2H-1-Benzopyran-2-one, 3,4-dihydro-6-methyl-4-phenyl-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26-Jan-2016 to 11-May-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

6-methyl-4-phenyl-3,4-dihydro-2H-1-benzopyran-2-one

EC Number:

609-846-0

Cas Number:

40546-94-9

Molecular formula:

C16H14O2

IUPAC Name:

6-methyl-4-phenyl-3,4-dihydro-2H-1-benzopyran-2-one

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

Identification: PNU-0181776
Appearance: Off-white powder (determined by WIL Research Europe)
Batch: L22216
Purity/Composition: 98%
Test item storage: At room temperature
Stable under storage conditions until: 04 May 2016 (retest date) (taken from label)

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

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, LeGenest-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. 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.

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:

dimethylformamide

Concentration:

In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 25, 50 or 80% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (N,N-Dimethyl formamide).

No. of animals per dose:

20 females (5f/dose)

Details on study design:

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 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 50% and 80% concentration. The highest concentration 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 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.

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.

Allocation
Group1 animal numbers induction (test item; % w/w)
1 01 - 05 0 (N,N-Dimethyl formamide)
2 06 - 10 25
3 11 - 15 50
4 16 - 20 80

1. five females per group

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 NL/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.

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-methylthymidine(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.

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.

Radioactivity Measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of UltimaGold 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).

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.

Interpretation
DPM values are presented for each animal and for each dose group. A Stimulation Index(SI) is calculated for each group using the individualSI values. The individualSI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. If the results indicate aSI ≥ 3, the test item 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 (2015) (including all amendments) 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 items and mixtures, including all amendments. Consideration was given to the EC3 value (the estimated test item concentration that will give aSI =3)

Statistics:

Radioactivity Measurements - Day 7
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).

Interpretation
In case of borderline results, statistical analysis may be performed to determine the dose response relationship and pair wise comparisons between dose groups versus negative control. The methods used will be specified in the raw data and report. The EC3 value (the estimated item concentration that will give a SI=3) may be determined if possible, based on the dose response relationship or calculated using linear interpolation (reference 1).

Results and discussion

Positive control results:

No positive control group.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 4.8

Test group / Remarks:

25 %

Results 2

Key result

Parameter:

SI

Value:

ca. 2.8

Test group / Remarks:

50 %

Results 3

Key result

Parameter:

SI

Value:

ca. 1.2

Test group / Remarks:

80 %

Cellular proliferation data / Observations:

Pre-screen Test
No irritation and no signs of systemic toxicity were observed in the pre-screen animals, except for the very slight erythema noted for one animal treated at 50% on Day 3. White staining of test item remnants on the dorsal surface of the ears did not hamper scoring for 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 80% concentration.

Main Study
Skin Reactions / Irritation
No irritation was observed in any of the animals except for the very slight erythema noted for one animal treated at 50% on Day 3. White staining of test item remnants on the dorsal surface of the ears did not hamper scoring for erythema. One control animal showed bald spots on the nose on Day 6.

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.

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.

Radioactivity Measurements and SI Values
Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 80% were 2652, 1512 and 642 DPM, respectively. The mean DPM/animal value for the vehicle control group was 547 DPM. The SI values calculated for the test item concentrations 25, 50 and 80% were 4.8, 2.8 and 1.2, respectively.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

These results show that the test item elicits a SI ≥ 3. The EC3 value (the estimated test item concentration that will give a SI =3) was established to be between >0 and 25%. The results did not show the expected dose-response relationship which more often seen in these kind of studies. The response might be lower at 50% and 80% due to differences in skin penetration (less vehicle present).
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

Based on these results:
- according to the recommendations made in the test guidelines (including all amendments), PNU-0181776 would be regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS)
of the United Nations (2015) (including all amendments), PNU-0181776 should be classified as skin sensitizer (Category 1).
- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), PNU-0181776 should be classified as skin sensitizer (Category 1) and labeled as H317: May cause an allergic skin reaction.

Executive summary:

Assessment of skin sensitization to PNU-0181776 in the Mouse (Local Lymph Node Assay). The study was carried out based on the guidelines described in: OECD, Section 4, Health Effects, No.429 (2010), EC, No 440/2008; B42: "Skin Sensitization: Local Lymph Node Assay" EPA, OPPTS 870.2600 (2003) “Skin Sensitization”. Test item concentrations selected for the main study were based on the results of a pre-screen test. In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 25, 50 or 80% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (N,N-Dimethyl formamide). Three days after the last exposure, all animals were injected with 3H-methylthymidineand after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index(SI) was subsequently calculated for each group. No irritation was observed in any of the animals except for the very slight erythema noted for one animal treated at 50% on Day 3. White staining of test item remnants on the dorsal surface of the ears did not hamper scoring for erythema. One control animal showed bald spots on the nose on Day 6. 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. Mean DPM/animal values for the experimental groups treated with test item concentrations 25, 50 and 80% were 2652, 1512 and 642 DPM, respectively. The mean DPM/animal value for the vehicle control group was 547 DPM. TheSI values calculated for the test item concentrations 25, 50 and 80% were 4.8, 2.8 and 1.2, respectively. These results show that the test item elicits aSI ≥ 3. The EC3 value (the estimated test item concentration that will give aSI =3) was established to be between >0 and 25%. The results did not show the expected dose-response relationship which more often seen in these kind of studies. The response might be lower at 50% and 80% due to differences in skin penetration (less vehicle present). 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. Based on these results: - according to the recommendations made in the test guidelines (including all amendments), PNU-0181776 would be regarded as skin sensitizer. - according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), PNU-0181776 should be classified as skin sensitizer (Category 1). - according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments), PNU-0181776 should be classified as skin sensitizer (Category 1) and labeled as H317: May cause an allergic skin reaction.