Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 October 1988 - 1 December 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to OECD 406 and under GLP
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1989
Report Date:
1989

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
other grading scale
Principles of method if other than guideline:
In stead of the Magnusson/Kligman grading scale a (other) numerical grading system was used:
- Erythema and eschar formation
0 - No erythema
1 - Slight erythema (barely perceptible)
2 - Well-defined erythema
3 - Moderate erythema
4 - Severe erythema (beet redness) to slight eschar formation (injuries in depth)
- Edema formation
0 - No edema
1 - Slight edema (barely perceptible)
2 - Well-defined edema (edges of area well-defined by definite raising)
3 - Moderate edema (raised approximately 1 millimeter)
4 - Severe edema (raised more than 1 millimeter and extending beyond the area of exposure)
GLP compliance:
yes
Remarks:
with statement of compliance, but without certificate
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Physical state: Liquid
- Stability under test conditions: 2 hours

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Himalayan White spotted (outbred)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Institute of Biomedical Research, CH 4414 Füllinsdorf, Switzerland.
- Age at study initiation: 8 weeks.
- Weight at study initiation: 336 - 378 g.
- Housing: Individually in Makrolon type-3 cages with standard softwood bedding.
- Diet (e.g. ad libitum): Ad libitum, pelleted standard guinea pig breeding/maintenance diet.
- Water (e.g. ad libitum): Ad libitum, community tap water.
- Acclimation period: 1 week under test conditions.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
olive oil
Concentration / amount:
Induction:
intradermal injection - 5%
epidermal application - 100%

Challenge and re-challenge: 30%
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
olive oil
Concentration / amount:
Induction:
intradermal injection - 5%
epidermal application - 100%

Challenge and re-challenge: 30%
No. of animals per dose:
10 control, 20 test substance.
Details on study design:
RANGE FINDING TESTS:

PRELIMINARY STUDY
Identification of irritant test article concentrations suitable for the induction phase of the main study.
INTRADERMAL injection:
6 concentrations (0.1, 0.3, 0.5, 1, 3 and 5%) of test article in olive oil injected into the flanks of 2 guinea-pigs. Dermal reactions assessed at 24 hours after injection.
EPIDERMAL application:
(Occlusive) Patches of filter paper (2x2 cm) saturated with 4 concentrations (3, 10, 30 and 100%) of test article in olive oil were applied to the flanks of each of four guinea-pigs. Period of exposure: 24 hours. Dermal reactions were assessed at 0, 24 and 48 hours after removal of patches.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epidermal).
- Exposure period: instant (intradermal) and 48 hours (epidermal).
- Test groups: 1
- Control group: 1
- Site: dorsal skin from the scapular region.
- Frequency of applications: epidermal application one week after intradermal injection.
- Duration: 48 hr (epidermal application)
- Concentrations:
INTRADERMAL (three pairs of injections)
1) Freunds' complete adjuvant 50:50 with distilled water for injection.
2) The test article, dilted to 5% with olive oil.
3) The test article, ate the concentration used in (2), emulsified in a 50:50 mixture of Freunds' complete adjuvant, and the vehicle used in (2).
EPIDERMAL
2x4 cm patch of filter paper saturated with the undiluted free test article (100%).
Reaction sites were assessed for erythema and edema at 0, 24 and 48 hours after removal of patches.

Remark: The control group was treated accordingly with the omission of the test article.
B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: two weeks after epidermal induction application.
- Exposure period: approx. 24 hours
- Test groups: 1
- Control group: 1
- Site: left and right flank (5x5 cm area)
- Concentrations:
Treatment and control group (patches, 2x2 cm):
a) non-irritant concentration of test article (30% in olive oil) on left flank.
b) vehicle alone (olive oil) on right flank.
- Evaluation (hr after challenge):
Assessment of sites for erythema and edema at 0, 24 and 48 hours after removal of patch.

OTHER:
C. RE-CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: two weeks after first challenge.
- Exposure period: approx. 24 hours
- Test groups: 1
- Control group: 1
- Site: left and right flank (5x5 cm area)
- Concentrations:
Treatment group (patches, 2x2 cm):
a) vehicle alone (olive oil) on left flank.
b) non-irritant concentration of test article (30% in olive oil) on right flank.
Control group (patch, 2x2 cm):
a) vehicle alone (olive oil) on left flank.
b) -
- Evaluation (hr after challenge):
Assessment of sites for erythema and edema at 0, 24 and 48 hours after removal of patch.
Challenge controls:
The control guinea-pigs were challenged in the same way as the substance treated animals. At re-challenge control animals were treated with the vehicle alone.
Positive control substance(s):
yes
Remarks:
dinitro-chloro-benzene, tested twice a year on a control group as a sensitivity check of the guinea-pig strain (most recent test: September 1988)

Study design: in vivo (LLNA)

Vehicle:
other: Not relevant
Concentration:
Not relevant
No. of animals per dose:
Not relevant
Details on study design:
Not relevant
Statistics:
Not relevant

Results and discussion

Positive control results:
Clear positive testing results were observed in the DNCB treated animals after the epidermal challenge application (September 1988).
10 Dunkin-Hartley albino guinea-pigs (from DUHA Kleintierfarm Madoerin, Switzerland) were used, of which 6 reacted positive and 3 negative (1 diedspontaneously on day 11 of test). This results in a 67% positive reaction.
Induction by 0.5% DNCB in ethanol, challenge by 0.3% DNCB. Substance: DNCB (1-chloro-2,4-dinitro-benzol) from Fluka (Switzerland) frabricated under No. 24440 with a purity of 98%.
Substance possesses a strong skin sensitizing potential according to the test.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
2nd reading
Hours after challenge:
24
Group:
negative control
Dose level:
Vehicle (olive oil)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Vehicle (olive oil). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Vehicle (olive oil)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Vehicle (olive oil). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.
Reading:
2nd reading
Hours after challenge:
24
Group:
negative control
Dose level:
30% Vitamin-A-Palmitate
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 30% Vitamin-A-Palmitate. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
30% Vitamin-A-Palmitate
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 30% Vitamin-A-Palmitate. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.
Reading:
2nd reading
Hours after challenge:
24
Group:
test group
Dose level:
Vehicle (olive oil)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: test group. Dose level: Vehicle (olive oil). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: None.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Vehicle (olive oil)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Vehicle (olive oil). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: None.
Reading:
2nd reading
Hours after challenge:
24
Group:
test group
Dose level:
30% Vitamin-A-Palmitate
No. with + reactions:
4
Total no. in group:
20
Clinical observations:
None
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 24.0. Group: test group. Dose level: 30% Vitamin-A-Palmitate. No with. + reactions: 4.0. Total no. in groups: 20.0. Clinical observations: None.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
30% Vitamin-A-Palmitate
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
None
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 30% Vitamin-A-Palmitate. No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: None.
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
Vehicle (olive oil)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: negative control. Dose level: Vehicle (olive oil). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
Vehicle (olive oil)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: Vehicle (olive oil). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None.
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
Vehicle (olive oil)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: Vehicle (olive oil). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: None.
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
Vehicle (olive oil)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: Vehicle (olive oil). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: None.
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
30% Vitamin-A-Palmitate
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 30% Vitamin-A-Palmitate. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: None.
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
30% Vitamin-A-Palmitate
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
None
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 30% Vitamin-A-Palmitate. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: None.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Not relevant
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Not relevant

Any other information on results incl. tables

Overview of important test results:

Reading Hours after challenge Group Dose level No. with + reactions Total No. in group Positive reactions (%) Clinical observations
        Erythema (E) Edema (O)    
2nd reading 24 Negative control Vehicle (olive oil) 0 0 10 0 None
2nd reading 48 Negative control Vehicle (olive oil) 0 0 10 0 None
2nd reading 24 Negative control 30% Vitamin-A-Palmitate 0 0 10 0 None
2nd reading 48 Negative control 30% Vitamin-A-Palmitate 0 0 10 0 None
2nd reading 24 Test group Vehicle (olive oil) 0 0 20 0 None
2nd reading 48 Test group Vehicle (olive oil) 0 0 20 0 None
2nd reading 24 Test group 30% Vitamin-A-Palmitate 4 0 20 20 None
2nd reading 48 Test group 30% Vitamin-A-Palmitate 2 0 20 10 None
Re-challenge 24 Negative control Vehicle (olive oil) 0 0 10 0 None
Re-challenge 48 Negative control Vehicle (olive oil) 0 0 10 0 None
Re-challenge 24 Test group Vehicle (olive oil) 0 0 20 0 None
Re-challenge 48 Test group Vehicle (olive oil) 0 0 20 0 None
Re-challenge 24 Test group 30% Vitamin-A-Palmitate 0 0 20 0 None
Re-challenge 48 Test group 30% Vitamin-A-Palmitate 0 0 20 0 None

In test group after first challenge: 20% (after 24 hours) and 10% (after 48 hours) positively reacting guinea-pigs.

On the rating of allergenicity (see below, Magnusson B. and Kligman A.M. 1969) this means that Vitamin-A-Palmitate 1.7 mIU/g is considered to be of a mild grade in this test.

Sensitization rate (%) Grade Classification
0-8 1 Weak
9-28 2 Mild
29-64 3 Moderate
65-80 4 Strong
81-100 5 Extreme

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
According to the results described above the allergenic potency of the test article Vitamin-A-Palmitate is considered to be of a mild grade in this test when followed the rating of allergenicity described by Magnusson B. and Kligman A.M. (1969). However, according to the criteria for the classification of a substance as sensitiser as set in Annex VI of EU Directive 67/548/EEC, this test article is not a sensitiser.
Executive summary:

The purpose of this skin sensitization study was to assess the allergenic potential of Vitamin-A-Palmitate when administered to the skin of male and female albino guinea pigs.

For this purpose the Maximization-Test of B. Magnusson and A.M. Kligman (1969) was used. Ten animals (10 females) were treated with the vehicle alone (i.e. olive oil) and 20 animals (20 females) were treated with the test article.

The study was conducted between October 17th and December 1st, 1988 at the RCC laboratories in 4452 Itingen/Switzerland.

If necessary prior to the first reading of the reactions, the skin was flushed with the vehicle or other solvents to clean the application site from staining produced by the test article, so that the reactions (erythema) were clearly visible at that time.

Results show that in 4 of 20 (20%) Vitamin-A-Palmitate treated guinea-pigs the test was positive 24 hours after first challenge, and 2 of 20 (10%) animals after 48 hours. No positive reactions were observed after second challenge (24 and 48 hours) and in the control group (n=10) after both challenges. No toxic symptoms were evident in the guinea pigs of either the control nor test group. No death occured.

According to the rating of allergenicity described by Magnusson B. and Kligman A.M. (1969), the test article is considered to be of a mild grade. According to the criteria in Annex VI of the EU directive for classification (67/548/EEC), the test article is considered not to be a sensitizer (<30% positive reactions).