the test substance was not sensitising.