9beta,11beta-Epoxy-6alpha-fluoro-16alpha-methyl-21-valeryloxy-1,4-pregnadiene-3,20-dione

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

17 July 1992

Deviations:

no

GLP compliance:

yes

Remarks:

- but a QA check was not performed

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

the guinea pig maximisation test was an adequate in vivo skin sensitisation test at the time of performance in 1996

Test material

Test material information

Constituent 1

Reference substance name:

9beta, 11beta-Epoxy-6alpha-fluoro-16alpha-methyl-21-valeryloxy-1,4-pregnadiene-3,20-dione

Cas Number:

65535-29-7

Molecular formula:

C27 H35 F O5

IUPAC Name:

9beta, 11beta-Epoxy-6alpha-fluoro-16alpha-methyl-21-valeryloxy-1,4-pregnadiene-3,20-dione

Test material form:

solid

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Pirbright White

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: CharIes River Kislegg
- Weight at study initiation: local tolerance test: male: 285-388 g; female: 280-378 g; maximization test: male: 402-517 g; female: 367-512 g
- Housing: 1 or 2 in conventional Makrolon, type IV cages with embedding
- Diet (e.g. ad libitum): pell. Altromin® MS, apple, hay ad libitum
- Water (e.g. ad libitum): demineralized, acidified tap water, pH 2-3, ad libitum
- Acclimation period: ≥ 19 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-23
- Humidity (%): 50-60
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

paraffin oil

Concentration / amount:

1.25 %

Day(s)/duration:

day 1

Induction 2

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

25 % and 12.5%

Day(s)/duration:

day 9

Challenge

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Concentration / amount:

25 %

Day(s)/duration:

day 23

No. of animals per dose:

control group: 10 (5/sex)
test substance group: 10 (5/sex)

Details on study design:

RANGE FINDING TESTS: A local tolerance study (pretest) was performed to ensure an intracutaneously tolerable concentration without any necrosis for the induction procedure (reddenings and swellings were accepted). The concentration used in the present test for intracutaneous application (1.25%) was the highest concentration that could be applied, as 5% [recommended as maximum concentration when tolerated] and 2.5% could not be formulated in a form that allowed intracutaneous application. The reactions were recorded 24 hours after application. Furthermore, an epicutaneously tolerable concentration for epidermal application in the induction procedure and for epicutaneous challenge was determined. Filter papers with 0.2 mL of the test formulations (25% and 12.5%) were epidermally applied under occlusive conditions for 24 hours. 25% as high concentration is the recommended maximum concentration for induction procedures, provided that the treatment produces no excessive irritation. As a concentration is recommended which is tolerated without any local reactions for the challenge, a second concentration of 12.5% was tested. The reactions were recorded 24 hours after removal of the filter papers.

MAIN STUDY
A. INDUCTION EXPOSURE
Intradermal (day 0)
- No. of exposures: 3 pairs of intradermal injections 0.1 mL
Test group
a) 0.1 mL FCA + vehicle (1 + 1)
b) 0.1 mL 1.25% (w/v) test item
c) 0.1 mL 2.5% (w/v) test item + FCA (1 + 1) ≙ 1.25% (w/v) of test item
Control group
a) 0.1 mL FCA + vehicle (1 + 1 )
b) 0.1 mL vehicle
c) 0.1 mL FCA + vehicle (1 + 1)

Epidermal application
The same area of skin (4 x 6 cm) in which intradermal injections were performed was reshaved on day 8 of the test and was epidermally treated with approximately 1 mL sodium lauryl sulphate (test item) [10% (w/v)] in liquid paraffin to cause superficial irritation to enhance the sensitivity for the subsequent second induction. 24 hours later, on day 9, 0.2 mL of a 25% (w/v) suspension of the test item in liquid paraffin was spread over a 2 x 4 cm filter paper (Whatmann, no. 3MM) for use in the test group, whereas for the control group 0.2 mL of liquid paraffin only was used. This filter paper was subsequently applied to the above-mentioned area pretreated with sodium lauryl sulphate. The patch was bandaged occlusively for 48 hours.

- Test groups: treatment and vehicle control group
- Site: In the neck region (right to left side of the spine) of each animal an overall area of 4 x 6 cm.

B. CHALLENGE EXPOSURE
On day 23 (two weeks after the epidermal administration) a skin area of 5 x 5 cm was shorn on the right flank of each animal in both groups. Approximately 0.1 mL of the test item [25% (w/v)] in liquid paraffin, which previously proved to be locally tolerated without reactions, was spread over a 2 x 2 cm piece of filter paper. This patch was placed in the middle of the shorn area of skin and bandaged occlusively for 24 hours. Reactions were recorded 24 and 48 hours after removal of the bandage.

Positive control substance(s):

yes

Remarks:

The reference substance mercaptobenzothiazol was tested in Aug./Sep. 96 in the testing facility.

Results and discussion

Positive control results:

The sensitivity of the used method was proven by performance of a maximization test with mercaptobenzothiazol, a positive reference substance recommended in OECD guideline 406. This reference substance (mercaptobenzothiazol) was tested in Aug./Sep. 96 at the test facility. In the test all animals reacted positively to the challenge with mercaptobenzothiazol.

In vivo (non-LLNA)

Results

Results 1

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

positive control

Remarks on result:

positive indication of skin sensitisation

Remarks:

The sensitivity of the used method was proven by performance of a maximization test with mercaptobenzothiazol, a positive reference substance recommended in OECD guideline 406. This reference substance (mercaptobenzothiazol) was tested in Aug./Sep. 96 at the test facility. In the test all animals reacted positively to the challenge with mercaptobenzothiazol. The number of animals used was not reported.

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

positive control

Remarks on result:

not measured/tested

Remarks:

The number of animals used for the positive control was not reported.

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

challenge 25 %

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

slight reddening: 5/10, moderate reddening: 2/10, slight swelling: 3/10

Remarks on result:

positive indication of skin sensitisation

Results 4

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

challenge 25 %

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Results 5

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

challenge 25 %

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

slight reddening: 5/10, moderate reddening: 2/10, slight swelling: 4/10

Remarks on result:

positive indication of skin sensitisation

Results 6

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

challenge 25 %

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

In a preceding local tolerance test, the concentration of 1.25 % (w/v) of the test item was found to be the highest intradermally applicable concentration and to be tolerated without necrosis. The concentration of 25 % (w/v) of the test item was tolerated epidermally without any local findings. Therefore, these concentrations were chosen to be used in the maximization test.

 

In the test group a positive reaction in the form of slight to moderate reddening and/or slight swelling was observed in 7 of the 10 animals 24 to 48 hours after the end of challenge with 25 % (w/v) of the test item, whereas no animal in the control group showed any reactions.

Applicant's summary and conclusion

Conclusions:

In a guinea pig maximization test (GPMT) according to OECD TG 406 a contact-sensitizing potential of the test substance could be observed based on the results after intradermal induction with 1.25 % of the test item and epicutaneous challenge with 25 % of the test item. As 70 % of the animals reacted positively, thus, the test item should be classified as a moderate skin sensitizer.

Executive summary:

In a dermal sensitization study according to OECD TG 406 (17 July 1992) with Epoxide-Valerate 1.25%(w/v), young adult female/male guinea pigs (Dunkin Harley Pirbright White) (5/sex/dose) were tested in the GPMT (OECD TG 406). For induction the animals were treated either intracutaneous with 0.1 mL of diluted (1 + 1 with vehicle) Freund´s complete adjuvant (FCA), test item [1.25% (w/v)] or test item [2.5% (w/v)] diluted 1 + 1 with FCA into the right and left dorsal neck region on study day 1. In the same way, the control group (5 male, 5 female) received 0.1 mL of the vehicle [paraffin oil] with and without FCA. On day 9, the same area of skin was covered with a filter paper impregnated with the test item [25% (w/v) in liquid paraffin] for the test group, or impregnated with liquid paraffin for the controls. On day 23, as a challenge, the same procedure was followed as on day 9, but the controls also received the filter paper impregnated with the test item [25% (w/v)] in liquid paraffin. The application was made in the more sensitive flank region.

In the test group a positive reaction in the form of slight to moderate reddening and/or slight swelling was observed in 7 of the 10 animals 24 to 48 hours after the end of challenge with 25 % (w/v) of the test item, whereas no animal in the control group showed any reactions.

Due to the moderate incidence of findings (70% of the animals reacted), this finding is regarded as a moderate contact-sensitizing potential of the test item.