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 and guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Cited as Directive 84/449/EEC, B.6
GLP compliance:
yes (incl. certificate)
Remarks:
testing lab.
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): 3-Methyl-2-butenal, CAS: 107-86-8; Substance-No. 90/734
- Physical state: liquid, achromatic
- Analytical purity: 96.3%
- Lot/batch No.: 90/11
- Storage condition of test material: room temperature under nitrogen

In vivo test system

Test animals

Species:
guinea pig
Strain:
Pirbright-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Lippische Versuchstierzucht, Hagemann GmbH & Co KG
- Weight at study initiation: 300-350 g
- Housing: 5 per cage type IV Macrolon
- Diet: Kliba 341.4 mm rabbit-guinea pig laboratory diet, ad libitum
- Water: ad libitum
- Acclimation period: at least 7 days


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

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
0.2% intradermal, 10% epicutaneous (induction); 5% (challenge)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
0.2% intradermal, 10% epicutaneous (induction); 5% (challenge)
No. of animals per dose:
20
Details on study design:
RANGE FINDING TESTS:
In the preliminary test after two 24-hour percutaneous occlusive applications within 96 hours the minimum irritant concentration was found to be a 10 % test substance preparation in aqua bidest, and the maximum nonirritant concentration a 5% test substance preparation in aqua bidest. (48 hours after the beginning of application).

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 6 intradermal injections in groups of two per animal (Freunds adjuvant alone, 0.1 mt test substance alone, Freunds adjuvant plus test substance); percutaneous induction (one week after intradermal injections): 2x4 cm filter paper strips containing 0.3g of the test substance formulation
- Exposure period: 24 hrs
- Test groups: 10
- Control group: 5 (left untreated)
- Site: shoulder
- Frequency of applications:
- Duration: 24 hrs (intradermal); 48 hrs (percutaneous)
- Concentrations: 0.3 g per patch (filter papers were soaked with test substance); intradermal: 0.2%; percutaneous: 10%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 21
- Exposure period: 24 hrs
- Test groups: 2 x2 cm filter paper strips applied under occlusive conditions
- Control group: group 1: receiving test substance formulation (5%); group 2: left untreated
- Site: flank skin at non-irritant concentration (5%)
- Evaluation (hr after challenge): 24, 48 hrs after removal of the patch

EVALUATION: The evaluation "sensitizing" results if at least 30 % of the experiment animals exhibit skin reactions
Challenge controls:
Untreated animals and animals challenged with 5% of test substance in aqua bidest in previously NaCl or Freund's adjuvant treated animals
Positive control substance(s):
yes
Remarks:
1-chlor-2,4-dinitro-benzol (historical control)

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
other: challenge
Hours after challenge:
24
Group:
test group
Dose level:
5% in aqua dest.
No. with + reactions:
3
Total no. in group:
9
Clinical observations:
one animal died 8 days after beginning of application
Remarks on result:
other: Reading:
Reading:
other: challenge
Hours after challenge:
24
Group:
negative control
Dose level:
5% in aqua dest.
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading:

Any other information on results incl. tables

Mortalities: One animal of the test group was found dead on day 8 after application.
Skin effects: (1) Well defined erythema and slight edema were seen 
at the injection sites of Freund's complete adjuvans 
(FCA)/saline (1:1) in both test and  control animals. 
Well defined erythema and slight edema were also 
seen at the injection sites of the test substance in 
FCA/saline in test animals. (2) Well defined erythema, slight edema and incrustation 
(partially open from interadermal injections) were noted 
after percutaneous induction in the test animals. (3) Positive skin reactions, i.e. erythema, 
were observed in 3/9 test animals after the challenge 
procedure at both readings. No reaction (0/5 animals) 
was seen in the control group. 
The positive control substance led to 
positive skin reactions (erythema) in 10/10 animals 
at both readings after the challenge

Readings

Animal

Exposure period: 4 h

Erythema

Edema

Additional findings

24 h

1

0

0

2

2

0

3

2

0

4

-

-

Exitus, April 3 1991

5

0

0

6

0

0

7

0

0

8

1

0

9

0

0

10

0

0

48 h

1

0

0

2

1

0

3

2

0

4

-

-

Exitus, April 3 1991

5

0

0

6

0

0

7

0

0

8

1

0

9

0

0

10

0

0

Mean

24, 48 h

1

0

0

2

1.5

0

3

2

0

4

-

-

Exitus, April 3 1991

5

0

0

6

0

0

7

0

0

8

1

0

9

0

0

10

0

0

Mean 24, 48 h

1-10

0.5

0

Results of the control group: all animals were scored 0/0 for erythema/edema formation

Results of the test group: 3/9 positive (equal to 33.3%)

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
3-methyl-2-butenal had a slight, borderline sensitizing
effect in the guinea pig maximation test.