Androstane-3,17-diol, 2-(4-morpholinyl)-16-(1-pyrrolidinyl)-, 17-acetate, (2.beta.,3.alpha.,5.alpha.,16.beta.,17.beta.)-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Deviations from the minimum level of daily mean relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations. The study integrity was not adversely affected by the deviation.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Deviations from the minimum level of daily mean relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations. The study integrity was not adversely affected by the deviation.

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

yes

Remarks:

Deviations from the minimum level of daily mean relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations. The study integrity was not adversely affected by the deviation.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Androstan-3,17-diol,2-(4-morpholinyl)-16-(1-pyrrolidinyl)-, 17-acetate (2α,3α,5α,16α,17β)-

EC Number:

601-596-0

Cas Number:

119302-24-8

Molecular formula:

C29H48N2O4

IUPAC Name:

Androstan-3,17-diol,2-(4-morpholinyl)-16-(1-pyrrolidinyl)-, 17-acetate (2α,3α,5α,16α,17β)-

Test material form:

solid: flakes

Details on test material:

- Name of test material (as cited in study report): Pymorolac-SZ
- Molecular formula (if other than submission substance): C29H48N2O4
- Molecular weight (if other than submission substance): 488.7
- Physical state: White flakes (determined at WIL Research Europe B.V.)
- Analytical purity: 85-95
- Lot/batch No.: DGN410K1A
- Expiration date of the lot/batch: 01 April 2014 (Retest date) (taken from label)
- Stability under test conditions: At room temperature protected from light
- Storage condition of test material: Stable

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J, inbred, SPF-Quality

Sex:

female

Details on test animals and environmental conditions:

Species Mouse: CBA/J strain, inbred, SPF-Quality. (Recognized by the international guidelines as the recommended test system (e.g. OECD, EC, EPA)).
Source: Janvier, Le Genest-Saint-Isle, France
Number of animals 20 females (nulliparous and non-pregnant), five females per group.
Age and body weight Young adult animals (approx. 9 weeks old) were selected.
Body weight variation was within +/- 20% of the sex mean.

Conditions
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.

Accommodation
Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) and shelters (disposable paper corner home,
MCORN 404, Datesand Ltd, USA) were supplied as cage-enrichment. The acclimatization period was at least 5 days before the start of treatment under laboratory conditions. On Day 6, the animals were group housed in Makrolon MII type cages with a sheet of paper instead of sawdust and cage
enrichment.

Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).

Water
Free access to tap water.

Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

10, 25, and 50% w/w

No. of animals per dose:

5

Details on study design:

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance concentration, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.

The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test substance.

Excision of the nodes - Day 6
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany) containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US).

After approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands). The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS.

Tissue processing for radioactivity - Day 6
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and stored in the refrigerator until the next day.

Radioactivity measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail (PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

Observations
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.
Necropsy: No necropsy was performed according to protocol.

Statistics:

Interpretation
DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group.
If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.
The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labelling and
packaging of substances and mixtures, including all amendments.
Consideration was given to the EC3 value (the estimated test substance concentration that will give a SI =3)

Results and discussion

In vivo (LLNA)

Results

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 9719, 10365 and 2727 DPM respectively. The mean DPM/animal value for the vehicle control group was 490 DPM.

Any other information on results incl. tables

Pre-screen test:

No irritation and no signs of systemic toxicity were observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values.

White test substance remnants were present on the dorsal surface of the ears of both animals at 25 and 50% during the observation period, which did not hamper scoring of the skin reactions. Based on these results, the highest test substance concentration selected for the main study was a 50% concentration.

Systemic toxicity

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Macroscopy of the auricular lymph nodes and surrounding area

All auricular lymph nodes of the animals at 10, 25 and 50% were considered larger in size as compared to the control treated animals. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The SI values calculated for the substance concentrations 10, 25 and 50% were 19.9, 21.2 and 5.6 respectively.

These results show that the test substance elicits an SI ≥ 3. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between 0 and 10%.

The response of the 50% group did not follow the expected dose-response relationship. The response might be less due to differences in viscosity of the test formulation.

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.

Based on these results:
- according to the recommendations made in the test guidelines, PYMOROLAC-SZ would be regarded as skin sensitizer.

- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011), PYMOROLAC-SZ should be classified as skin sensitizer (Category 1).

- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, PYMOROLAC-SZ should be classified as skin sensitizer (Category 1 and labeled as H317: May cause an allergic skin reaction).
A category 1A is suggested since the EC3 is considered to be <2% based on the high SI value at 10% test substance concentration.