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: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report Date:
1996

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes
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): 625-Triketone
- Substance No.: 95/225-1
- Physical state: Powder, beige
- Analytical purity: 99.4 %
- Lot/batch No.: LJ 27881/1
- Date of manufacturing: 18-27 Jul 1995
- Stability under test conditions: guaranteed by the sponsor and verified by reanalysis
- Storage condition of test material: refrigerator

In vivo test system

Test animals

Species:
guinea pig
Strain:
Pirbright-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dr. K. Thomae GmbH, Biberach, FRG
- Strain/Quality: Pirbright White, Dunkin Hartley Crl: (HA) BR [SPF]
- Age at study initiation: young adult animals
- Weight at study initiation: animals of comparable weight; 255 - 344 g
- Fasting period before study: none
- Housing: single housing
- Diet: KLIBA-Labordiaet 341, Klingenthalmuehle AG Kaiseraugst, Switzerland, ad libitum
- Water: tap water ad libitum
- Acclimation period: 6 days before the beginning of the study in the laboratory for dermal toxicity


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

Analysis of drinking water
The drinking water is regularly assayed for chemical contaminants by the municipal authorities of Frankenthal and the technical services of the BASF AG as well as for the presence of germs by a contract laboratory.

Analysis of feed:
The feed used in the study was assayed for chemical and microbial contaminates.

Analysis of the bedding :
The bedding is regularly assayed for contaminants (chlorinated hydrocarbons, heavy metals).

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
olive oil
Concentration / amount:
Intradermal induction:
5 % test substance in olive oil DAB 10
5 % test substance in Freund’s adjuvant/0.9 % aqueous NaCl-solution (1:1)
Freund’s adjuvant/0.9 % aqueous NaCl-solution (1:1)
olive oil DAB 10

Percutaneous induction:
50 % test substance in olive oil DAB 10
olive oil DAB 10

Challenge:
25 % test substance in olive oil DAB 10
olive oil DAB 10
Challengeopen allclose all
Route:
epicutaneous, open
Vehicle:
olive oil
Concentration / amount:
Intradermal induction:
5 % test substance in olive oil DAB 10
5 % test substance in Freund’s adjuvant/0.9 % aqueous NaCl-solution (1:1)
Freund’s adjuvant/0.9 % aqueous NaCl-solution (1:1)
olive oil DAB 10

Percutaneous induction:
50 % test substance in olive oil DAB 10
olive oil DAB 10

Challenge:
25 % test substance in olive oil DAB 10
olive oil DAB 10
No. of animals per dose:
Test group: 10 animals
Control group 1: 5 animals
Control group 2: 5 animals
Details on study design:
RANGE FINDING TEST
The concentrations of the test substance suitable for use in the main experiment were determined in a pretest. After two 24-hour percutaneous occlusive applications within 96 hours, the minimum irritant concentration was found to be a 50 % test substance preparation in olive oil DAB 10. The maximum non-irritant concentration was found to be a 25 % test substance preparation in olive oil DAB 10 (24 and 48 hours after application) .

Applicability: it was possible to inject a 5 % test substance preparation in olive oil DAB 10 resp. in
Freund's adjuvant / 0.9% aqueous NaCl-solution (1:1) with a syringe. The following concentration for induction and the challenge were selected on the basis of the pretests:

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal induction
- No. of exposures: single exposure with 6 intradermal injections in groups of two per animal
- Test groups: 2 injections each of 0.1 ml Freund's adjuvant without test substance emulsified with 0.9% aqueous NaCl-solution in a ratio of 1 : 1/ 2 injections each of 0.1 ml of the test substance fomulation/ 2 injections each of 0.1 ml Freund's adjuvant emulsified with 0.9% aqueous NaCl-solution (1 : 1) with test substance
- Control groups: same injections but without test substance
- Site: shoulder
- Readings: 24 h after beginning of the application

Percutaneous induction
Percutanous induction was carried out one week after intradermal induction
- No. of exposures: single exposure
- Exposure period: 48 h
- Test groups: test substance in olive oil DAB10 under occlusive dressing
- Control groups: olive oil DAB10 under an occlusive dressing
- Site: shoulder, same area as for intradermal injection
- Readings: 48 h after beginning of the application

B. CHALLENGE EXPOSURE
Challenge was performed 21 days after intradermal exposure.
- No. of exposures: single exposure
- Exposure period: 24 h
- Test groups: test substance in olive oil DAB10 under an occlusive dressing
- Control groups: olive oil DAB10 under an occlusive dressing
- Site: intact flank of the animals
- Evaluation (hr after challenge): 24 h and 48 h after removal of the patch
Challenge controls:
Control groups only received olive oil DAB 10 during the induction phase. At challenge control group 1 was treated analogous to the test group, control group 2 only received olive oil DAB 10.
Positive control substance(s):
yes
Remarks:
alpha-Hexylcinnamaldehyde 85 % (A positive control with a known sensitizer is not included in this study. However, a separate study is performed twice a year in the laboratory.)

Results and discussion

Positive control results:
Control group 1:
1. challenge (50 % test substance in olive oil DAB10): 2/10
2. challenge (50 % test substance in olive oil DAB10): 3/10
Control group 2:
1. challenge (olive oil DAB10): 2/10
2. challenge (50 % test substance in olive oil DAB10): 1/10
test group:
1. challenge (50 % test substance in olive oil DAB10): 13/19
2. challenge (50 % test substance in olive oil DAB10): 14/19

x/y = number of animals showing symptoms / total number of treated animals in the respective dose group

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25 %
No. with + reactions:
4
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25 %. No with. + reactions: 4.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
3
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: 3.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25 %
No. with + reactions:
0
Total no. in group:
4
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25 %. No with. + reactions: 0.0. Total no. in groups: 4.0.

Any other information on results incl. tables

MORTALITIES 2 animals each of control group 1 and 2 died 20, 21, 22 and 24 days after the beginning of the study. Macroscopic examination revealed, that the animals suffered from pneumonia. The deaths were not related to the test substance treatment. BODY WEIGHTS Body weights developed within the expected range.
treatment group formulation symptoms number of positive reactions/number of animals tested
Intradermal induction Test group Freund's adjuvant/0.9% aqueous NaCl-solution (1 : 1) well-defined erythema and slight edema 10/10
5 % 625-Triketone in olive oil DAB 10 well-defined erythema and slight edema 10/10
5 % 625-Triketone in Freund's adjuvant/0.9% aqueous NaC1-solution (1 : 1) well-defined erythema and slight edema 10/10
Control group 1+2  Freund's adjuvant/0.9% aqueous NaC1-solution (1 : 1) well-defined erythema and slight edema 10/10
Olive oil DAB 10 well-defined erythema 10/10
Percutanous induction Test group 50 % 625-Triketone in olive oil DAB 10 incrustation (partially open), well-defined erythema and slight edema 10/10
Control group 1+2  Olive oil DAB 10 incrustation (partially open), well-defined erythema and slight edema 10/10
Challenge Test group:  25 % test substance in olive oil DAB10 very slight to well-defined erythema 4/10
Olive oil DAB 10 - 0/10
Control group 1 25 % test substance in olive oil DAB10 - 0/4
Olive oil DAB 10 - 0/4
Control group 2 Olive oil DAB 10 - 0/3

Applicant's summary and conclusion