1,2,4-Triazin-3-amine, 6-bromo-

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 days

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Principles of method if other than guideline:

In the main study, six Balb/c female mice per group received topical test or control items on
the dorsum of both ears once a day on 3 consecutive days. Auricular lymph nodes were taken
24 hours after the last application. Endpoints: visual examination ears and sizes ear-draining
lymph nodes, body weight at treatment start and day of necropsy, ear weight (skin irritation),
ear-draining lymph node weights and cell counts (LN hyperplasia). Concentrations: vehicle
control Propylene glycol/water (7/3, v/v), positive control DNCB (1-Chloro-2,4-
Dinitrobenzene): 0.5% (w/w), and INC280-M3A: 5%, 0.5%, 0.05% (w/w). The vehicle was
selected on the basis of maximizing the solubility and the formulations appeared visually as
clear (0.05% or turbid (0.5% and 5%) solutions.

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:

6-bromo-1,2,4-triazin-3-amine

Cas Number:

69249-22-5

Molecular formula:

C3H3BrN4

IUPAC Name:

6-bromo-1,2,4-triazin-3-amine

Test material form:

solid: crystalline

Specific details on test material used for the study:

Identification INC280-M3A
Appearance Slightly yellowish powder
Batch 1010006035

In vivo test system

Test animals

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

Location: Specific Pathogen Free area: animal rooms A0.18.
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
cage-enrichment.
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 WIL Research Europe archives.

Study design: in vivo (LLNA)

Vehicle:

other: Propylene glycol/water (7/3, v/v)

Concentration:

5%, 0.5%, 0.05% (w/w).

No. of animals per dose:

6

Details on study design:

In the main study, six Balb/c female mice per group received topical test or control items on
the dorsum of both ears once a day on 3 consecutive days. Auricular lymph nodes were taken
24 hours after the last application. Endpoints: visual examination ears and sizes ear-draining
lymph nodes, body weight at treatment start and day of necropsy, ear weight (skin irritation),
ear-draining lymph node weights and cell counts (LN hyperplasia).

Positive control substance(s):

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

Statistics:

The results of the LLNA were evaluated according to the following criteria. The following
thresholds were used: ear weight index: 1.05, LN weight: 1.2, LN cell count: 1.3 as advised by
the Sponsor.
1. Values which exceed these thresholds were considered positive
with no statistical significance, but a clear concentration-dependence
or when a statistically significant increase in one of the parameters occurs and a clear
concentration-dependence can be derived.
2. Values which are below these thresholds were considered positive when a statistical
significance occurs in one of the parameters together with a clear concentrationdependence.
3. Values were considered negative
in case of being below the thresholds and without a statistical significance.
in case of being below the thresholds, with a statistical significance, but without a clear
concentration-dependence.
in case of being above the thresholds, without statistical significance and without a clear
concentration-dependence.

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.
INC280-M3A meeted the ear weight threshold at the highest concentration with a statistically
significant difference when compared to vehicle. No clear dose response was found.
INC280-M3A exceeded the LN weight threshold at 0.5% and 5% with a statistically
significant difference when compared to vehicle. No clear dose response was found.
INC280-M3A exceeded the LN count threshold at all concentrations with a statistically
significant difference when compared to vehicle and showed a clear dose response.

In vivo (LLNA)

Results

Results 1

Parameter:

other: Ear weight index

Value:

1.05

Test group / Remarks:

5% INC280-M3A

Results 2

Parameter:

other: LN weight index

Value:

1.31

Test group / Remarks:

5% INC280-M3A

Results 3

Key result

Parameter:

other: Cell count index

Value:

2.71

Test group / Remarks:

5% INC280-M3A

Remarks on result:

other: P<0.01 treated groups vs control group

Cellular proliferation data / Observations:

INC280-M3A appeared to be a strong sensitizer, since the cell count index
exceeded the threshold of 1.3 at the lowest concentration of 0.05% (1.62), in the murine
LLNA TIER I.

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

According to the Globally Harmonized System of Classification and Labelling of Chemicals
(GHS) of the United Nations (2011) (including all amendments), INC280-M3A should be
classified as skin sensitizer (Category 1A).

Executive summary:

INC280-M3A: Assessment of contact sensitizing potential with the murine local lymph node

assay (TIER I).

The purpose of this study is to evaluate whether the test item induces contact hypersensitivity

in mice after three epidermal exposures of the animals under the conditions described in this

report. This study should provide a rational basis for risk assessment in man. The proposed

study complies with the test principle outlined in OECD Guideline 429 (2010), EC No

440/2008 Part B42 (2008) and EPA OPPTS 870.2600 (2003), except that these guidelines

describe a study plan using radioactive methods for the determination of cell proliferation.

Test item concentrations selected for the main study were based on results of a pre-screen test.

In the main study, six Balb/c female mice per group received topical test or control items on

the dorsum of both ears once a day on 3 consecutive days. Auricular lymph nodes were taken

24 hours after the last application. Endpoints: visual examination ears and sizes ear-draining

lymph nodes, body weight at treatment start and day of necropsy, ear weight (skin irritation),

ear-draining lymph node weights and cell counts (LN hyperplasia). Concentrations: vehicle

control Propylene glycol/water (7/3, v/v), positive control DNCB (1-Chloro-2,4-

Dinitrobenzene): 0.5% (w/w), and INC280-M3A: 5%, 0.5%, 0.05% (w/w). The vehicle was

selected on the basis of maximizing the solubility and the formulations appeared visually as

clear (0.05% or turbid (0.5% and 5%) solutions.

Formulation analyses confirmed that formulations of INC280-M3A in Propylene glycol/water

(7/3, v/v) were prepared accurately and homogenously, and were stable over at least 5 hours.

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.

INC280-M3A meeted the ear weight threshold at the highest concentration with a statistically

significant difference when compared to vehicle. No clear dose response was found.

INC280-M3A exceeded the LN weight threshold at 0.5% and 5% with a statistically

significant difference when compared to vehicle. No clear dose response was found.

INC280-M3A exceeded the LN count threshold at all concentrations with a statistically

significant difference when compared to vehicle and showed a clear dose response.

In conclusion, INC280-M3A appeared to be a strong sensitizer, since the cell count index

exceeded the threshold of 1.3 at the lowest concentration of 0.05% (1.62), in the murine

LLNA TIER I. Weak irritating potential was ascribed to INC280-M3A.

Based on these results according to the recommendations made in the test guidelines

(including all amendments), INC280-M3A would be regarded as skin sensitizer.

According to the Globally Harmonized System of Classification and Labelling of Chemicals

(GHS) of the United Nations (2011) (including all amendments), INC280-M3A should be

classified as skin sensitizer (Category 1A).