4-(1-piperidinyl)-2-(E)-butenoicacid hydrochloride

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26th October 2011 to 26th January 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

1-[(2E)-3-carboxyprop-2-en-1-yl]piperidin-1-ium chloride

EC Number:

690-933-5

Cas Number:

197892-69-6

Molecular formula:

C9H16ClNO2

IUPAC Name:

1-[(2E)-3-carboxyprop-2-en-1-yl]piperidin-1-ium chloride

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- Housing: Animals were group housed in labeled Makrolon cages (MIII type; height 18 cm) containing sterilised sawdust as bedding material. Paper and shelters (disposable paper corner home) were supplied as cage-enrichment. 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
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.
- Indication of any skin lesions: A health inspection was performed prior to treatment, to ensure that the animals were in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.1 – 22.3
- Humidity (%): 44 - 65
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.

- IN-LIFE DATES: 9th November 2011 to 28th November 2011

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

10, 25 and 50% w/w

No. of animals per dose:

Five

Details on study design:

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 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) containing 20 μCi of 3H-methyl thymidine. After approximately five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) with Euthasol® 20%. 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
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) and 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 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).

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. Furthermore, a description of all other (local) effects was recorded.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:

5% Hexylcinnamicaldehyde SI = 1.4
10% Hexylcinnamicaldehyde SI = 2.0
25% Hexylcinnamicaldehyde SI = 7.4

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 13.9, 16.0, 11.9, 16.9, 10.7 and 14.4%.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

10% w/w

Results 2

Key result

Parameter:

SI

Value:

0.8

Test group / Remarks:

25% w/w

Results 3

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

50% w/w

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The SI values calculated for the substance concentrations 10, 25 and 50% were 0.8, 0.8 and 0.9 respectively.

Since there was no indication that the test substance elicited an SI ≥ 3 when tested up to 50%, PF-00818977-01 was considered to be a non skin sensitizer

Executive summary:

Assessment of Contact Hypersensitivity to PF-00818977-01 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 (Propylene glycol).
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 irritation of the ears was observed in any of the animals examined.
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 in any of the animals.
Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 102, 99 and 115 DPM respectively. The mean DPM/animal value for the vehicle control group was 124 DPM.
The SI values calculated for the substance concentrations 10, 25 and 50% were 0.8, 0.8 and 0.9 respectively.
Since there was no indication that the test substance elicited an SI ≥ 3 when tested up to 50%, PF-00818977-01 was considered to be a non skin sensitizer. 

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, PF-00818977-01 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance 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 (2007) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.