2-Amino-3-bromopyridine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2016 - June 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:

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

Test guideline 2

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-bromopyridin-2-amine

EC Number:

629-620-5

Cas Number:

13534-99-1

Molecular formula:

C5 H5 Br N2

IUPAC Name:

3-bromopyridin-2-amine

Test material form:

solid

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.
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 Charles River Den Bosch archives.

Study design: in vivo (LLNA)

Vehicle:

other: DAE433

Concentration:

Initially, four young adult animals were selected and two test item concentrations were tested, each on two animals. Concentrations of 25% and 60% were tested and the highest concentration was the maximum concentration that could be prepared homogeneously.
Based on the results of the initially treated animals, four additional animals were treated in a similar manner with two lower concentrations (2% and 5%) at a later stage. Based on these results, two additional animals were treated in a similar manner (10%) at a later stage.
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.
Correction of the purity/composition of the test substance is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be dosed.

No. of animals per dose:

GROUP* INDUCTION
1 (1-6) Vehicle control Vehicle: DAE433
2 (7-12) Positive control 0.5% 1-Chloro-2,4-Dinitrobenzene (DNCB)
3 (13-18) Experimental 0.05% test item concentration
4 (19-24) Experimental 0.5% test item concentration
5 (25-30) Experimental 5% test item concentration
*. Six females per group, animal numbers given between brackets.

Details on study design:

Pre-screen test
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.
Initially, four young adult animals were selected and two test item concentrations were tested, each on two animals. Concentrations of 25% and 60% were tested and the highest concentration was the maximum concentration that could be prepared homogeneously.
Based on the results of the initially treated animals, four additional animals were treated in a similar manner with two lower concentrations (2% and 5%) at a later stage. Based on these results, two additional animals were treated in a similar manner (10%) at a later stage.
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, the animals were 8 weeks old, no examinations were performed after the animals were sacrificed on Day 4 and scoring for irritation included scoring for oedema according the scheme below:
Oedema formation:
No oedema .................................................................................……………………….……………….. 0
Slight oedema (barely perceptible) ......................................................………………………………..... 1
Moderate oedema....................................……………………................................................................. 2
Severe oedema…..................................................................................................................................... 3

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.

Positive control substance(s):

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

Statistics:

In case of a positive finding, a linear regression was used to calculate the concentration corresponding to the respective threshold. The calculation for the LN hyperplasia threshold concentration was performed with the respective LN cell count threshold index. In case of inconclusive cell count results, LN weight data were used to calculate the threshold concentration. This classification scheme (see Table) is derived from results obtained with standard irritants and contact allergens (Ulrich P, Streich J, Suter W (2001)). Calculations were performed in MS EXCEL and statistical analysis was performed with GraphPad Prism 4 (Kruskal-Wallis test, followed by the Mann Whitney test).

Table Classification of LLNA results
Mode Strong Moderate Weak
Rules for ear skin irritation TC < 0.1 % 0.1% ≤ TC < 5% 5% ≤ TC
Rules for LN hyperplasia TC < 1 % 1% ≤ TC < 5 % 5% ≤ TC

TC: threshold concentration.
The results were evaluated according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015).

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:

0.05% QAW039-C1

Remarks on result:

not measured/tested

Results 2

Key result

Parameter:

SI

Test group / Remarks:

0.5% QAW039-C1

Remarks on result:

not measured/tested

Results 3

Key result

Parameter:

SI

Test group / Remarks:

5% QAW039-C1

Remarks on result:

not measured/tested

Results 4

Key result

Parameter:

other: Ear weight index

Value:

1.01

Test group / Remarks:

0.05% QAW039-C1

Results 5

Key result

Parameter:

other: Ear weight index

Value:

1.03

Test group / Remarks:

0.5% QAW039-C1

Results 6

Key result

Parameter:

other: Ear weight index

Value:

1.02

Test group / Remarks:

5% QAW039-C1

Results 7

Key result

Parameter:

other: LN weight index

Value:

1.03

Test group / Remarks:

0.05% QAW039-C1

Results 8

Key result

Parameter:

other: LN weight index

Value:

1.06

Test group / Remarks:

0.5% QAW039-C1

Results 9

Key result

Parameter:

other: LN weight index

Value:

1.09

Test group / Remarks:

5% QAW039-C1

Results 10

Key result

Parameter:

other: Cell count index

Value:

1.2

Test group / Remarks:

0.05% QAW039-C1

Results 11

Key result

Parameter:

other: Cell count index

Value:

1.2

Test group / Remarks:

0.5% QAW039-C1

Results 12

Key result

Parameter:

other: Cell count index

Value:

1.33

Test group / Remarks:

5% QAW039-C1

Cellular proliferation data / Observations:

No irritation of the ears was noted after visual examination in the majority of animals, except for the very slight irritation of two animals treated at 0.05% and one animal treated at 5% on Day 3. The very slight irritation was considered not to have a toxicologically significant effect on the activity of the nodes. No further relevance was attached to this finding. Visual examination of the nodes revealed that all the nodes of the experimental animals were considered normal in size when compared to the vehicle control group. No macroscopic abnormalities of the surrounding areas were noted in any of the animals.
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. There were no clinical observations attributable to treatment with QAW039-C1.
QAW039-C1 did not cause any relevant changes in ear weight or LN weight up to a concentration of 5% in DAE433.
QAW039-C1 exceeded the LN count threshold at 5% with a statistically significant difference when compared to vehicle but did not show a clear dose response curve.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

In conclusion, QAW039-C1 appeared to be a moderate sensitizer (based on a threshold concentration for cell counts of 4.3%) without irritating potential in the murine LLNA TIER I.
Based on these results:
according to the recommendations made in the test guidelines (including all amendments), QAW039-C1 would be regarded as skin sensitizer.
- according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments), QAW039-C1 should be classified as skin sensitizer (Category 1B).