9β,11β-epoxy-17,21-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Between 11 October 2011 and 01 November 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Done by OECD and GLP standards

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Principles of method if other than guideline:

The methodology for the LLNA is detailed in the OECD Guideline for the Testing of Chemicals, No. 429, and Method B.42 of Commission Regulation (EC) No. 440/2008. The study described in this document is based on these test methods but has been refined in order to reduce the number of animals required. The reduced LLNA (rLLNA) has been endorsed by the non-Commission members of the European Centre for the Validation of Alternative Methods (ECVAM) Scientific Advisory Committee (ESAC) at its 26th meeting held on 26 – 27 April 2007 at ECVAM, Ispra, Italy.

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

9β,11β-epoxy-17,21-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione

EC Number:

246-529-2

EC Name:

9β,11β-epoxy-17,21-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione

Cas Number:

24916-90-3

Molecular formula:

C22H28O5

IUPAC Name:

9β,11β-epoxy-17,21-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione

Test material form:

solid: crystalline

Details on test material:

Sponsor's identification : GR46708X
Description : pale yellow crystalline solid
Purity : >98%
Date received : 04 July 2011
Expiry date : 30 June 2012
Storage conditions : room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

Supplied by Harlan Laboratories UK Ltd., Oxon, UK.
Acclimatization period of at least five days
Weight range of 15 to 23 g, and were eight to twelve weeks old
Free access to mains tap water and food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 19 to 25°C and 30 to 70%, respectively
Air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours continuous light (06.00 to 18.00) and twelve hours darkness.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

other: topical application

Vehicle:

other: dimethyl formamide

Induction 2

Concentration / amount:

The mice were treated by daily application of 25 µl of the test item

Challenge

Challenge 1

Route:

other: injected via the tail vein

Vehicle:

other: dimethyl formamide

Challenge 2

Concentration / amount:

The mice were treated by daily application of 25 µl of the test item

No. of animals per dose:

5

Details on study design:

Clinical Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.


Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Challenge controls:

A further group of five mice received the vehicle alone in the same manner

Positive control substance(s):

not specified

Study design: in vivo (LLNA)

Vehicle:

other: phosphate buffered saline

Concentration:

3HTdR: 80 µCi/ml, specific activity 2.0 Ci/mmol, ARC UK Ltd) giving a total of 20 µCi to each mouse

No. of animals per dose:

All test and contol mice

Details on study design:

The mice were treated by daily application of 25 μl of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3) by using a micropipette whose tip is used to spread the formulation over the dorsal surface of each ear. On Day 6 all mice were injected with 250 ul of a phophate buffered saline solution containing 3H-methyl thymidine (3HTdR ) to the tail vein giving a total of 20 uCi to each mouse. All animals were observed twice daily on Day 1, 2 and 3 and once daily on days 4 ,5, and 6. Any signs of toxicity or ill health were noted. Body weights of each mouse were recorded before dosing and before termination. Five hours after administration of 3HTdR all mice were killed by carbon dioxide asphyxiation and their auricular lymph nodes were excised and a 1 ml of phosphate buffered saline was added to each set of lymph nodes. Preparation of a single cell suspension was completed for the lymph nodes cells for each individual animal following standard procedures. Determination of the 3HTdR incorporation was completed by measuring radioactive disintegration for each animal's lymph node cells by using a beta-scintillation counter.

Statistics:

Analysis of variance (ANOVA) was carried out on the data followed by Barlett's test for analysis of homogenicity of variance. Comparisons were made beween the control and treatment groups using Dunnett's test.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Concentration (10% w/w) in dimethyl formamide: SI = 0.89

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Concentration (10% w/w) in dimethyl formamide: mean dpm/animal 1843.90 (±941.38)

Any other information on results incl. tables

Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item was considered to be a non-sensitiser under the conditions of the test.


The test item did not meet the criteria for classification as a sensitiser according to EU labelling regulations Commission Directive 2001/59/EC. No symbol and risk phrase are required