Methyl 3-amino-5,6-dichloropyrazine-2-carboxylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 January - 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-amino-5,6-dichloropyrazine-2-carboxylate

EC Number:

215-947-7

EC Name:

Methyl 3-amino-5,6-dichloropyrazine-2-carboxylate

Cas Number:

1458-18-0

Molecular formula:

C6H5Cl2N3O2

IUPAC Name:

methyl 3-amino-5,6-dichloropyrazine-2-carboxylate

Test material form:

solid: particulate/powder

Details on test material:

Identification: Amiloride Compound 7
Molecular formula: C6H5Cl2N3O2
Molecular weight: 222.03
CAS Number:1458-18-0
Description: Tan powder
Batch: 206-2
Purity/Composition: 99.7 area%
Test substance storage: In refrigerator (2-8°C) protected from light, desiccated
Stability under storage conditions: Stable
Expiry date: 11 May 2014 (Retest date)

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 labeled 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, 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.

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. 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

Value:

6.7

Test group / Remarks:

@ 10% conc.

Results 2

Parameter:

SI

Value:

3.1

Test group / Remarks:

@ 25% conc.

Results 3

Parameter:

SI

Value:

5.1

Test group / Remarks:

@ 50% conc.

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.

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

Other results - main study:

The very slight irritation of the ears as shown by some animals treated at 25 and 50% was considered not to have a toxicologically significant effect on the activity of the nodes. Brown staining by the test substance was noted in animals at 25 and 50%, which did not hamper scoring of the skin reactions.

 

The auricular lymph nodes of all animals treated at 10% and 50% and two animals treated at 25% appeared larger in size as compared to the control animals.

No macroscopic abnormalities of the surrounding area 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.

    

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines, Amiloride Compound 7 was considered to be a skin sensitiser, as the SI was ≥ 3 when tested up to 50%.

Executive summary:

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

Amiloride Compound 7 concentrations of 10, 25 or 50% w/w on three consecutive days, by open application on the ears. The very slight irritation of the ears as shown by some animals treated at 25 and 50% was considered not to have a toxicologically significant effect on the activity of the nodes. Brown staining by the test substance was noted in animals at 25 and 50%, which did not hamper scoring of the skin reactions. 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 auricular lymph nodes of all animals treated at 10% and 50% and two animals treated at 25% appeared larger in size as compared to the control animals. 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 3928, 1798 and 3018 DPM respectively. The mean DPM/animal value for the vehicle control group was 588 DPM. The SI values calculated for the substance concentrations 10, 25 and 50% were 6.7, 3.1 and 5.1 respectively. These results show that the test substance elicits an SI ≥ 3. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between 0 and 25%. According to the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures, Amiloride Compound 7 should be classified as skin sensitizer (Category 1B) and labeled as H317: May cause an allergic skin reaction.