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Description of key information

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 06 to 19 Jul 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
equivalent or similar to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, Horst, The Netherlands.
- Age at study initiation: approx. 10 weeks old.
- Weight at study initiation: Body weight variation was within ±20% of the sex mean.
- Housing: Individual housing in labeled Macrolon cages containing sterilized sawdust as bedding material.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: at least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0±3.0℃ (actual range: 20.8-23.2℃ )
- Humidity (%): 40-70% (actual range: 43-81%)
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day

IN-LIFE DATES: From: 2010-07-06 To: 2010-07-19
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
In preliminary study: 25 and 50% w/w
In main study: 0, 10, 25 and 50% w/w
No. of animals per dose:
In preliminary study:
Two groups of one animal were treated with one test substance concentration per group.
In main study:
One group of five animals was treated with vehicle.
Three groups of five animals were treated with one test substance concentration per group.
Details on study design:
Preliminary irritation study
A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (maximally grade 2) at the highest concentration.
Two test substance concentrations were tested; a 25% and 50% concentration. The highest concentration was the highest concentration that could be prepared homogeneously. The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study unless otherwise specified. Two young adult animals were selected. Each animal was treated with one concentration on three consecutive days. Approximately 3-4 hours after the last exposure, the irritation of the ears was assessed. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation and no necropsy was performed.

Main study
1) Allocation
Three groups of five animals were treated with one test substance concentration per group (10, 25 and 50% w/w). One group of five animals was treated with vehicle.
2) 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 mixed thoroughly using a vortex mixer 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.
3) Excision of the nodes - Day 6
All animals:
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 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.
4) 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.
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. 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).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
None stated
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
at 10% substance concentration
Parameter:
SI
Value:
3.1
Test group / Remarks:
at 25% substance concentration
Key result
Parameter:
SI
Value:
3.7
Test group / Remarks:
at 50% substance concentration
Key result
Parameter:
EC3
Value:
ca. 23.6
Test group / Remarks:
dose reponse noted
Cellular proliferation data / Observations:
The mean DPM/animal value for the vehicle control group was 1023 DPM. The mean DPM/animal value for the experimental group treated with test substance concentrations 10% was 2087 DPM. The mean DPM/animal value for the experimental group treated with test substance concentrations 25% was 3215 DPM. The mean DPM/animal value for the experimental group treated with test substance concentrations 50% was 3820 DPM.

The slight irritation of the ears as shown by all animals treated at 25% and 50% was considered not to have a toxicologically significant effect on the activity of the nodes. No oedema was observed in any of the animals examined.

One animal at 25% and all animals at 50% showed one or two auricular lymph nodes, which were enlarged in size. The auricular lymph nodes of the other animals were considered normal in size. One animal at 25% showed many dark red foci in the muscle of the neck. No macroscopic abnormalities of the surrounding area were noted in any of the other animals.

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, was considered not toxicologically significant.

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Disintegrations Per Minute (DPM) and Stimulation Index (SI)

Group

Test substance

(% w/w)

Mean

DPM

SI

2

10%

2087

2.0

3

25%

3215

3.1

4

50%

3820

3.7

1

0% (vehicle)

1023

1.0

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
It was concluded that the test substance elicited SI values of >3 and hence is considered as a skin sensitiser under this studies conditions.The data showed a dose response and an EC3 value (the estimated test substance concentration that will give a SI =3) of 23.6% was calculated.

Executive summary:

Assessment of Contact Hypersensitivity to PF-00968603 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 (2002),  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 preliminary study. 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.

The slight irritation of the ears as shown by all animals treated at 25% and 50% was considered not to have a toxicologically significant effect on the activity of the nodes. No oedema was observed in any of the animals examined.

One animal at 25% and all animals at 50% showed one or two auricular lymph nodes, which were enlarged in size. The auricular lymph nodes of the other animals were considered normal in size. One animal at 25% showed many dark red foci in the muscle of the neck. No macroscopic abnormalities of the surrounding area were noted in any of the other animals.

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 2087, 3215 and 3820 DPM respectively. The mean DPM/animal value for the vehicle control group was 1023 DPM.

The SI values calculated for the substance concentrations 10, 25 and 50% were 2.0, 3.1 and 3.7 respectively (Table 4 and Figure 1).

These results indicate that the test substance could elicit an SI ≥ 3. The data showed a doseresponse and an EC3 value (the estimated test substance concentration that will give a SI =3) of 23.6% was calculated.

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

Based on these results: - according to the recommendations made in the test guidelines, PF-00968603 would be regarded as skin sensitizer. - according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2007), PF-00968603 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, PF-00968603 should be classified as skin sensitizer (Category 1) and labeled as H317: May cause an allergic skin reaction.

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

A LLNA study was conducted according to OECD 429 using mouse (Beerens-Heijnen, 2010). Key study. This study indicate that the test substance could elicit an SI ≥ 3, hence the test substance should be classfied as a skin sensitizer.

Short description of key information:

A LLNA (Beerens-Heijnen, 2010) study was available which is key study. This study showed that the test substance is skin sensitising.

Justification for selection of skin sensitisation endpoint:

Study run to a method comparable with current guidelines and to GLP

Justification for classification or non-classification

Skin sensitisation: Animal tests gave positive results (LLNA SI ≥ 3 (actual value 3.7)).

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.4.2 the substance is classified as a skin sensitizer (Category 1).