(S)-5-fluoro-3-methylisobenzofuran-1(3H)-one

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

22 March 2016 to 26 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 B: Methods for the Determination of Toxicity and other Health Effects; B42: "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

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

(3S)-5-fluoro-3-methyl-1,3-dihydro-2-benzofuran-1-one

EC Number:

943-180-4

Cas Number:

1803573-19-4

Molecular formula:

C9H7FO2

IUPAC Name:

(3S)-5-fluoro-3-methyl-1,3-dihydro-2-benzofuran-1-one

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Specific details on test material used for the study:

Purity 100% (HPLC %area)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

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.

6.4. Weight of Evidence Analysis
In the interest of animal welfare and to minimize any testing likely to produce severe responses in animals, a weight of evidence analysis was performed prior to the start of this study. All available information was evaluated (e.g. existing human and animal data, literature, item data supplied by the Sponsor, analysis of structure activity relationships (SAR), physicochemical properties and reactivity (pH, buffering capacity)). It was concluded by the Study Director that no severe effects were to be expected.

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Remarks:

From Merck, Darmstadt, Germany

Concentration:

25% and 50%

No. of animals per dose:

5 females/group

Details on study design:

6.5. Pre-screen Test
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2 (see section 7.8) and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can technically be applied.

Two test item concentrations were tested; a 25% and 50% concentration. The highestconcentration was the maximum concentration as required in the test guidelines.

The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 11 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.

Animals were sacrificed after the final observation.

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 (Propylene glycol)
2 06 - 10 10
3 11 - 15 25
4 16 - 20 50
* five females per group

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.

Positive control substance(s):

other: vehicle administered instead of test item

Statistics:

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 (PerkinElme 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).

6.8. Interpretation
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, the test item may be regarded as a skin sensitizer.

Results and discussion

Positive control results:

The mean SI value for al the animals administered the control was 0.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 1.4

Variability:

± 0.4

Test group / Remarks:

10 %

Results 2

Key result

Parameter:

SI

Value:

ca. 1.8

Variability:

± 0.4

Test group / Remarks:

25 %

Results 3

Key result

Parameter:

SI

Value:

ca. 0.9

Variability:

± 0.2

Test group / Remarks:

50 %

Cellular proliferation data / Observations:

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.

6.8. Interpretation
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, the test item may be regarded as a skin sensitizer.

Classification of results
UN-GHS 2015; EC-CLP 2008 EC Hazard statement
SI < 3 No sensitizer
SI > 3 Cat 1 Skin sensitizer
EC3 value £ 2%: sub-category 1A H317: May cause an allergic skin reaction
EC3 value > 2%: sub-category 1B

Consideration was given to the EC3 value (the estimated test item concentration that will give
a SI =3) (Ref. 1).

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 when tested up to 50%, PF-06811569 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-06811569 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).