Registration Dossier

Administrative data

Description of key information

Sensitizing to skin in a mouse LLNA (GLPs and OECD 429 compliant. Stimulation Index (SI) values calculated for the registerered substance concentrations 10, 25 and 50% were 2.3, 4.1 and 9.2, respectively. Ab EC3 value (the estimated test substance concentration that will give a SI =3) of 15.8% was calculated.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 7 October to 24 December 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Batch No.of test material: MR92143651
- Expiration date of the lot/batch: 01 January 2017
- Purity: 86.4% (No correction was made for purity of the test substance)
- Purity test date: 18 June 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Yes, maximum temperature: 50°C, maximum duration: unknown (until liquefaction).
- Solubility and stability of the test substance in the solvent/vehicle: The vehicle, Acetone/Olive oil (4:1 v/v), was selected on the basis of maximizing the solubility using the test substance data provided by the Sponsor and trial preparation results performed at WIL Research Europe (the vehicle was chosen from the vehicles specified in the test guideline: Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol and dimethylsulfoxide).
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not assessed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance preparations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was assessed by visual inspection of the solutions. Prior to weighing, the test substance was heated in a water bath with a maximum temperature of 40 ºC for 2 hours and 18 minutes. Correction of the purity/composition of the test substance is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be dosed.
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: N/A
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material): N/A

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF-Quality.
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: between 19.1 and 24.0g
- Housing: 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.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water
- Acclimation period: at least 5 days before the start of treatment
- Indication of any skin lesions: At least prior to dosing. It was ensured that the animals were healthy and that the ears were intact and free from any abnormality.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 12-hour light/12-hour dark cycle
- IN-LIFE DATES: From: 07 October 2015 To: 9 November 2015
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 10, 25 or 50% w/w
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Acetone/Olive oil (4:1 v/v) was selected on the basis of maximizing the solubility of the test substance.
- Irritation: Very slight erythema (score 1) was noted for one animal treated at 25% and both animals treated at 50% on Day 4.
- Systemic toxicity: No signs of systemic toxicity were observed in any of the pre-screen animals.
- Ear thickness measurements: Variations in ear thickness during the observation period were less than 25% compared to Day 1 pre-dose values.
- Erythema scores: see above.

MAIN STUDY
- Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the pre-screen test. One group of five animals was treated with vehicle.

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

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.

- Criteria used to consider a positive response:
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 substance may be regarded as a skin sensitizer. The EC3 value (the estimated test substance concentration that will give a SI =3) was determined using linear interpolation. If EC3 value ≤ 2%: sub-category 1A; if EC3 value > 2%: sub-category 1B.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No
Positive control results:
A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by WIL Research Europe. In this study, performed in November 2015, females of the CBA/J mouse strain (Janvier, Le Genest-Saint-Isle, France) were checked for sensitivity to Hexylcinnamaldehyde. The females were approx. 10 weeks old at commencement of the study. The study was based on the OECD Guideline No. 429, EC No 440/2008, Part B.42 and EPA, OPPTS 870.2600 “Skin Sensitization”. Alpha- Hexylcinnamaldehyde, technical grade (CAS no. 101-86-0) was fabricated under lot no. MKBJ8846V (Sigma- Aldrich, Steinheim, Germany). Concentrations used for this study were 5, 10 and 25% in Acetone/Olive oil (4:1 v/v).
The SI values calculated for the item concentrations 5, 10 and 25% were 1.0, 1.5 and 4.4 respectively. An EC3 value of 17.8% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5%. The results of the 6 monthly HCA reliability checks of the recent years were 14.5, 13.4, 14.1, 17.3, 9.8% and 10%.
Based on the results, it was concluded that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.
Key result
Parameter:
EC3
Remarks:
(%)
Value:
15.8
Key result
Parameter:
SI
Value:
1
Variability:
± 0.2
Test group / Remarks:
Vehicle control
Key result
Parameter:
SI
Value:
2.3
Variability:
± 0.7
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
4.1
Variability:
± 0.6
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
9.2
Variability:
± 1.1
Test group / Remarks:
50%
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 1832, 3293 and 7402 DPM, respectively.

DETAILS ON STIMULATION INDEX CALCULATION:
The SI values calculated for the substance concentrations 10, 25 and 50% were 2.3, 4.1 and 9.2, respectively.

EC3 CALCULATION:
An EC3 value (the estimated test substance concentration that will give a SI =3) of 15.8% was calculated.

CLINICAL OBSERVATIONS:
Systemic toxicity:
No mortality occurred. Piloerection was noted for the animals treated at 25% and 50% on Day 3 only.
Skin reactions / Irritation:
The slight irritation of the ears as shown by three animals treated at 25% and four animals treated at 50% on Days 3 and/or 4, was considered not to have a toxicologically significant effect on the activity of the nodes.

BODY WEIGHTS
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:
The auricular lymph nodes of the animals treated at 10% and 25% and of the control animals were considered normal in size. The nodes of the animals treated at 50% were considered enlarged. No macroscopic abnormalities of the surrounding area were noted for any of the animals.
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Based on the results of the murine LLNA (SI ≥ 3 and EC3 value > 2%), 1-Phenyldecane-1,3-dione is considered as skin sensitizer and should be classified as skin sensitizer Category 1B.
Executive summary:

The objective of this study was to evaluate the skin sensitization potential of the test item, 1-Phenyldecane-1,3-dione, 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 substance 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 substance concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)).

Three days after the last exposure, all animals were injected with 3H-methyl thymidine and 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 mortality occurred. Piloerection was noted for the animals treated at 25% and 50% on Day 3 only.

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

The slight irritation of the ears as shown by three animals treated at 25% and four animals treated at 50% on Days 3 and/or 4, was considered not to have a toxicologically significant effect on the activity of the nodes.

The auricular lymph nodes of the animals treated at 10% and 25% and of the control animals were considered normal in size. The nodes of the animals treated at 50% were considered enlarged.

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 substance concentrations 10 25 and 50% were 1832, 3293 and 7402 DPM, respectively. The mean DPM/animal value for the vehicle control group was 803 DPM. The SI values calculated for the substance concentrations 10, 25 and 50% were 2.3, 4.1 and 9.2, respectively.

Since the animals in the middle and highest dose groups showed signs of systemic toxicity it cannot be excluded that this influenced the activity of the lymph nodes. However, since the test item elicits a SI ≥ 3 and the data show a clear dose-response, it was concluded that based on these results the test item should be considered as a skin sensitizer.

An EC3 value (the estimated test substance concentration that will give a SI =3) of 15.8% was calculated.

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),

1-Phenyldecane-1,3-dione would be regarded as skin sensitizer.

- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS of the United Nations (2011) (including all amendments), 1-Phenyldecane-1,3-dione should be classified as skin sensitizer (Category 1B).

- according to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments), 1-Phenyldecane-1,3-dione should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

One Klimisch score 1 study is available to assess the skin sensitizing potential of the registered substance and was used as key study:

In this study, performed according to OECD Test Guideline No.429 (Mouse Local Lymph Node Assay), three experimental groups of five female CBA/J mice were treated with the registered substance 1-Phenyldecane-1,3-dione at concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with vehicle alone (Acetone/Olive oil (4:1 v/v)).

No mortality occurred. Piloerection was noted for the animals treated at 25% and 50% on Day 3 only.

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight irritation of the ears as shown by three animals treated at 25% and four animals treated at 50% on Days 3 and/or 4, was considered not to have a toxicologically significant effect on the activity of the nodes. The auricular lymph nodes of the animals treated at 10% and 25% and of the control animals were considered normal in size. The nodes of the animals treated at 50% were considered enlarged. 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 substance concentrations 10 25 and 50% were 1832, 3293 and 7402 DPM, respectively. The mean DPM/animal value for the vehicle control group was 803 DPM. The SI values calculated for the substance concentrations 10, 25 and 50% were 2.3, 4.1 and 9.2, respectively.

Since the animals in the middle and highest dose groups showed signs of systemic toxicity it cannot be excluded that this influenced the activity of the lymph nodes. However, since the test item elicits a SI ≥ 3 and the data show a clear dose-response, it was concluded that based on these results the test item should be considered as a skin sensitizer.

An EC3 value (the estimated test substance concentration that will give a SI =3) of 15.8% was calculated.

Based on these results, 1-Phenyldecane-1,3-dione would be regarded as skin sensitizer and should be classified as skin sensitizer Category 1B, H317 according to CLP and GHS-UN regulations.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In a LLNA, performed according to OECD guideline no. 429 and in compliance with GLP, the Stimulation Index (SI) values calculated for the registerered substance concentrations 10, 25 and 50% were 2.3, 4.1 and 9.2, respectively. An EC3 value (the estimated test substance concentration that will give a SI =3) of 15.8% was calculated.

Since the test item elicits a SI ≥ 3 and the data show a clear dose-response, it was concluded that based on these results the test item should be considered as a skin sensitizer.

Based on these results, 1-Phenyldecane-1,3-dione would be regarded as skin sensitizer and should be classified as skin sensitizer Category 1B, H317 according to CLP and GHS-UN regulations.