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Toxicological information

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
Buehler test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Retinol 50 C and Retinol 10 CM, two commercial samples of retinol (CAS No. 68-26-8)
- Retinol 50 C (retinol); purity 47.1% (UV), in Polysorbate 20, designated test substance (A)
- Retinol 10 CM (retinol); purity ca. 10%, in Miglyol 812 N, designated test substance (B)
At induction, first challenge, and second challenge, test substance (A) was applied. At third challenge, test substance (B) or Polysorbate 20 Lutrol E400 was applied.

Physical state / appearance : At room temperature solid, yellow; after heating at about 50 degrees centigrade liquid, yellow
Storage conditions : Refrigerator, exclusion of light and oxygen (under nitrogen)

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: Harlan
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen (Germany) Hsd Poc: DH guinea pigs.
- Age at study initiation: young adults
- Weight at study initiation: 328 - 388 g (range)
- Acclimation period: 8 days before the beginning of the study
- Housing: Stainless steel wire mesh cages with plastic -coated grating , floor area 3000 square-cm, 5 animals per cage.
- Diet: Kliba Labordiät (Kaninchen-Meerschweinchen-Haltungsdiät) ad libitum
- Water: Tap water ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-25
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12


Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: Lutrol E 400, Miglyol 812 N
Concentration / amount:
Induction: 25 % test substance occlusive epicutaneous
Challenge:
1st application: 10 % test substance occlusive epicutaneous
2nd application: 10%, 5 % test substance occlusive epicutaneous
3rd application: 2.5 % test substance occlusive epicutaneous
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: Lutrol E 400, Miglyol 812 N
Concentration / amount:
Induction: 25 % test substance occlusive epicutaneous
Challenge:
1st application: 10 % test substance occlusive epicutaneous
2nd application: 10%, 5 % test substance occlusive epicutaneous
3rd application: 2.5 % test substance occlusive epicutaneous
No. of animals per dose:
20
Details on study design:
I) Pretest:
The pretest was carried out to determine the test concentrations for the  main test. The pretest was divided into 3 subtests. The respective test  article was applied to the left and right flank for 6 hours under  occlusive conditions. The test material was removed with Lutrol at the  end of the application period. Application sites were read at 6 and 30  hours after beginning of application.
1st pretest (8 females): Eight females were assigned to 2 groups of four. The test substance was  applied undiluted or at concentrations of 75%, 50% , and 25%, 0.5 ml per  animal and dose each. The first group was applied the undiluted test  substance to the right flank and the 75% dilution to the left flank, The  second group was applied the 50% and 25% dilution to the right and left  flank, respectively. All animals received two applications within 5 days.

2nd pretest (4 females): The animals used for the high concentrations (100% and 75%) in the 1st  pretest were used. A 10% and 5% dilution of the test substance was  applied to the right and left flank, respectively. All animals received  two applications within 5 days.

3rd pretest (5 females): The animals of this test had not been used in pretests 1 or 2. Three  animals were applied the test substance at 5% and 1% to the right flank  and at 1% and 0.5% to the left flank. Two animals were applied Polysorbat  at 5% and 2.5% to the right and left flank, respectively.

II) Main test:
Fifty animals were randomly assigned to a test group of 20 animals and to  three control groups of 10 animals each (designated controls groups 1, 2,  and 3). The test substance/vehicle (0.5 ml) was applied for 6-hour  intervals under occlusive conditions. The application sites were scored  at 24 and 48 hours after removal of the test patches. Each animal  receiving the test substance was also applied the corresponding vehicle.  Two solutions of retinol were used (see freetext "Test substance").

1) Induction: For induction, the animals of the test group received 3 weekly  applications of the test substance (A) (0.5 ml of a 25% preparation in  Lutrol E 400). The test substance was  applied to the left flank on study days 0, 7, 14. Control animals were  applied the vehicles only.

2) Challenge: Three challenges were carried out at 14, 21, and 56 days after the last  induction. The test substance was applied to the right, previously  untreated flank.
1st challenge: At 14 days after the last induction, animals of the test group and of  control group 1 were applied 10% of test substance (A) in Lutrol E 400 . Animals of control groups 2 and 3 were applied  the vehicles only.
2nd challenge: Due to the skin reaction observed in control group 1, a second challenge  was performed. At 21 days after the last induction (7 days after 1st  challenge), animals of the test group and of control groups 1 and 2 were  applied 5% and 10% of test substance (A) in Lutrol E 400. Animals of control group 3 were applied the vehicles  only.
3rd challenge: At 56 days after the last induction (42 days after 1st challenge and 35 days after the 2nd challenge), animals of the test group and of all  control groups were applied a 2.5% preparation of the test substance (B)  in Miglyol 812 N or 2.5% Polysorbate 20 in Lutrol E400.
Positive control: A positive control group was not included in this study. According to the  authors, a separate positive control study using technical-grade  alpha-hexylcinnamaldehyde (purity 85%) was conducted routinely twice a  year, proving that the guinea pig strain was able to detect sensitizing compounds under the laboratory conditions chosen.
Challenge controls:
see above
Positive control substance(s):
no
Remarks:
A positive control is not included in this study. In a seperate study a positive control with Alpha-Hexylcinnamaldehyde techn . 85% showed that the chosen guinea pig strain was able to detect sensitizing compounds under the laboratory conditions chosen .

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
other: 1st challenge
Hours after challenge:
24
Group:
other: challenge control group 1
Dose level:
10% TS in Lutrol E400
No. with + reactions:
3
Total no. in group:
10
Remarks on result:
other: Reading:
Reading:
other: 1st challenge
Hours after challenge:
48
Group:
other: challenge control group 1
Dose level:
10% TS in Lutrol E400
No. with + reactions:
3
Total no. in group:
10
Remarks on result:
other: Reading:
Reading:
other: 1st challenge
Hours after challenge:
24
Group:
test group
Dose level:
10% TS in Lutrol E400
No. with + reactions:
17
Total no. in group:
20
Remarks on result:
other: Reading:
Reading:
other: 1st challenge
Hours after challenge:
48
Group:
test group
Dose level:
10% TS in Lutrol E400
No. with + reactions:
17
Total no. in group:
20
Remarks on result:
other: Reading:
Reading:
other: 2nd challenge
Hours after challenge:
24
Group:
other: challenge control group 2
Dose level:
5% in Lutrol E 400
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
other: Reading:
Reading:
other: 2nd challenge
Hours after challenge:
48
Group:
other: challenge control group 2
Dose level:
5% in Lutrol E 400
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading:
Reading:
other: 2nd challenge
Hours after challenge:
24
Group:
test group
Dose level:
5% in Lutrol E 400
No. with + reactions:
13
Total no. in group:
20
Remarks on result:
other: Reading:
Reading:
other: 2nd challenge
Hours after challenge:
48
Group:
test group
Dose level:
5% in Lutrol E 400
No. with + reactions:
15
Total no. in group:
20
Remarks on result:
other: Reading:
Reading:
other: 3rd challenge
Hours after challenge:
24
Group:
other: challenge control group 3
Dose level:
2.5% in Miglyol 812
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading:
Reading:
other: 3rd challenge
Hours after challenge:
48
Group:
other: challenge control group 3
Dose level:
2.5% in Miglyol 812
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading:
Reading:
other: 3rd challenge
Hours after challenge:
24
Group:
test group
Dose level:
2.5% in Miglyol 812
No. with + reactions:
18
Total no. in group:
20
Remarks on result:
other: Reading:
Reading:
other: 3rd challenge
Hours after challenge:
48
Group:
test group
Dose level:
2.5% in Miglyol 812
No. with + reactions:
18
Total no. in group:
20
Remarks on result:
other: Reading:

Any other information on results incl. tables

I) Pretest
----------
A concentration of 5% was found to be the minimal irritating concentration. No irritation was observed at 2.5% or less.

1st test:
All concentrations used in this test resulted in skin irritation reactions. Draize scores were  0-2 after the first application (6- and 30-hour reading).

 After the  second application, skin irritation was more severe: Draize scores 1-3 at  100%, 75%, and 50%, Draize score 1-2 at 25%; edema and 

scabbing was observed at 100%, 75%, and 50%.

2nd test:
Very slight irritaion (Draize score 1) was noted after the first  application of 10% at the 6-hour reading. After the second application,  Draize scores 

were 0-2 at both readings for both concentrations.

3rd test:
No skin response was noted after application of the test substance (0.5-5%). Very  slight irritation (Draize score 1) was observed after application of  

5% Polysorbat 20 (No reactions after application of 2.5% Polysorbat 20).

II) Main test
-------------
 Induction:
No signs of skin irritation were observed after the first induction.  After the 2nd and 3rd induction application, discrete or patchy erythema  to intense 

erythema, swelling and scaling was observed in animals of the  test group.

Tables 1-4: number of animals with skin findings after challenge (CG =  control 

group; TC = test group)

Table 1: 1st challenge (Test substance (A): Retinol/ Polysorbate 20 formulation;

 vehicle: Lutrol E 400)
                10%          *       vehicle
       24h     48 h   total  *  24h    48h    total
------------------------------------------------------
CG 1   3/10    3/10    3/10  *  0/10   0/10   0/10
CG 2      no application     *  0/10   0/10   0/10
TC    17/20   17/20   17/20  *  0/20   0/20   0/20

Table 2: 2nd challenge (Test substance (A): Retinol/ Polysorbate 20 formulation;

 vehicle: Lutrol E 400)
                10%          *          5%           ...
       24h     48 h   total  *  24h    48h    total  ...
---------------------------------------------------- ...
CG 1   9/10    8/10    9/10  *  4/10   6/10   6/10   ...
CG 2   4/10    4/10    5/10  *  2/10   0/10   2/10   ...
TC    19/20   19/20   19/20  * 13/20  15/20  16/20   ...

...             vehicle
...        24h    48h    total
... ----------------------------
... CG 1   0/10   0/10   0/10
... CG 2   0/10   0/10   0/10
... TC     0/20   0/20   0/20

Table 3: 3rd challenge (Test substance (B): Retinol; vehicle: Miglyol 812)
                2.5%         *       vehicle
       24h     48 h   total  *  24h    48h    total
------------------------------------------------------
CG 1   2/10    3/10    3/10  *  0/10   0/10   0/10
CG 2   2/10    1/10    2/10  *  0/10   0/10   0/10
CG 3   0/10    0/10    0/10  *  0/10   0/10   0/10
TC    18/20   18/20   19/20  *  0/20   0/20   0/20

Table 4: 3rd challenge (Polysorbate 20; vehicle: Lutrol E400)
                2.5%         *       vehicle
       24h     48 h   total  *  24h    48h    total
------------------------------------------------------
CG 1   0/10    0/10    0/10  *  0/10   0/10   0/10
CG 2   0/10    0/10    0/10  *  0/10   0/10   0/10
CG 3   0/10    0/10    0/10  *  0/10   0/10   0/10
TC     0/20    0/20    0/20  *  0/20   0/20   0/20

Further observations:
The expected body weight gain was generally observed in the course of the study.


Applicant's summary and conclusion