Imidodicarbonic acid, 2-[5-bromo-3-[[(4-methylphenyl)sulfonyl]oxy]-2-pyridinyl]-, 1,3-bis(1,1-dimethylethyl) ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 March 2016 to 04 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Organization for Economic Co-operation and Development (OECD), OECD Guidelines for Testing of Chemicals, Section 4, Health Effects, No.429, "Skin Sensitization: Local Lymph Node Assay", Paris Cedex, July 2010.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Commission Regulation (EC) No 440/2008 Part В: Methods for the Determination of Toxicity and other Health Effects; В42: "Skin Sensitization: Local Lymph Node Assay". Official Journal of the European Union No. L142, May 2008, including most recent amendments.

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

Environmental Protection Agency (EPA): Health Effects Test Guidelines OPPTS 870.2600. “Skin Sensitization”, March 2003.

Deviations:

no

GLP compliance:

yes

Remarks:

Exception: Trial formulation preparation (for optimal vehicle selection) had a non-OLP status but was carried out in the quality assured environment of WIL Research Europe's OLP test facility.

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

tert-butyl N-{5-bromo-3-[(4-methylbenzenesulfonyl)oxy]pyridin-2-yl}-N-[(tert-butoxy)carbonyl]carbamate

EC Number:

810-844-9

Cas Number:

1803573-21-8

Molecular formula:

C22H27BrN2O7S

IUPAC Name:

tert-butyl N-{5-bromo-3-[(4-methylbenzenesulfonyl)oxy]pyridin-2-yl}-N-[(tert-butoxy)carbonyl]carbamate

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Purity by HPLC, % a/a: 99.7

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:JN

Sex:

female

Details on test animals and environmental conditions:

6.2. Test System
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 (main study only).

Age and body weight
Young adult animals (approx. 10 weeks old) were selected. Body weight variation was within +/- 20% of the sex mean.

Identification
Tail mark with a marker pen.

Health inspection
At least prior to dosing. It was ensured that the animals were healthy and that the ears were intact and free from any
abnormality.

Reliability check
The results of a reliability test with three concentrations of Hexylcinnamaldehyde (CAS No. 101-86-0) in Acetone/Olive oil (4:1 v/v), performed not more than 6 months previously and using the same materials, animal supplier, animal strain and essential procedures are summarized in APPENDIX 2 of this report. For both scientific and animal welfare reasons, no concurrent positive control group was included in the study.
An extensive data base is available with reliability checks performed at half year intervals during at least the past 9 years showing reproducible and consistent positive results.

6.3. Animal Husbandry
Conditions
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 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 (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany). 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 evaluations for contaminants and/or nutrients were 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, 50 % w/w

No. of animals per dose:

five females

Details on study design:

6.6. Main Study
Three groups of five animals were treated with one test item concentration per group. The highest test item concentration was selected from the pre-screen test.
One group of five animals was treated with the vehicle.

6.6.1. Allocation
Group animal numbers induction (test item; % w/w)
1 01 - 05 0 (Methyl ethyl ketone)
2 06 - 10 10
3 11 - 15 25
4 16 - 20 50

6.6.2. Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, 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 item.

6.6.3. 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 five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of 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

6.6.4. Tissue Processing for Radioactivity - Day 6
Following excision of the nodes, 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 then stored in the refrigerator until the next day.

6.6.5. 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. Radioactivity 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).

6.7. 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. In addition, a description of all other (local) effects was recorded.
Grading Irritation Reactions:
Erythema and eschar formation:
No erythema .... 0
Very slight erythema (barely perceptible) 1
Well-defined erythema . 2
Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth) ... 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema . 4

Necropsy No necropsy for gross macroscopic examination was performed according to study plan.

Positive control substance(s):

other: vehicle

Statistics:

DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
If the results indicate a SI ³ 3, the test item may be regarded as a skin sensitizer.

Results and discussion

Positive control results:

SI value of 1.0 +/- 0.2 for 0% concentration of test substance in vehicle.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

ca. 1.4

Variability:

+/- 0.3

Test group / Remarks:

10%

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Results 2

Parameter:

SI

Value:

ca. 1.2

Variability:

+/- 0.3

Test group / Remarks:

25%

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Results 3

Parameter:

SI

Value:

ca. 0.9

Variability:

+/- 0.2

Test group / Remarks:

25%

Remarks on result:

no indication of skin sensitisation based on QSAR/QSPR prediction

Any other information on results incl. tables

8.1. Pre-screen Test

No irritation and no signs of systemic toxicity were observed in any of the pre-screen animals. Scaliness was noted for both animals treated at 25% and one animal treated at 50% on Days 5 and/or 6. White staining of test item remnants on the dorsal surface of the ears of all animals, did not hamper scoring of erythema. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values.

Based on these results, the highest test item concentration selected for the main study was a 50% concentration.

8.2. Main Study

8.2.1. Skin Reactions / Irritation

No irritation was observed in any of the animals. Scaliness was noted for all experimental animals and some control animals. White staining of test item remnants on the dorsal surface of the ears of the animals treated at 25% and 50%, did not hamper scoring for erythema.

8.2.2. 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.

8.2.3. Macroscopic Examination of the Auricular Lymph Nodes and Surrounding Area

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size.

No macroscopic abnormalities of the surrounding area were noted for any of the animals.

8.2.4. Radioactivity Measurements and SI Values

Mean DPM/animal values for the experimental groups treated with test item concentrations 10, 25 and 50% were 1006, 902 and 681 DPM, respectively. The mean DPM/animal value for the vehicle control group was 725 DPM. The SI values calculated for the test item concentrations 10, 25 and 50% were 1.4, 1.2 and 0.9, respectively.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test item elicited a SI ³ 3 when tested up to 50%, PF-06841215 was not considered to be a skin sensitizer.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity (see APPENDIX 2).

Based on these results, PF-06841215 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test item does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).