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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:
24 October 1988 - 2 December 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted according to OECD guideline 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 used
Principles of method if other than guideline:
In stead of the Magnusson/Kligman grading scale an 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
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study was performed before LLNA was formally validated and available as OECD guideline in 2002.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Vitamin-A-Acetate
- Physical state: Liquid
- Composition of test material, percentage of components: 1.5 million IU/g (diluted with arachidis oil)
- Stability under test conditions: At least 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: 328 - 386 g
- Housing: Individually in cages
- 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: One 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

IN-LIFE DATES: From: To: 2 December 1988

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
olive oil
Concentration / amount:
Induction:
- Intradermal: 5%
- Epicutaneous: 30%
Challenge: 10%
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
olive oil
Concentration / amount:
Induction:
- Intradermal: 5%
- Epicutaneous: 30%
Challenge: 10%
No. of animals per dose:
Control group: 10
Test article-treated group: 20
Details on study design:
RANGE FINDING TESTS:
Intradermal: injections made into shaved flank of two guinea-pigs: 0.1, 0.3, 0.5, 1, 3 and 5% test article in olive oil. Dermal reactions assessed 24 hours later.
Epicutaneous: occlusive patches with 3, 10, 30 and 100% test article in olive oil applied (24 hours) to shaved flanks of each of four guinea-pigs. At 0, 24 and 48 hours after treatment the reactions were assessed.

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 (6x8 cm)
- Frequency of applications: epidermal application one week after intradermal injection
- Concentrations:
INTRADERMAL (three pairs of injections)
1) Freunds' complete adjuvant 50:50 with distilled water for injection.
2) The test article, diluted to 5% with olive oil.
3) The test article at 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 30% test article in olive oil.
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 (10% 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 (10% 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 as sensitivity check (most recent test: September 1988)

Results and discussion

Positive control results:
For the induction period a 0.5 % dilution of DNCB in ethanol, and for the challenge procedure a 0.3 % dilution of DNCB was used.
According to the results observed it is considered that DNCB possess a strong skin sensitizing (contact allergenic) potential in the guinea pig strain used (Dunkin-Hartley albino guinea pigs; DUHA KFM. Kleintierfarm Madoerin AG, CH 4414 Fullinsdorf // Switzerland).

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
2nd reading
Hours after challenge:
24
Group:
negative control
Dose level:
10%
No. with + reactions:
5
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: 10%. No with. + reactions: 5.0. Total no. in groups: 10.0. Clinical observations: None.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10%
No. with + reactions:
5
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: 10%. No with. + reactions: 5.0. Total no. in groups: 10.0. Clinical observations: None.
Reading:
2nd reading
Hours after challenge:
24
Group:
test group
Dose level:
10%
No. with + reactions:
3
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: 10%. No with. + reactions: 3.0. Total no. in groups: 20.0. Clinical observations: None.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
10%
No. with + reactions:
4
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: 10%. No with. + reactions: 4.0. Total no. in groups: 20.0. Clinical observations: None.
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle
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. 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
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. 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:
10%
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: 10%. 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:
10%
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: 10%. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: None.

Any other information on results incl. tables

Table 1: VEHICLE CONTROL
SKIN RESPONSE AFTER THE FIRST CHALLENGE PROCEDURE
VEHICLE, OLIVE OIL (RIGHT FLANK)
Animal
Number
Sex Erythema/Edema
immediately
readings after
24 hours
removal of badage
48 hours
    E  O E O E O
431 female 0 0 0 0 0 0
432 female 0 0 0 0 0 0
433 female 0 0 0 0 0 0
434 female 0 0 0 0 0 0
435 female 0 0 0 0 0 0
436 female 0 0 0 0 0 0
437 female 0 0 0 0 0 0
438 female 0 0 0 0 0 0
439 female 0 0 0 0 0 0
440 female 0 0 0 0 0 0
E = Erythema
O = Oedema

Table 2:  VEHICLE CONTROL
SKIN RESPONSE AFTER THE FIRST CHALLENGE PROCEDURE
TEST ARTICLE-TRATED, 10% IN OLIVE OIL (LEFT FLANK)
Animal
Number
Sex Erythema/Edema
immediately
readings after
24 hours
removal of badage
48 hours
    E  O E O E O
431 female 0 0 0 0 0 0
432 female 1 0 1 0 1 0
433 female 1 0 1 0 1 0
434 female 1 0 1 0 1 0
435 female 0 0 0 0 0 0
436 female 0 0 0 0 0 0
437 female 1 0 1 0 1 0
438 female 0 0 0 0 0 0
439 female 0 0 0 0 0 0
440 female 1 0 1 0 1 0

Table 3:  TEST ARTICLE GROUP
SKIN RESPONSE AFTER THE FIRST CHALLENGE PROCEDURE
VEHICLE, OLIVE OIL (RIGHT FLANK)
Animal
Number
Sex Erythema/Edema
immediately
readings after
24 hours
removal of badage
48 hours
    E  O E O E O
441 female 0 0 0 0 0 0
442 female 0 0 0 0 0 0
443 female 0 0 0 0 0 0
444 female 0 0 0 0 0 0
445 female 0 0 0 0 0 0
446 female 0 0 0 0 0 0
447 female 0 0 0 0 0 0
448 female 0 0 0 0 0 0
449 female 0 0 0 0 0 0
450 female 0 0 0 0 0 0
451 female 0 0 0 0 0 0
452 female 0 0 0 0 0 0
453 female 0 0 0 0 0 0
454 female 0 0 0 0 0 0
455 female 0 0 0 0 0 0
456 female 0 0 0 0 0 0
457 female 0 0 0 0 0 0
458 female 0 0 0 0 0 0
459 female 0 0 0 0 0 0
460 female 0 0 0 0 0 0

Table 4:  TEST ARTICLE GROUP
SKIN RESPONSE AFTER THE FIRST CHALLENGE PROCEDURE
TEST ARTICLE-TREATED, 10% IN OLIVE OIL (LEFT FLANK)
Animal
Number
Sex Erythema/Edema
immediately
readings after
24 hours
removal of badage
48 hours
    E  O E O E O
441 female 0 0 0 0 0 0
442 female 0 0 0 0 0 0
443 female 0 0 0 0 0 0
444 female 0 0 0 0 0 0
445 female 0 0 0 0 0 0
446 female 0 0 1 0 1 0
447 female 0 0 0 0 0 0
448 female 0 0 0 0 0 0
449 female 0 0 0 0 0 0
450 female 0 0 0 0 0 0
451 female 1 0 0 0 0 0
452 female 0 0 0 0 0 0
453 female 0 0 0 0 0 0
454 female 0 0 0 0 0 0
455 female 1 0 0 0 0 0
456 female 1 0 1 0 0 0
457 female 0 0 1 0 1 0
458 female 0 0 0 0 1 0
459 female 0 0 0 0 0 0
460 female 0 0 0 0 1 0

Both the negative control and article-treated group respond positive to a challenge with vitamin-A-acetate. This indicates that the substance is rather irritating than sensitizing. At re-challenge, none of the animals reacts positively to the test substance.

 

Considering the article-treated group, 3 and 4 out of 20 animals responded positively at challenge: this is 15-20%. 50% of the controls (5 out of 10) responded positive to the substance.

In accordance with the OECD-guidelines, a rechallenge was performed to clarify the results (as also mentioned in the REACH guidance on Information Requirements). As no skin reactions were noted at the rechallenge, the results of the test can be used for classification.

Table 5:

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-Acetate 1.5 millionIU/g is considered to be of a mild to moderate grade in this test when followed the rating of allergenicity described by Magnusson B. and Kligman A.M. (1969).

According to EEC (European Economic Community) classification criteria described in guidelines 83/467, September 16, 1984 however, this test article is not a sensitiser.
Executive summary:

In this skin sensitization study Vitamin-A-Acetate 1.5 millionIU/g was used at concentrations of 5% for intradermal and 30% for epicutaneous induction. After the first challenge with 10% test article, 20% of experimental and 50% of control animals showed skin responses, while no skin reactions were noted after rechallenge.

According to the data of this study it can be concluded that Vitamin-A-Acetate 1.5 millionIU/g possesses slight allergenic potential for guinea pigs. It can be assumed that occasional or intended and conditioned exposure of the human skin to this test article will, with high probability, be harmless for human health.