4-[(3,5-Dichlorobenzoyl)amino]-3-hydroxybenzoic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

4-(3,5-dichlorobenzamido)-3-hydroxybenzoic acid

EC Number:

832-365-4

Cas Number:

1184581-58-5

Molecular formula:

C14H9Cl2NO4

IUPAC Name:

4-(3,5-dichlorobenzamido)-3-hydroxybenzoic acid

Test material form:

solid: particulate/powder

Details on test material:

Batch E010014831, re-test date: 30 June 2015

Specific details on test material used for the study:

Description White to off white powder
Batch E010014831
Purity/Composition 99.5%
Test substance storage At room temperature in the dark
Stable under storage conditions until 30 June 2015 (Retest date)
pH (1% in water, indicative range) 5.3 – 5.0 (determined by WIL Research Europe B.V.)
Stability in vehicle:
• N,N-Dimethylformamide Unknown
Solubility in vehicle:
• N,N-Dimethylformamide 172 mg/g

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 (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
- Age at study initiation: Young adult animals (approx. 9 weeks old)
- Weight at study initiation: 18-23g 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 (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: 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.
- Indication of any skin lesions:

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: 19 March 2014 To: 14 April 2014

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Pre-screen 25 and 50% w/v
Main test 0, 10, 25 and 50% w/v

No. of animals per dose:

5 (five)

Details on study design:

PRE-SCREEN TESTS:
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 (see section 6.7) and/or an increase in ear thickness < 25%) and is the highest possible concentration that can technically be applied.
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).
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.
- Compound solubility: 172 mg/g
- Irritation: none noted
- Systemic toxicity: none noted
- Ear thickness measurements: <25% variation from the Day 1 pre-dose values
- Erythema scores: no erythema noted

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: SI >3

TREATMENT PREPARATION AND ADMINISTRATION:
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.

Positive control substance(s):

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

Results and discussion

Positive control results:

The SI values calculated for the substance concentrations 5, 10 and 25% were 1.2, 1.1 and 8.0
respectively. An EC3 value of 14.1% was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 2 and 20%. The results of the 6
monthly HCA reliability checks of the recent years were 11.9, 16.9, 14.4, 16.5, 14.5 and 13.4%.
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.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

ca. 1

Variability:

SEM 0.2

Test group / Remarks:

0% w/v

Results 2

Parameter:

SI

Value:

ca. 1.2

Variability:

SEM 0.2

Test group / Remarks:

10% w/v

Results 3

Parameter:

SI

Value:

ca. 1.1

Variability:

SEM 0.2

Test group / Remarks:

25% w/v

Results 4

Parameter:

SI

Value:

ca. 1.1

Variability:

SEM 0.2

Test group / Remarks:

50% w/v

Results 5

Parameter:

EC3

Remarks on result:

not determinable

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA - DPM
0% w/v 187 ± 24
10% w/v 224 ± 20
25% w/v 204 ± 34
50% w/v 209 ± 30

DETAILS ON STIMULATION INDEX CALCULATION
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 DPM/vehicle control group.

EC3 CALCULATION
Not Applicable

CLINICAL OBSERVATIONS:
No irritation of the ears was observed in any of the animals examined. White test substance remnants were present on the dorsal surface of the ears of the experimental animals during the observation period but did not hamper scoring of the skin reactions.
No test substance related mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

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

Any other information on results incl. tables

Macroscopic findings

No abnormalities were found at macroscopic post  mortem  examination of the anima, which died prior

to 3H-methyl  thymidine  injection.

Macroscopy of the auricular lymph nodes and surrounding area

All auricular lymph nodes were considered normal in size.

No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Radioactivity measurements and  SI  values

Mean DPM/animal values for the experimental groups treated with test substance concentrations 10,

25 and 50% were 224, 204 and 209 DPM respectively. The mean DPM/animal value for the vehicle

control group was 187 DPM. The  SI  values calculated for the substance concentrations 10, 25 and

50% were 1.2, 1.1 and 1.1, respectively.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Since there was no indication that the test substance elicited an SI ≥ 3 when tested up to 50%, PF06410251
was not considered to be a skin sensitizer.
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, PF-06410251 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 (2011)
(including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and
packaging of substances and mixtures (including all amendments).

Executive summary:

Assessment of Contact Hypersensitivity to  PF-06410251 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 (Dimethyl formamide).

Three days after the last exposure, the 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 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 224, 204 and 209 DPM respectively. The mean DPM/animal value for the vehicle

control group was 187 DPM. The  SI  values calculated for the substance concentrations 10, 25 and

50% were 1.2, 1.1 and 1.1, respectively.

Since there was no indication that the test substance elicited an  SI  ≥ 3 when tested up to 50%,  PF06410251

was not considered to be a skin sensitizer.

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,  PF-06410251 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 (2011)

(including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and

packaging of substances and mixtures (including all amendments).