Retinyl acetate

Skin sensitisation

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-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

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

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

Test material information

Constituent 1

Reference substance name:

Retinyl acetate

EC Number:

204-844-2

EC Name:

Retinyl acetate

Cas Number:

127-47-9

Molecular formula:

C22H32O2

IUPAC Name:

(2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraen-1-yl acetate

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)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

olive oil

Induction 2

Concentration / amount:

Induction:
- Intradermal: 5%
- Epicutaneous: 30%
Challenge: 10%

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

olive oil

Challenge 2

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)

Results

Results 1

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.

Results 2

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.

Results 3

Reading:

2nd reading

Hours after challenge:

24

Group:

test chemical

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.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

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.

Results 5

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.

Results 6

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.

Results 7

Reading:

rechallenge

Hours after challenge:

24

Group:

test chemical

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.

Results 8

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

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.