Methyl 3-aminopyrazinecarboxylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 January, 2013 - 28 January, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

Methyl 3-aminopyrazinecarboxylate

EC Number:

240-387-5

EC Name:

Methyl 3-aminopyrazinecarboxylate

Cas Number:

16298-03-6

Molecular formula:

C6H7N3O2

IUPAC Name:

methyl 3-aminopyrazine-2-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): Amiloride Compound 6
- Description: Tan powder
- Storage condition of test material: In refrigerator (2-8°C) protected from light, desiccated
- Other: Reactive to light, moisture and oxygen

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. 9-11 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)

Vehicle:

propylene glycol

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 was the maximum concentration as required in the test guidelines (undiluted for liquids, 50% for solids).
The test system, procedures and techniques were identical to those used in the main study except that assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). 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 prior to dosing on Days 1 and 3, and on Day 6.
Animals were sacrificed after the final observation.

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.

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, while protected from light as much as possible by using aluminum foil. 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 following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.

Necropsy: No necropsy was performed.

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 Europe B.V. is an appropriate model for testing for contact hypersensitivity. See attached document 'Reliability check'.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.7

Test group / Remarks:

@10% concentration

Results 2

Parameter:

SI

Value:

1

Test group / Remarks:

@25% concentration

Results 3

Parameter:

SI

Value:

1.1

Test group / Remarks:

@ 50% concentration

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, 3 and/or 4), 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 all test substance treated animals (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. 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 animal was considered not toxicologically significant since it was an incidental finding and the change was slight in nature.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an LLNA skin sensitisation study, performed according to OECD 429 test guideline and GLP principles, the substance was considered not be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 50%.

Executive summary:

Amiloride Compound 6 was assessed in an LLNA skin sensitisation study, performed according to OECD 429 test guideline and GLP principles.

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 all test substance treated animals (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. 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.

The SI values calculated for the substance concentrations 10, 25 and 50% were 1.7, 1.0 and 1.1 respectively. Since there was no indication that the test substance elicits an SI ≥ 3 when tested up to 50%, Amiloride Compound 6 was considered not to be a skin sensitizer.

Based on the results, the test substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures.