Registration Dossier

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:
other: Guideline-Studie (GLP, QAU)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (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): dibutyl phthalate (test substance number 89/449)
- Physical state: liquid, clear, achromatic (the sample appeared to be homogeneous)
- Analytical purity: 99.4%
- Lot/batch No.: P. 66 A/89
- Stability under test conditions: on completion of all tests the stability of the test substance will be verified by a repeated analysis. The stability of the test substance in olive oil DAB 9 was confirmed by analysis
- Storage condition of test material: room temperature

In vivo test system

Test animals

Species:
guinea pig
Strain:
Pirbright-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Pirbright White, Dunkin Hartley HOE DHPK [SPF-LAC] BO from Lippische Versuchstierzucht, Hagemann GmbH & Co. KG, D-4923 Extertal 1, FRG
- Weight at study initiation: 252 – 313 g
- Housing: the animals, identified by ear tag numbering, were housed 5 per cage in Makrolon, type IV
- Diet (e.g. ad libitum): Kliba 341.4 mm (Kaninchen-Meerschweinchen-Haltungsdiät) from Firma Klingentalmühle AG, CH-4303 Kaiseraugst, Switzerland
- Water (e.g. ad libitum): tap water; about 2 g of ascorbic acid per 10 l water was added to the drinking water twice a week
- Acclimation period: at least 8 days before the beginning of the study in the laboratory for dermal toxicity

ENVIRONMENTAL CONDITIONS
The animals were housed in fully air-conditioned rooms in which a central air-conditioning system
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Air changes (per hr): - Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: olive oil
Concentration / amount:
5% at intradermal induction, 75% at epicutaneous induction and 50% at challenge
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: olive oil
Concentration / amount:
5% at intradermal induction, 75% at epicutaneous induction and 50% at challenge
No. of animals per dose:
10 in the control groups, 20 in the test group (see Table 1)
Details on study design:
RANGE FINDING TESTS:
The test substance was applied 2 times for 24 hours within a period of 96 hours in order to detect non-specific phenomena that are not caused by a sensitization reaction but could possibly be attributed to a shift in the irritation threshold. (1) Site of application: flank, respective on the same area; (2) number of test animals: 4 per test concentration; (3) readings: about 24 and 48 h after the beginning of application.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 6 intradermal injections in groups of two per animal
- Exposure period: at the beginning for intradermal injection and one week thereafter for epicutaneous treatment
- Test groups: a) front row: 2 injections each of 0.1 ml Freund's adjuvant without test substance emulsified with water in a ratio of 1:1; b) middle row: 2 injections each of 0.1 ml of the test substance formulation; c) back row: 2 injections each of 0.1 ml Freund's adjuvant/water (1:1) with test substance. A percutaneous induction was carried out about one week after intradermal induction, when 2x4 cm filter paper strips were applied to the skin of the shoulder under an occlusive dressing. In the case of liquids the filter paper strip was soaked in the test substance formulation; thus, the animals were exposed to about 0.3 g of the test substance formulation.
- Control group: the animals were given the same injections (a, b, c) as the test groups but without test substance, only with the formulating agent. At epicutaneous induction, the control groups were treated analogously to the test group but only with the solvent without the test substance.
- Site: shoulder at intradermal induction; shoulder at epicutaneous induction, same area as in the case of the previous intradermal application
- Duration: reading was done 24 h after the beginning of intradermal application. The epicutaneous patch was maintained for 48 hours
- Concentrations:

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: first challenge 14 days after percutaneous application, second challenge one week later
- Exposure period: 24 hours
- Test groups and control group: 1) first challenge; treatment of the test group and of control group 1 with the test substance formulation. Additionally, olive oil DAB 9 was applied as a vehicle. Control group 2 only received olive oil DAB 9. 2) 2nd challenge; treatment of the test group and of control groups 1 and 2 with the test substance formulation. Analogous to the first challenge olive oil DAB 9 was applied as a vehicle.
- Site: intact clipped flank
- Concentrations: non-irritant concentration; 2 x 2 cm filter paper strips were applied to the skin of the flank under an occlusive dressing. In the case of liquids the test filter paper strip was soaked in the test substance formulation; thus the animals were exposed to about 0 .15 g of the test substance formulation.
- Evaluation (hr after challenge): 24, 48 and 72 hours after the beginning of application

OTHER: erythema and the edema were scored according to the OECD guideline. The evaluation was based on the criteria of Annex VI/II D of the Council Directive of July 29, 1983 for the 5th Amendment of the Directive 67/548 EEC (= 83/467 EEC)
Positive control substance(s):
no
Remarks:
the maximization test is often conducted in the testing facility

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50%
No. with + reactions:
15
Total no. in group:
20
Clinical observations:
8 animals with erythema score of 2 and 7 animals with erythema score of 1; maximum erythema score: 4
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 15.0. Total no. in groups: 20.0. Clinical observations: 8 animals with erythema score of 2 and 7 animals with erythema score of 1; maximum erythema score: 4.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50%
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
both animals with erythema score of 1; all animals showed negative reactions at the 72 hours reading
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: both animals with erythema score of 1; all animals showed negative reactions at the 72 hours reading.
Reading:
other: 1st readingd after rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
50%
No. with + reactions:
18
Total no. in group:
20
Clinical observations:
7 animals with erythema score of 2 and 11 animals with erythema score of 1
Remarks on result:
other: see Remark
Remarks:
Reading: other: 1st readingd after rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 18.0. Total no. in groups: 20.0. Clinical observations: 7 animals with erythema score of 2 and 11 animals with erythema score of 1.
Reading:
other: 2nd readingd after rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no irritation was observed at this reading time point and 24 hours thereafter
Remarks on result:
other: see Remark
Remarks:
Reading: other: 2nd readingd after rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: no irritation was observed at this reading time point and 24 hours thereafter.

Any other information on results incl. tables

- The injection of test substance preparation in olive oil DAB 9 resp. in Freund's adjuvant/aqua dest. (1 : 1) caused distinct erythema and edema in the test animals. The control animals, injected with olive oil DAB 9 (vehicle) exhibited distinct erythema.

 

- After percutaneous induction incrustation, partially open (caused by the intradermal induction) was observed in the test animals in addition to distinct erythema and edema. Control group 1 and 2 which were applied with the vehicle (olive oil DAB 9) exhibited the same reactions as the test animals.

 

Table 1: Summary of number of animals with skin findings after the 1stchallenge (20 days after intradermal induction) and after the 2nd challenge (27 days after intradermal induction)

 

1stchallenge

2nd challenge

 

50% TS in olive oil DAB 9

Olive oil DAB 9

50% TS in olive oil DAB 9

Olive oil DAB 9

Control group 1

0/10

0/10

0/10

0/10

Control group 2

-

1/10

0/10

0/10

Test group

2/20

0/20

0/20

0/20

TS: Test substance; -: no application of test substance; x/y : number of positive reactions/number of animals tested; readings 48 h after the beginning of application

 

 

- After the first challenge (48 hours after the beginning of application) the 50% test substance preparation in olive oil DAB 9 caused slight erythema in 2 of 20 test animals. Control group 1 did not show any skin reactions.

 

- After the second challenge (48 hours after the beginning of application) with the 50 % test substance preparation in olive oil DAB 9 no skin reactions could be observed neither in the test group nor in the control groups.

 

- Olive oil DAB 9 which was applied as a vehicle caused slight erythema in 1 of 10 animals of control group 2 after the first challenge (48 hours after the beginning of application).

 

- Olive oil DAB 9 caused no skin reactions in all animals after the second challenge (48 hours after the beginning of application).

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information