3-aminopyrazine-2-carboxylic acid

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 January - 21 January, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2010)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2008)

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:

3-aminopyrazine-2-carboxylic acid

EC Number:

226-558-7

EC Name:

3-aminopyrazine-2-carboxylic acid

Cas Number:

5424-01-1

Molecular formula:

C5H5N3O2

IUPAC Name:

3-aminopyrazine-2-carboxylic acid

Details on test material:

- Name of test material (as cited in study report): Amiloride Compound 5
- Description: Tan powder

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: Young adult animals (approx. 9 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- Housing: Animals were group housed in labeld makrolon cages.
- 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

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Concentration:

0, 10, 25 and 50%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
Two test substance concentrations were tested; a 25% and 50% concentration. The highest concentration should cause no systemic toxicity and may give well-defined irritation at the most.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. The pre-weighed test substance and the formulations were wrapped in tin foil to protect from light. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.
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 applied.
Rationale for vehicle: The vehicle was selected based on trial formulations performed at WIL Research Europe and on test substance data supplied by the sponsor.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

Observations:
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 within 1 hour after dosing) according to the numerical scoring system. Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: No necropsy was performed according to protocol.

Positive control substance(s):

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

Statistics:

Not performed.

Results and discussion

Positive control results:

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research is an appropriate model for testing for contact hypersensitivity. See attached document 'Reliability check'.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: The SI values calculated for the substance concentrations 10, 25 and 50% were 0.8, 1.2 and 1.1 respectively.

Remarks:

No irritation observed

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 266, 392 and 359 DPM respectively. The mean DPM/animal value for the vehicle control group was 332 DPM.

Any other information on results incl. tables

Results Pre-screen test:

No irritation and no signs of systemic toxicity were observed in any of the animals examined. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Brown test substance remnants were present on the dorsal surface of the ears of both animals at 25 and 50% (Days 1,2 and 3), which did not hamper scoring of the skin reactions.

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

Other results - main study:

No irritation of the ears was observed in any of the animals examined. Brown test substance remnants were present on the dorsal surface of the ears of animals at 10, 25 and 50% (Days 1,2 and/or 3), which did not hamper scoring of the skin reactions.

 

All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. Except that one control animal showed one slightly smaller lymph node (animal excluded for evaluation).

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

 

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 for one control animal was considered not toxicologically significant since the change was slight in nature. However, since this animal showed also one smaller lymph node and a very low DPM value, this animal was excluded for evaluation of the sensitization potential.    

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines, Amiloride Compound 5 was considered not be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 50%.

Executive summary:

In an LLNA skin sensitisation study, performed according to OECD 429 test guidelines, female CBA/J mice were treated with

Amiloride Compound 5 concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. No irritation of the ears was observed in any of the animals examined. Brown test substance remnants were present on the dorsal surface of the ears of animals at 10, 25 and 50% (Days 1,2 and/or 3), which did not hamper scoring of the skin reactions. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. Except that one control animal showed one slightly smaller lymph node (DPM value was too low and considered to be an outlier). 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 266, 392 and 359 DPM respectively. The mean DPM/animal value for the vehicle control group was 332 DPM. The SI values calculated for the substance concentrations 10, 25 and 50% were 0.8, 1.2 and 1.1 respectively. There was no indication that the test substance elicits an SI ≥ 3 when tested up to 50%. Based on these results, Amiloride Compound 5 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 Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.