3-chloro-4-fluoroaniline

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 24th 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:

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

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:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

3-chloro-4-fluoroaniline

EC Number:

206-682-8

EC Name:

3-chloro-4-fluoroaniline

Cas Number:

367-21-5

Molecular formula:

C6H5ClFN

IUPAC Name:

3-chloro-4-fluoroaniline

In vivo test system

Test animals

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
- 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 (Litalabo, S.P.P.S., Argenteuil, France). 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. 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: 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 – 22.6ºC
- Humidity (%): 44 - 68%)
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.
- IN-LIFE DATES: From: 09 November 2011 To: 05 December 2011

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Remarks:

Acetone/Olive oil (4:1 v/v) (Acetone p.a.: Merck, Darmstadt, Germany); Olive oil: Sigma-Aldrich, Steinheim, Germany

Concentration:

2.5, 5 and 10 %w/w

No. of animals per dose:

5 animals per dose

Details on study design:

Pre-screen test
A pre-screen test was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation at the most (maximum grade 2) and/or an increase in ear thickness < 25%) and is the highest possible concentration that can technically be applied.
Initially, two test substance concentrations were tested; a 25% and 50% concentration. The highest concentration was the maximum concentration as required in the test guidelines (50% for solids).
Based on the results of the initially treated animals, two lower concentrations (2.5% and 5%) were tested at a later stage. Based on the results of the additionally treated animals, a concentration of 10% was also tested at a later stage.
The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). Each surviving animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages. Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 for all animals and on Days 3 and Day 6 for surviving animals.
The initially treated animals were sacrificed for humane reasons on Day 1 and the additionally treated animals were sacrificed after the final observation.

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 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. 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)

Results and discussion

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 NOTOX. In this study, performed in October 2011, females of the CBA/J mouse strain) 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. 04012JE. Concentrations used for this study were 5, 10 and 25% in Acetone/Olive oil (4:1 v/v).

The SI values calculated for the substance concentrations 5, 10 and 25% were 1.4, 2.0 and 7.4 respectively. An EC3 value of 12.8% 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 13.9, 16.0, 11.9, 16.9, 10.7 and 14.4%. Based on the results, it was concluded that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

2.5% w/w

Results 2

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

5% w/w

Results 3

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

10% w/w

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The SI values calculated for the substance concentrations 2.5, 5 and 10% were 0.9, 1.0 and 1.1 respectively.
Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 10%, PF- 01458762 was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 10%.
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-01458762 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.

Executive summary:

Assessment of Contact Hypersensitivity to PF-01458762 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. Animals treated at 25 and 50% were sacrificed for humane reasons on Day 1 based on signs of systemic toxicity. No irritation and no signs of systemic toxicity were observed for any of the animals treated at 2.5, 5 or 10%. Based on these results, the highest test substance concentration selected for the main study was a 10% concentration.
In the main study, three experimental groups of five female CBA/J mice were treated with test substance concentrations of 2.5, 5 or 10% 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 irritation of the ears was observed in any of the animals examined.
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. The body weight loss noted across the dose groups was considered not toxicologically significant since the changes were slight in nature and no concentration-related incidence was apparent.
The auricular lymph nodes of the majority of animals treated at 5 and 10% appeared larger in size when compared to nodes of the control animals. Auricular lymph nodes of one animal at 10%, one animal at 5% and all animals at 2.5% 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 2.5, 5 and 10% were 118, 122, and 143 DPM respectively. The mean DPM/animal value for the vehicle control group was 127.

The SI values calculated for the substance concentrations 2.5, 5 and 10% were 0.9, 1.0 and 1.1 respectively.
Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 10%, PF- 01458762 was considered not to be a skin sensitizer. It was established that the EC3 value (the estimated test substance concentration that will give a SI =3) (if any) exceeds 10%.
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-01458762 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.