4-Methanesulfonyl-2-(trifluoromethyl)benzaldehyde

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 2016 - Sep 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

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:

4-methanesulfonyl-2-(trifluoromethyl)benzaldehyde

EC Number:

812-681-9

Cas Number:

1215310-75-0

Molecular formula:

C9H7F3O3S

IUPAC Name:

4-methanesulfonyl-2-(trifluoromethyl)benzaldehyde

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

Mouse, Balb/c strain, inbred, SPF-Quality. 30 females (nulliparous and non-pregnant), six females per group.

Conditions:
Environmental controls for the animal room were set to maintain
18 to 24°C, a relative humidity of 40 to 70%, at least 10 air
changes/hour, and a 12-hour light/12-hour dark cycle. Any
variations to these conditions were maintained in the raw data
and had no effect on the outcome of the study.
Animal caging:
Group housing in Makrolon cages (MIII type; height 18 cm)
containing sterilized sawdust as bedding material (Lignocel S 8-
15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany).
The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.
Cage enrichment:
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 cageenrichment.
Food:
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
Water:
Free access to tap-water.
Analysis of food, sawdust, paper, shelters and water:
Results of analysis for diet (nutrients and contaminants), sawdust, paper, shelters and water were assessed and did not
reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are
retained in the Charles River Den Bosch archives.

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

Preparations (w/w) were prepared within 4 hours prior to dosing and homogeneity was assessed by visual inspection of the solutions. No adjustment was made for specific gravity of the vehicle.

No. of animals per dose:

Three groups of six animals were treated with one test item concentration per group. The highest
test item concentration was selected from the pre-screen test. One group of six animals was
treated with vehicle and one group with the positive control item.

GROUP* INDUCTION
1 (1-6) Vehicle control Vehicle: Propylene glycol/water (7/3, v/v)
2 (7-12) Positive control 0.5% 1-Chloro-2,4-Dinitrobenzene (DNCB)
3 (13-18) Experimental 0.5% test item concentration
4 (19-24) Experimental 5% test item concentration
5 (25-30) Experimental 50% test item concentration
*. Six females per group, animal numbers given between brackets.

Details on study design:

A pre-screen test was conducted in order to select the highest test item concentration to be used
in the main study. In principle, this concentration should cause no systemic toxicity and may
give well-defined irritation (maximally grade 2) at the highest.
Four young adult animals were selected and two test item concentrations were tested, each on
two animals. Concentrations of 25% and 50% were tested and the highest concentration was
the maximum concentration as required in the test guidelines.
The test system, procedures and techniques were the same to those used in the main study, with
the exceptions that Makrolon MII type cages (height 14 cm) were used for group housing, no
examinations were performed after the animals were sacrificed on Day 4 and scoring for
irritation included scoring for oedema.

Main study
Three groups of six animals were treated with one test item concentration per group. The highest
test item concentration was selected from the pre-screen test. One group of six animals was
treated with vehicle and one group with the positive control item.

Induction - Days 1, 2 and 3
For the experimental animals, the dorsal surface of both ears was epidermally treated
(25 μL/ear) with the test item, approximately the same time each day. The concentrations were
mixed thoroughly using a magnetic stirrer immediately prior to dosing. The control animals
were treated as described for the experimental animals, except that, instead of the test item, the
vehicle or positive control item was administered.

Weighing of ear punches and lymph nodes - Day 4
Approximately 24 hours after the last treatment, all animals were sacrificed by intra-peritoneal
injection with pentobarbital (0.2 mL/animal Euthasol® 20% (AST Farma BV, Oudewater, The
Netherlands)).
Both ears (left and right) were punched in the apical area using a biopsy punch (Stiefel, Ø 8 mm
=> 0.5 cm2). For each animal both punches were immediately weighed pooled per animal using
an analytical balance after which the punches were discarded.
Both auricular draining lymph nodes (left and right) of mice were excised. The relative sizes of
the nodes (as compared to normal) were estimated by visual examination and abnormalities of
the nodes and surrounding area were recorded. For each animal both lymph nodes were pooled
and immediately weight using an analytical balance.

Determination of total cell-counts of lymph nodes – Day 4
Following excision and weighing of the nodes, single cell suspensions of lymph node cells
(LNC) were prepared in phosphate buffered saline (PBS) by gentle separation through stainless
steel gauze (diameter 200 μm mesh). LNC were collected in approximately 0.7 mL of PBS in
a 24 wells plate that was kept on ice as much as possible.
Cell counts were determined using a Coulter Counter (Beckman Coulter, The Netherlands).

Positive control substance(s):

other: 0.5% 1-Chloro-2,4-Dinitrobenzene (DNCB)

Statistics:

Calculations were performed in MS EXCEL and statistical analysis was performed with
GraphPad Prism 4 (Kruskal-Wallis test, followed by the Mann Whitney test).

Results and discussion

Positive control results:

The positive control item DNCB elicited a reaction pattern with increased LN hyperplasia, which was in congruence with the expected mode of action of a contact allergen.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Test group / Remarks:

0.5% QAW039-C2

Remarks on result:

not measured/tested

Results 2

Key result

Parameter:

SI

Test group / Remarks:

5% QAW039-C2

Remarks on result:

not measured/tested

Results 3

Key result

Parameter:

SI

Test group / Remarks:

50% QAW039-C2

Remarks on result:

not measured/tested

Results 4

Key result

Parameter:

other: Ear weight index

Value:

1

Test group / Remarks:

0.5% QAW039-C2

Results 5

Key result

Parameter:

other: Ear weight index

Value:

1

Test group / Remarks:

5% QAW039-C2

Results 6

Key result

Parameter:

other: Ear weight index

Value:

1

Test group / Remarks:

50% QAW039-C2

Results 7

Key result

Parameter:

other: LN weight index

Value:

1.26

Test group / Remarks:

0.5% QAW039-C2

Results 8

Key result

Parameter:

other: LN weight index

Value:

0.98

Test group / Remarks:

5% QAW039-C2

Results 9

Key result

Parameter:

other: LN weight index

Value:

1.05

Test group / Remarks:

50 % QAW039-C2

Results 10

Key result

Parameter:

other: Cell count index

Value:

1.38

Test group / Remarks:

0.5% QAW039-C2

Results 11

Key result

Parameter:

other: Cell count index

Value:

1.05

Test group / Remarks:

5% QAW039-C2

Results 12

Key result

Parameter:

other: Cell count index

Value:

1.22

Test group / Remarks:

50 % QAW039-C2

Any other information on results incl. tables

Pre-screen test

Erythema was noted for one animal at 50% on Day 3 only. No signs of systemic toxicity were noted.

Based on the results, the highest test item concentration selected for the main study was a 50% concentration.

No erythema of the ears was noted after visual examination in all of animals. Yellow

testsubstance remnants on the dorsal surface of the ears did not hamper scoring for erythema.

Visual examination of the nodes revealed that the nodes of two animals treated at 0.5% and

two animals treated at 50% were enlarged when compared to the vehicle control group. No

macroscopic abnormalities of the surrounding areas were noted in any of the animals.

Statistically significant body weight loss was noted in the 50% group in comparison to the

vehicle control. Since there were no clinical observations attributable to treatment with

QAW039-C2 and the body weight loss was only slight this was considerd not to have affected

the study.

The positive control item DNCB elicited a reaction pattern with increased LN hyperplasia,

which was in congruence with the expected mode of action of a contact allergen.

QAW039-C2 did not cause any relevant changes in ear weight up to a concentration of 50% in

Propylene glycol/water (7/3, v/v).

QAW039-C2 did no show a statistically significant difference when compared to vehicle and

did not show a dose response for LN weights or LN counts. The threshold for LN weights and

LN counts was exceeded at the lowest concentration (0.5%). These high values of the lowest

dose groups were mostly caused by one animal.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Since the high values of the lowest dose groups for LN weights and LN counts were mostly caused by one animal and no dose reponse or statistical significant difference was shown, no sensitizing was ascribed to QAW039-C2 in the murine LLNA TIER I.
Further, based on the ear weight results, no irritating potential as ascribed to QAW039-C2 in the murine LLNA TIER I.
Based on these results QAW039-C2 would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. It 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 (2015) (including all amendments).